RNAi targeting <i>Caenorhabditis elegans</i> α-arrestins marginally affects lifespan

Sangsoon Park; Yoonji Jung; Seon Woo A. An; Heehwa G. Son; Wooseon Hwang; Dongyeop Lee; Murat Artan; Hae-Eun H. Park; Dae-Eun Jeong; Yujin Lee; Seung-Jae V. Lee

doi:10.12688/f1000research.12337.1

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Research Article

RNAi targeting Caenorhabditis elegans α-arrestins marginally affects lifespan

[version 1; peer review: 2 approved with reservations]

Sangsoon Park¹, Yoonji Jung¹, Seon Woo A. An¹, [...] Heehwa G. Son¹, Wooseon Hwang¹, Dongyeop Lee¹, Murat Artan¹, Hae-Eun H. Park¹, Dae-Eun Jeong¹, Yujin Lee¹, Seung-Jae V. Lee ^1,2

Sangsoon Park¹, Yoonji Jung¹, [...] Seon Woo A. An¹, Heehwa G. Son¹, Wooseon Hwang¹, Dongyeop Lee¹, Murat Artan¹, Hae-Eun H. Park¹, Dae-Eun Jeong¹, Yujin Lee¹, Seung-Jae V. Lee ^1,2

PUBLISHED 18 Aug 2017

Author details Author details

¹ Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk, 37673, South Korea
² School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, 37673, South Korea

Sangsoon Park
Roles: Conceptualization, Formal Analysis, Investigation, Project Administration, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Yoonji Jung
Roles: Investigation, Validation, Writing – Review & Editing

Seon Woo A. An
Roles: Investigation

Heehwa G. Son
Roles: Investigation

Wooseon Hwang
Roles: Investigation

Dongyeop Lee
Roles: Investigation

Murat Artan
Roles: Investigation

Hae-Eun H. Park
Roles: Investigation

Dae-Eun Jeong
Roles: Investigation

Yujin Lee
Roles: Investigation

Seung-Jae V. Lee
Roles: Conceptualization, Funding Acquisition, Project Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: α-arrestins are a family of proteins that are implicated in multiple biological processes, including metabolism and receptor desensitization. Methods: Here, we sought to examine the roles of α-arrestins in the longevity of Caenorhabditis elegans through an RNA interference screen. Results: We found that knocking down each of 24 out of total 29 C. elegans α-arrestins had small or no effects on lifespan. Thus, individual C. elegans α-arrestins may have minor effects on longevity. Conclusions: This study will provide useful information for future research on the functional role of α-arrestins in aging and longevity.

Keywords

C. elegans, α-arrestin, insulin/IGF-1 signaling, aging, lifespan

Corresponding author: Seung-Jae V. Lee

Competing interests: No competing interests were disclosed.

Grant information: This work was supported by the Korean Government (MSIP) through the National Research Foundation of Korea (NRF) [NRF-2016R1E1A1A01941152] funded to S-J.V.L.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2017 Park S et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

How to cite: Park S, Jung Y, An SWA et al. RNAi targeting Caenorhabditis elegans α-arrestins marginally affects lifespan [version 1; peer review: 2 approved with reservations]. F1000Research 2017, 6:1515 (https://doi.org/10.12688/f1000research.12337.1) First published: 18 Aug 2017, 6:1515 (https://doi.org/10.12688/f1000research.12337.1) Latest published: 08 Dec 2017, 6:1515 (https://doi.org/10.12688/f1000research.12337.4)

Introduction

α-arrestins are a family of proteins that contain arrestin domains whose sequences and structures have similarities with those of classical visual and β-arrestins^1–3. α-arrestins are considered as ancestral forms of arrestins because their orthologs exist in fungi, including yeast, which do not have visual or β-arrestins^1,4. Several mammalian α-arrestins modulate metabolism and receptor desensitization^5,6, but much remains to be elucidated concerning the functions of α-arrestins in many organisms. The human genome encodes 6 α-arrestins and 4 visual or β-arrestins. Interestingly, the simple roundworm Caenorhabditis elegans genome contains 29 α-arrestin and 1 β-arrestin genes^1,7. Therefore, the C. elegans system provides opportunities for the genetic analysis of α-arrestins in various aspects of physiology both individually and combinatorially. However, information regarding the functions of C. elegans α-arrestins is limited^8,9.

C. elegans is an excellent genetic model organism that has been exploited for studying conserved biological processes, including apoptosis, behavior, development and aging. In particular, its short lifespan in combination with genetic amenability has made C. elegans one of the most popular models for research on aging and longevity^10,11. Many factors, including components in insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS), have been identified as lifespan and aging regulators in C. elegans^11–13. For example, genetic inhibition of IIS components, such as DAF-2/insulin/IGF-1 receptor, robustly extends lifespan and delays physiological aging through up-regulating transcription factors, including DAF-16/FOXO^11–13. Importantly, the roles of these aging-regulatory factors in C. elegans have been shown to be conserved in other species, including Drosophila and mammals^12,13. One of the powerful ways to identify novel factors that influence aging is by employing genetic screens, such as an RNA interference (RNAi) screen. We previously identified several genetic factors, including RNA helicases, that modulate longevity in C. elegans, through targeted or genome-wide RNAi screens^14–17. Because of its robust longevity phenotype that confers sensitivity to changes in lifespan, daf-2/insulin/IGF-1 receptor mutants serve as an excellent platform for the identification of novel lifespan-regulating factors^17,18.

In this study, we aimed to determine whether any α-arrestins played a role in the lifespan regulation of wild-type or daf-2 mutants. We performed a lifespan assay-based RNAi screen targeting 24 out of 29 C. elegans α-arrestins. We found that knocking down each of the α-arrestins had small or no effects on the lifespan of wild-type and daf-2 mutants. Thus, C. elegans α-arrestins may play minor or modulatory roles in lifespan regulation. Based on our results, it will be important to test the roles of α-arrestins in combinatorial manners and/or by using strong loss-of-function mutations in future research.

Methods

Caenorhabditis elegans strains

All strains were maintained as previously described¹⁹. The following strains were used in this study: N2 wild-type, CF1041 daf-2(e1370) III outcrossed six times to N2.

Phylogenetic analysis

The protein sequences of 27 α-arrestins, except arrd (arrestin domain protein)-20 and arrd-21, were obtained from Wormbase (www.wormbase.org, version WS259). The protein sequences of arrd-20 and arrd-21 were obtained from Ensembl (http://www.ensembl.org, release 89). The phylogenetic tree of 29 α-arrestins in C. elegans was generated using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/)²⁰ and re-visualized using the Dendroscope 3 (version 3.5.9)²¹. For the α-arrestins that have multiple isoforms, isoform a was used for the analysis.

RNAi clones

Twenty one RNAi clones that target C. elegans α-arrestin genes were used from two commercial C. elegans RNAi feeding libraries. Specifically, RNAi clones targeting arrd-2, arrd-6, arrd-7, arrd-8, arrd-9, arrd-10, arrd-13, arrd-16, arrd-18, arrd-23, arrd-24, arrd-25, arrd-28 and ttm-2 (toxin-regulated targets of MAPK 2) were obtained from Ahringer laboratory library (Geneservice Ltd., Cambridge, UK), while those against arrd-1, arrd-3, arrd-4, arrd-5, arrd-14, arrd-15 and arrd-19 were from Vidal laboratory library (Source BioScience, Nottingham, UK). Three RNAi clones targeting arrd-11, arrd-17, and arrd-26 were generated by molecular cloning using infusion recombinase (EZ-Fusion^TM Cloning Kit, Enzynomics, Daejeon, South Korea). The N2 genomic DNA was obtained through the lysis of worms using proteinase K (Invitrogen, Carlsbad, CA, USA) and N2 complementary DNA was obtained from RNA extraction using RNAiso Plus (Takara, Shiga, Japan) followed by reverse transcription using ImProm-II reverse transcriptase (Promega, Madison, WI, USA, USA). The infusion reaction between PCR products and pL4440 plasmids (Fire lab C. elegans vector kit, 1999) digested with HindIII (New England Biolabs, Ipswich, MA, USA) and Acc65I (New England Biolabs) was followed by transformation of in-house competent E. coli HT115 cells and by selection of positive clones on ampicillin (USB, Santa Clara, CA, USA)-containing LB plates. Primers (CosmoGenetech, Seoul, South Korea) that were used to amplify coding regions of arrd-11 from N2 genomic DNA, and those of arrd-17 and arrd-26 from N2 complementary DNA are as follows: forward primer 5’-GAATTCGATATCAAGCTCCCTCGTGCAAATTAGGAAA-3’ and reverse primer 5’-CTATAGGGCGAATTGGGGTTCCTCCCACTCCATACA-3’ for arrd-11; forward primer 5’-GAATTCGATATCAAGCTATGGTGCAGTTAGATCGTTTTG-3’ and reverse primer 5’-CTATAGGGCGAATTGGTTAATCGGTATAAAATGG-3’ for arrd-17; forward primer 5’-GAATTCGATATCAAGCTATGAAGGTCGATTACTTCG-3’ and reverse primer 5’- CTATAGGGCGAATTGGCTACTTCTCGGAGCCATTTG-3’ for arrd-26. The sequences of all these 24 α-arrestin RNAi clones were confirmed using DNA sequencing (Solgent, Daejeon, South Korea) before lifespan assays. RNAi clones targeting remaining five α-arrestin genes were not generated.

RNAi screen using lifespan assay

The RNAi screen employing lifespan assay was performed as previously described¹⁷. Briefly, E. coli HT115 bacteria that expressed specific double-stranded RNAs (dsRNAs) were cultured overnight at 37°C in LB media containing 50 μg/ml ampicillin (USB). The cultured bacteria were seeded onto nematode growth media plates containing 50 μg/ml ampicillin and incubated overnight at 37°C. The seeded bacteria were treated with 1 mM isopropyl β-D-1-thiogalactopyranoside (Gold Biotechnology, St. Louis, MO, USA) and incubated at room temperature for approximately 24 h to induce dsRNAs. Age-synchronized wild-type N2 and daf-2(e1370) mutant animals were grown on RNAi plates from embryo to L4 stage at 20°C. Worms were then transferred onto RNAi plates containing 5 μM 5-fluoro-2’deoxyuridine (FUdR; Sigma, St. Louis, MO, USA), which prevents eggs from hatching, at young (day 1) adult stage, and transferred again to new FUdR-containing RNAi plates after 1 or 2 days. All lifespan assays were performed at 20°C as duplicates by two independent researchers. The survival of worms was determined by gently touching worms with a platinum wire. Worms that did not respond were counted as dead worms and removed from the plates. Worms that crawled off the plates, burrowed into the agar media, or displayed internal hatching or vulval rupture were censored, but included in the statistical analysis. Lifespan data from two independent lifespan experiments within the experimental sets were pooled and used for statistical analysis (see Dataset 1–Dataset 3). OASIS 2 (Online Application for Survival Analysis 2; https://sbi.postech.ac.kr/oasis2)²² was used for the statistical analysis of lifespan results. P values were calculated using long-rank (Mantel-Cox method) test.

Results

Knocking down each of several C. elegans α-arrestins marginally influenced lifespan

We measured the lifespan of wild-type and long-lived daf-2 mutant animals upon knocking down each of 24 of 29 genes encoding putative α-arrestin proteins (Figure 1A). We used daf-16 RNAi that largely suppressed the longevity of daf-2(-) mutants as a positive control (Figure 1B, Figure S1A, Table S1)^17,18. We found that knockdown of each of individual α-arrestin genes tended to slightly reduce lifespan in wild-type or in daf-2(-) mutants (Figure 1B, Figure S1B–U, Table S1). Out of the 24 RNAi clones, RNAi targeting arrd (arrestin domain protein)-13, arrd-16, arrd-23, arrd-24 or arrd-25 in wild-type, and RNAi against arrd-1, arrd-2, arrd-5, arrd-24 or arrd-28 in daf-2(-) mutant animals decreased lifespan by more than 5% (Figure 1B–E, Figures S1B, D, K, Q, R, T, Table S1). Specifically, arrd-16 RNAi decreased lifespan in wild-type by 9%, and arrd-1 RNAi decreased lifespan in daf-2(-) mutants by 7% (Figure 1B and C). In addition, RNAi targeting arrd-24 decreased the lifespan of wild-type and daf-2 mutant animals by 11% and 6%, respectively (Figure 1E). In contrast, arrd-3 RNAi increased the lifespan of wild-type animals by 10% (Figure 1F). Overall, genetic inhibition of individual α-arrestin genes using RNAi appears to have minor effects on C. elegans lifespan.

Figure 1. RNAi targeting each of Caenorhabditis elegans α-arrestins had small or no effects on lifespan.

(A) A phylogenetic tree showing C. elegans α-arrestin family members. Asterisks (*) indicate α-arrestins that were not examined in this study and number signs (#) indicate predicted pseudogenes. (B) Each circle represents percent mean lifespan change by treatment with individual α-arrestin gene RNAi in wild-type (WT) and daf-2(e1370) (daf-2(-)) mutants. Red triangles indicate lifespan changes by daf-16 RNAi, which was used as a positive control. (C–F) Lifespan curves of WT and daf-2(-) animals treated with RNAi targeting arrd-16 (C), arrd-1 (D), arrd- 24 (E), and arrd-3 (F). See Table S1 for statistical analysis.

Set	Strain	Treatment	Days	At Risk	Dead	Censored	Percent Mortality	S_hat	Var S_hat	SE(S)	Survival Time	Percent Alive
1	N2	Control RNAi	0	240	0	0	0	1	0	0	0	100
1	N2	Control RNAi	1	240	0	0	0	1	0	0	0	100
1	N2	Control RNAi	2	240	0	0	0	1	0	0	0	100
1	N2	Control RNAi	4	240	1	15	0.42	0.996	0	0.004	0.017	99.6
1	N2	Control RNAi	5	224	0	2	0.42	0.996	0	0.004	0.017	99.6
1	N2	Control RNAi	6	222	2	19	1.31	0.987	0	0.008	0.07	98.7
1	N2	Control RNAi	7	201	0	1	1.31	0.987	0	0.008	0.07	98.7
1	N2	Control RNAi	8	200	2	10	2.3	0.977	0	0.01	0.149	97.7
1	N2	Control RNAi	10	188	6	2	5.42	0.946	0	0.016	0.461	94.6
1	N2	Control RNAi	11	180	3	10	7	0.93	0	0.018	0.635	93
1	N2	Control RNAi	12	167	10	1	12.56	0.874	0.001	0.024	1.303	87.4
1	N2	Control RNAi	14	156	44	4	37.23	0.628	0.001	0.036	4.756	62.8
1	N2	Control RNAi	16	108	28	2	53.5	0.465	0.001	0.038	7.36	46.5
1	N2	Control RNAi	18	78	21	1	66.02	0.34	0.001	0.036	9.613	34
1	N2	Control RNAi	20	56	14	1	74.51	0.255	0.001	0.033	11.312	25.5
1	N2	Control RNAi	22	41	5	0	77.62	0.224	0.001	0.032	11.996	22.4
1	N2	Control RNAi	23	36	17	0	88.19	0.118	0.001	0.025	14.426	11.8
1	N2	Control RNAi	24	19	5	0	91.3	0.087	0	0.022	15.172	8.7
1	N2	Control RNAi	25	14	7	0	95.65	0.044	0	0.016	16.26	4.4
1	N2	Control RNAi	26	7	3	0	97.51	0.025	0	0.012	16.745	2.5
1	N2	Control RNAi	28	4	4	0	100	0	0	0	17.441	0
1	N2	daf-16 RNAi	0	240	0	0	0	1	0	0	0	100
1	N2	daf-16 RNAi	1	240	0	0	0	1	0	0	0	100
1	N2	daf-16 RNAi	2	240	0	0	0	1	0	0	0	100
1	N2	daf-16 RNAi	4	240	2	28	0.83	0.992	0	0.006	0.033	99.2
1	N2	daf-16 RNAi	5	210	0	2	0.83	0.992	0	0.006	0.033	99.2
1	N2	daf-16 RNAi	6	208	1	20	1.31	0.987	0	0.008	0.062	98.7
1	N2	daf-16 RNAi	7	187	0	5	1.31	0.987	0	0.008	0.062	98.7
1	N2	daf-16 RNAi	8	182	4	7	3.48	0.965	0	0.013	0.235	96.5
1	N2	daf-16 RNAi	10	171	9	1	8.56	0.914	0	0.021	0.743	91.4
1	N2	daf-16 RNAi	11	161	37	9	29.57	0.704	0.001	0.034	3.055	70.4
1	N2	daf-16 RNAi	12	115	10	2	35.7	0.643	0.001	0.036	3.79	64.3
1	N2	daf-16 RNAi	14	103	48	5	65.66	0.343	0.001	0.037	7.985	34.3
1	N2	daf-16 RNAi	16	50	25	3	82.83	0.172	0.001	0.031	10.732	17.2
1	N2	daf-16 RNAi	18	22	12	0	92.2	0.078	0.001	0.023	12.418	7.8
1	N2	daf-16 RNAi	20	10	7	0	97.66	0.023	0	0.013	13.51	2.3
1	N2	daf-16 RNAi	22	3	3	0	100	0	0	0	14.025	0
1	N2	arrd-1 RNAi	0	240	0	0	0	1	0	0	0	100
1	N2	arrd-1 RNAi	1	240	0	0	0	1	0	0	0	100
1	N2	arrd-1 RNAi	2	240	0	0	0	1	0	0	0	100
1	N2	arrd-1 RNAi	4	240	0	12	0	1	0	0	0	100
1	N2	arrd-1 RNAi	5	228	0	3	0	1	0	0	0	100
1	N2	arrd-1 RNAi	6	225	1	38	0.44	0.996	0	0.004	0.027	99.6
1	N2	arrd-1 RNAi	7	186	0	1	0.44	0.996	0	0.004	0.027	99.6
1	N2	arrd-1 RNAi	8	185	0	12	0.44	0.996	0	0.004	0.027	99.6
1	N2	arrd-1 RNAi	10	173	2	0	1.6	0.984	0	0.009	0.142	98.4
1	N2	arrd-1 RNAi	11	171	17	7	11.38	0.886	0.001	0.024	1.218	88.6
1	N2	arrd-1 RNAi	12	147	5	2	14.39	0.856	0.001	0.027	1.58	85.6
1	N2	arrd-1 RNAi	14	140	36	3	36.41	0.636	0.001	0.037	4.661	63.6
1	N2	arrd-1 RNAi	16	101	25	1	52.15	0.479	0.002	0.039	7.18	47.9
1	N2	arrd-1 RNAi	18	75	26	2	68.74	0.313	0.001	0.037	10.166	31.3
1	N2	arrd-1 RNAi	20	47	10	1	75.39	0.246	0.001	0.034	11.496	24.6
1	N2	arrd-1 RNAi	22	36	10	0	82.22	0.178	0.001	0.031	13.001	17.8
1	N2	arrd-1 RNAi	23	26	14	0	91.8	0.082	0.001	0.022	15.202	8.2
1	N2	arrd-1 RNAi	24	12	6	0	95.9	0.041	0	0.016	16.186	4.1
1	N2	arrd-1 RNAi	25	6	1	0	96.58	0.034	0	0.015	16.357	3.4
1	N2	arrd-1 RNAi	26	5	4	0	99.32	0.007	0	0.007	17.068	0.7
1	N2	arrd-1 RNAi	28	1	1	0	100	0	0	0	17.26	0
1	N2	arrd-2 RNAi	0	240	0	0	0	1	0	0	0	100
1	N2	arrd-2 RNAi	1	240	0	0	0	1	0	0	0	100
1	N2	arrd-2 RNAi	2	240	0	0	0	1	0	0	0	100
1	N2	arrd-2 RNAi	4	240	0	13	0	1	0	0	0	100
1	N2	arrd-2 RNAi	5	227	0	0	0	1	0	0	0	100
1	N2	arrd-2 RNAi	6	227	1	30	0.44	0.996	0	0.004	0.026	99.6
1	N2	arrd-2 RNAi	7	196	0	0	0.44	0.996	0	0.004	0.026	99.6
1	N2	arrd-2 RNAi	8	196	0	18	0.44	0.996	0	0.004	0.026	99.6
1	N2	arrd-2 RNAi	10	178	3	3	2.12	0.979	0	0.011	0.194	97.9
1	N2	arrd-2 RNAi	11	172	16	2	11.22	0.888	0.001	0.024	1.196	88.8
1	N2	arrd-2 RNAi	12	154	4	0	13.53	0.865	0.001	0.026	1.473	86.5
1	N2	arrd-2 RNAi	14	150	36	3	34.28	0.657	0.001	0.036	4.378	65.7
1	N2	arrd-2 RNAi	16	111	22	0	47.31	0.527	0.001	0.038	6.462	52.7
1	N2	arrd-2 RNAi	18	89	28	0	63.88	0.361	0.001	0.037	9.446	36.1
1	N2	arrd-2 RNAi	20	61	20	0	75.73	0.243	0.001	0.033	11.814	24.3
1	N2	arrd-2 RNAi	22	41	7	0	79.87	0.201	0.001	0.031	12.726	20.1
1	N2	arrd-2 RNAi	23	34	21	0	92.3	0.077	0	0.02	15.585	7.7
1	N2	arrd-2 RNAi	24	13	5	0	95.26	0.047	0	0.016	16.296	4.7
1	N2	arrd-2 RNAi	25	8	2	0	96.45	0.036	0	0.014	16.592	3.6
1	N2	arrd-2 RNAi	26	6	4	0	98.82	0.012	0	0.008	17.208	1.2
1	N2	arrd-2 RNAi	28	2	2	0	100	0	0	0	17.539	0
1	N2	arrd-3 RNAi	0	240	0	0	0	1	0	0	0	100
1	N2	arrd-3 RNAi	1	240	0	0	0	1	0	0	0	100
1	N2	arrd-3 RNAi	2	240	0	0	0	1	0	0	0	100
1	N2	arrd-3 RNAi	4	240	0	6	0	1	0	0	0	100
1	N2	arrd-3 RNAi	5	234	0	1	0	1	0	0	0	100
1	N2	arrd-3 RNAi	6	233	2	23	0.86	0.991	0	0.006	0.052	99.1
1	N2	arrd-3 RNAi	7	208	0	0	0.86	0.991	0	0.006	0.052	99.1
1	N2	arrd-3 RNAi	8	208	2	13	1.81	0.982	0	0.009	0.128	98.2
1	N2	arrd-3 RNAi	10	193	2	1	2.83	0.972	0	0.011	0.23	97.2
1	N2	arrd-3 RNAi	11	190	7	0	6.41	0.936	0	0.017	0.623	93.6
1	N2	arrd-3 RNAi	12	183	4	1	8.45	0.915	0	0.02	0.869	91.5
1	N2	arrd-3 RNAi	14	178	27	8	22.34	0.777	0.001	0.03	2.813	77.7
1	N2	arrd-3 RNAi	16	143	20	3	33.2	0.668	0.001	0.034	4.551	66.8
1	N2	arrd-3 RNAi	18	120	25	1	47.12	0.529	0.001	0.037	7.056	52.9
1	N2	arrd-3 RNAi	20	94	18	0	57.24	0.428	0.001	0.037	9.081	42.8
1	N2	arrd-3 RNAi	22	76	18	0	67.37	0.326	0.001	0.035	11.309	32.6
1	N2	arrd-3 RNAi	23	58	32	1	85.37	0.146	0.001	0.026	15.449	14.6
1	N2	arrd-3 RNAi	24	25	10	0	91.22	0.088	0	0.021	16.853	8.8
1	N2	arrd-3 RNAi	25	15	5	0	94.15	0.059	0	0.018	17.585	5.9
1	N2	arrd-3 RNAi	26	10	4	0	96.49	0.035	0	0.014	18.193	3.5
1	N2	arrd-3 RNAi	28	6	5	0	99.41	0.006	0	0.006	19.012	0.6
1	N2	arrd-3 RNAi	30	1	1	0	100	0	0	0	19.188	0
1	N2	arrd-4 RNAi	0	240	0	0	0	1	0	0	0	100
1	N2	arrd-4 RNAi	1	240	0	0	0	1	0	0	0	100
1	N2	arrd-4 RNAi	2	240	0	0	0	1	0	0	0	100
1	N2	arrd-4 RNAi	4	240	0	15	0	1	0	0	0	100
1	N2	arrd-4 RNAi	5	225	0	2	0	1	0	0	0	100
1	N2	arrd-4 RNAi	6	223	1	22	0.45	0.996	0	0.004	0.027	99.6
1	N2	arrd-4 RNAi	7	200	0	0	0.45	0.996	0	0.004	0.027	99.6
1	N2	arrd-4 RNAi	8	200	2	15	1.44	0.986	0	0.008	0.107	98.6
1	N2	arrd-4 RNAi	10	183	4	5	3.6	0.964	0	0.013	0.322	96.4
1	N2	arrd-4 RNAi	11	174	20	3	14.68	0.853	0.001	0.026	1.541	85.3
1	N2	arrd-4 RNAi	12	151	6	2	18.07	0.819	0.001	0.029	1.948	81.9
1	N2	arrd-4 RNAi	14	143	24	4	31.82	0.682	0.001	0.035	3.873	68.2
1	N2	arrd-4 RNAi	16	115	17	3	41.9	0.581	0.001	0.037	5.485	58.1
1	N2	arrd-4 RNAi	18	95	17	1	52.3	0.477	0.001	0.038	7.357	47.7
1	N2	arrd-4 RNAi	20	77	21	0	65.31	0.347	0.001	0.037	9.959	34.7
1	N2	arrd-4 RNAi	22	56	11	0	72.12	0.279	0.001	0.035	11.458	27.9
1	N2	arrd-4 RNAi	23	45	22	0	85.75	0.142	0.001	0.027	14.593	14.2
1	N2	arrd-4 RNAi	24	23	5	0	88.85	0.112	0.001	0.025	15.336	11.2
1	N2	arrd-4 RNAi	25	18	4	0	91.33	0.087	0	0.022	15.956	8.7
1	N2	arrd-4 RNAi	26	14	5	0	94.42	0.056	0	0.018	16.761	5.6
1	N2	arrd-4 RNAi	28	9	8	0	99.38	0.006	0	0.006	18.149	0.6
1	N2	arrd-4 RNAi	30	1	1	0	100	0	0	0	18.335	0
1	N2	arrd-5 RNAi	0	240	0	0	0	1	0	0	0	100
1	N2	arrd-5 RNAi	1	240	0	0	0	1	0	0	0	100
1	N2	arrd-5 RNAi	2	240	0	0	0	1	0	0	0	100
1	N2	arrd-5 RNAi	4	240	0	9	0	1	0	0	0	100
1	N2	arrd-5 RNAi	5	231	0	3	0	1	0	0	0	100
1	N2	arrd-5 RNAi	6	228	5	27	2.19	0.978	0	0.01	0.132	97.8
1	N2	arrd-5 RNAi	7	196	1	1	2.69	0.973	0	0.011	0.167	97.3
1	N2	arrd-5 RNAi	8	194	0	15	2.69	0.973	0	0.011	0.167	97.3
1	N2	arrd-5 RNAi	10	179	3	3	4.32	0.957	0	0.014	0.33	95.7
1	N2	arrd-5 RNAi	11	173	9	7	9.3	0.907	0	0.021	0.877	90.7
1	N2	arrd-5 RNAi	12	157	2	0	10.46	0.895	0	0.022	1.016	89.5
1	N2	arrd-5 RNAi	14	155	41	6	34.14	0.659	0.001	0.036	4.332	65.9
1	N2	arrd-5 RNAi	16	108	19	2	45.73	0.543	0.001	0.038	6.186	54.3
1	N2	arrd-5 RNAi	18	87	25	1	61.32	0.387	0.001	0.038	8.993	38.7
1	N2	arrd-5 RNAi	20	61	30	2	80.34	0.197	0.001	0.031	12.797	19.7
1	N2	arrd-5 RNAi	22	29	9	0	86.44	0.136	0.001	0.027	14.139	13.6
1	N2	arrd-5 RNAi	23	20	11	1	93.9	0.061	0	0.019	15.854	6.1
1	N2	arrd-5 RNAi	24	8	2	0	95.43	0.046	0	0.017	16.22	4.6
1	N2	arrd-5 RNAi	25	6	4	0	98.48	0.015	0	0.011	16.982	1.5
1	N2	arrd-5 RNAi	26	2	1	0	99.24	0.008	0	0.008	17.181	0.8
1	N2	arrd-5 RNAi	28	1	1	0	100	0	0	0	17.394	0
1	daf-2(e1370)	Control RNAi	0	210	0	0	0	1	0	0	0	100
1	daf-2(e1370)	Control RNAi	1	210	0	0	0	1	0	0	0	100
1	daf-2(e1370)	Control RNAi	2	210	0	0	0	1	0	0	0	100
1	daf-2(e1370)	Control RNAi	4	210	0	6	0	1	0	0	0	100
1	daf-2(e1370)	Control RNAi	7	204	0	8	0	1	0	0	0	100
1	daf-2(e1370)	Control RNAi	8	196	0	0	0	1	0	0	0	100
1	daf-2(e1370)	Control RNAi	10	196	0	4	0	1	0	0	0	100
1	daf-2(e1370)	Control RNAi	11	192	0	3	0	1	0	0	0	100
1	daf-2(e1370)	Control RNAi	13	189	0	4	0	1	0	0	0	100
1	daf-2(e1370)	Control RNAi	14	185	1	3	0.54	0.995	0	0.005	0.076	99.5
1	daf-2(e1370)	Control RNAi	16	181	2	1	1.64	0.984	0	0.009	0.252	98.4
1	daf-2(e1370)	Control RNAi	18	178	0	0	1.64	0.984	0	0.009	0.252	98.4
1	daf-2(e1370)	Control RNAi	19	178	1	0	2.19	0.978	0	0.011	0.357	97.8
1	daf-2(e1370)	Control RNAi	20	177	0	6	2.19	0.978	0	0.011	0.357	97.8
1	daf-2(e1370)	Control RNAi	22	171	1	0	2.76	0.972	0	0.012	0.482	97.2
1	daf-2(e1370)	Control RNAi	23	170	0	2	2.76	0.972	0	0.012	0.482	97.2
1	daf-2(e1370)	Control RNAi	25	168	2	17	3.92	0.961	0	0.015	0.772	96.1
1	daf-2(e1370)	Control RNAi	28	149	4	2	6.5	0.935	0	0.019	1.494	93.5
1	daf-2(e1370)	Control RNAi	30	143	0	2	6.5	0.935	0	0.019	1.494	93.5
1	daf-2(e1370)	Control RNAi	31	141	7	0	11.14	0.889	0.001	0.025	2.933	88.9
1	daf-2(e1370)	Control RNAi	33	134	1	0	11.81	0.882	0.001	0.026	3.152	88.2
1	daf-2(e1370)	Control RNAi	34	133	6	1	15.78	0.842	0.001	0.029	4.504	84.2
1	daf-2(e1370)	Control RNAi	36	126	2	1	17.12	0.829	0.001	0.03	4.986	82.9
1	daf-2(e1370)	Control RNAi	37	123	7	1	21.84	0.782	0.001	0.033	6.731	78.2
1	daf-2(e1370)	Control RNAi	38	115	4	1	24.56	0.754	0.001	0.035	7.764	75.4
1	daf-2(e1370)	Control RNAi	40	110	10	0	31.42	0.686	0.001	0.038	10.507	68.6
1	daf-2(e1370)	Control RNAi	41	100	3	0	33.47	0.665	0.001	0.038	11.351	66.5
1	daf-2(e1370)	Control RNAi	43	97	12	0	41.7	0.583	0.002	0.04	14.89	58.3
1	daf-2(e1370)	Control RNAi	44	85	6	1	45.82	0.542	0.002	0.041	16.701	54.2
1	daf-2(e1370)	Control RNAi	46	78	23	0	61.79	0.382	0.002	0.04	24.05	38.2
1	daf-2(e1370)	Control RNAi	49	55	26	0	79.86	0.201	0.001	0.033	32.9	20.1
1	daf-2(e1370)	Control RNAi	51	29	10	0	86.8	0.132	0.001	0.028	36.442	13.2
1	daf-2(e1370)	Control RNAi	52	19	6	0	90.97	0.09	0.001	0.024	38.61	9
1	daf-2(e1370)	Control RNAi	54	13	5	0	94.44	0.056	0	0.019	40.485	5.6
1	daf-2(e1370)	Control RNAi	55	8	3	0	96.53	0.035	0	0.015	41.631	3.5
1	daf-2(e1370)	Control RNAi	57	5	2	0	97.92	0.021	0	0.012	42.423	2.1
1	daf-2(e1370)	Control RNAi	60	3	2	0	99.31	0.007	0	0.007	43.257	0.7
1	daf-2(e1370)	Control RNAi	63	1	1	0	100	0	0	0	43.694	0
1	daf-2(e1370)	daf-16 RNAi	0	240	0	0	0	1	0	0	0	100
1	daf-2(e1370)	daf-16 RNAi	1	240	0	0	0	1	0	0	0	100
1	daf-2(e1370)	daf-16 RNAi	2	240	0	0	0	1	0	0	0	100
1	daf-2(e1370)	daf-16 RNAi	4	240	0	3	0	1	0	0	0	100
1	daf-2(e1370)	daf-16 RNAi	7	237	4	12	1.69	0.983	0	0.008	0.118	98.3
1	daf-2(e1370)	daf-16 RNAi	8	221	0	1	1.69	0.983	0	0.008	0.118	98.3
1	daf-2(e1370)	daf-16 RNAi	10	220	4	2	3.48	0.965	0	0.012	0.297	96.5
1	daf-2(e1370)	daf-16 RNAi	11	214	4	1	5.28	0.947	0	0.015	0.495	94.7
1	daf-2(e1370)	daf-16 RNAi	13	209	2	2	6.19	0.938	0	0.016	0.613	93.8
1	daf-2(e1370)	daf-16 RNAi	14	205	8	2	9.85	0.902	0	0.02	1.126	90.2
1	daf-2(e1370)	daf-16 RNAi	16	195	11	1	14.93	0.851	0.001	0.024	1.939	85.1
1	daf-2(e1370)	daf-16 RNAi	18	183	10	0	19.58	0.804	0.001	0.027	2.776	80.4
1	daf-2(e1370)	daf-16 RNAi	19	173	16	0	27.02	0.73	0.001	0.03	4.189	73
1	daf-2(e1370)	daf-16 RNAi	20	157	18	3	35.39	0.646	0.001	0.032	5.863	64.6
1	daf-2(e1370)	daf-16 RNAi	22	136	17	1	43.46	0.565	0.001	0.034	7.64	56.5
1	daf-2(e1370)	daf-16 RNAi	23	118	33	0	59.27	0.407	0.001	0.034	11.276	40.7
1	daf-2(e1370)	daf-16 RNAi	25	85	30	0	73.65	0.264	0.001	0.03	14.87	26.4
This is a portion of the data; to view all the data, please download the file.

Dataset 1.Kaplan-Meier estimator of RNAi lifespan experiments.

Kaplan-Meier estimate values were calculated from pooled lifespan data of two independent lifespan experiments using OASIS2 (https://sbi.postech.ac.kr/oasis2/). ‘At risk’ indicates the number of individuals at risk just prior to a specific time point. ‘S_hat’ indicates Kaplan-Meier estimate of survival function, ‘Var S_hat’ indicates variance of ‘S_hat’, and ‘SE(S)’ indicates standard error of ‘S_hat’.

Set	Strain	Treatment	Number of subjects	Mean survival days	Standard error of mean survival	95 percent confidence interval of mean	Age in days at 25 percent mortailty	Age in days at 50 percent mortailty	Age in days at 75 percent mortailty	Age in days at 90 percent mortailty	Age in days at 100 percent mortailty	95 percent median confidence interval	25 percent linear interpolation of mortality curve	50 percent linear interpolation of mortality curve	75 percent linear interpolation of mortality curve	90 percent linear interpolation of mortality curve	100 percent linear interpolation of mortality curve
1	N2	control RNAi	240	17.44	0.35	16.75 ~ 18.13	14	16	22	24	28	16.0 ~ 16.0	13.01	15.57	20.31	23.58	28
1	N2	daf-16 RNAi	240	14.03	0.25	13.54 ~ 14.51	11	14	16	18	22	- ~ -	10.78	12.95	15.09	17.53	22
1	N2	arrd-1 RNAi	240	17.26	0.34	16.60 ~ 17.92	14	16	20	23	28	16.0 ~ 16.0	12.96	15.73	19.88	22.81	28
1	N2	arrd-2 RNAi	240	17.54	0.33	16.89 ~ 18.19	14	18	20	23	28	16.0 ~ 16.0	13.11	16.32	19.88	22.81	28
1	N2	arrd-3 RNAi	240	19.19	0.34	18.53 ~ 19.85	16	20	23	24	30	18.0 ~ 18.0	14.49	18.57	22.42	23.79	30
1	N2	arrd-4 RNAi	240	18.34	0.39	17.56 ~ 19.11	14	18	23	25	30	18.0 ~ 18.0	13.01	17.56	22.21	24.46	30
1	N2	arrd-5 RNAi	240	17.39	0.32	16.76 ~ 18.02	14	18	20	23	28	16.0 ~ 16.0	13.23	16.55	19.44	22.48	28
1	daf-2(e1370)	control RNAi	210	43.69	0.69	42.35 ~ 45.04	40	46	49	52	63	44.0 ~ 44.0	38.13	44.52	48.19	51.77	63
1	daf-2(e1370)	daf-16 RNAi	240	22.52	0.37	21.79 ~ 23.25	19	23	28	28	34	22.0 ~ 22.0	18.73	22.41	25.22	27.63	34
1	daf-2(e1370)	arrd-1 RNAi	180	40.81	0.85	39.15 ~ 42.47	36	43	46	52	60	41.0 ~ 44.0	34.76	42.49	45.88	51.34	60
1	daf-2(e1370)	arrd-2 RNAi	210	41.47	0.66	40.18 ~ 42.76	36	44	46	51	57	43.0 ~ 43.0	35.75	43.17	45.9	50.15	57
1	daf-2(e1370)	arrd-3 RNAi	240	43.32	0.65	42.04 ~ 44.59	38	46	49	52	63	43.0 ~ 44.0	37.42	44.03	48.52	51.77	63
1	daf-2(e1370)	arrd-4 RNAi	240	41.74	0.65	40.45 ~ 43.02	37	43	49	52	60	41.0 ~ 43.0	36.34	42.36	47.09	51.55	60
1	daf-2(e1370)	arrd-5 RNAi	240	41.11	0.6	39.93 ~ 42.28	37	43	46	51	57	41.0 ~ 43.0	36.08	41.49	45.81	50.26	57
2	N2	control RNAi	240	21.66	0.35	20.97 ~ 22.36	18	22	24	27	34	21.0 ~ 21.0	17.01	21	23.94	26.51	34
2	N2	daf-16 RNAi	240	17.63	0.31	17.02 ~ 18.24	15	18	22	24	27	18.0 ~ 18.0	12.51	16.89	21.09	22.51	27
2	N2	arrd-6 RNAi	240	20.51	0.43	19.68 ~ 21.35	15	19	24	30	37	19.0 ~ 21.0	14.27	18.98	23.93	27.15	37
2	N2	arrd-7 RNAi	240	20.92	0.39	20.17 ~ 21.68	15	21	24	27	34	19.0 ~ 21.0	14.77	19.8	23.81	26.85	34
2	N2	arrd-8 RNAi	240	20.52	0.42	19.69 ~ 21.35	15	21	24	30	30	19.0 ~ 21.0	13.94	19.31	23.77	27.72	30
2	N2	arrd-9 RNAi	240	20.77	0.39	20.00 ~ 21.54	15	21	24	27	34	19.0 ~ 19.0	14.71	19.27	23.85	26.91	34
2	N2	arrd-10 RNAi	240	20.22	0.41	19.42 ~ 21.02	15	21	24	27	30	19.0 ~ 21.0	13.79	19.6	23.46	26.53	30
2	daf-2(e1370)	control RNAi	240	44.31	0.73	42.88 ~ 45.75	39	48	51	57	62	44.0 ~ 45.0	37.78	45.29	50.37	55.18	62
2	daf-2(e1370)	daf-16 RNAi	241	25.28	0.41	24.47 ~ 26.09	21	24	30	34	40	24.0 ~ 24.0	20.6	23.58	27.85	33.09	40
2	daf-2(e1370)	arrd-6 RNAi	240	44.25	0.66	42.95 ~ 45.54	39	45	51	55	62	44.0 ~ 45.0	38.29	44.79	49.25	54.51	62
2	daf-2(e1370)	arrd-7 RNAi	240	43.09	0.74	41.64 ~ 44.55	37	45	51	55	62	44.0 ~ 45.0	36.15	44.52	49.54	54.84	62
2	daf-2(e1370)	arrd-8 RNAi	240	43.33	0.66	42.04 ~ 44.61	39	45	48	54	62	44.0 ~ 45.0	38.9	44.06	47.99	52.18	62
2	daf-2(e1370)	arrd-9 RNAi	240	43.59	0.66	42.30 ~ 44.88	40	45	51	54	62	44.0 ~ 45.0	39.41	44.68	48.43	52.92	62
2	daf-2(e1370)	arrd-10 RNAi	240	46.02	0.75	44.56 ~ 47.48	40	48	55	59	66	45.0 ~ 45.0	39.45	46.23	54.18	57.44	66
3	N2	control RNAi	240	19.83	0.32	19.21 ~ 20.45	17	20	24	24	34	- ~ -	16.35	19.25	22.42	23.98	34
3	N2	daf-16 RNAi	240	14.82	0.22	14.40 ~ 15.25	12	14	17	18	22	14.0 ~ 14.0	11.53	13.92	16.71	17.98	22
3	N2	arrd-13 RNAi	210	18.67	0.3	18.08 ~ 19.27	16	18	22	24	30	18.0 ~ 18.0	15.38	17.96	20.24	23.29	30
3	N2	arrd-14 RNAi	240	19.13	0.3	18.53 ~ 19.72	16	20	22	24	28	18.0 ~ 18.0	15.93	18.58	21.53	23.73	28
3	N2	arrd-15 RNAi	240	18.86	0.32	18.23 ~ 19.49	14	20	22	26	30	18.0 ~ 18.0	13.76	18.24	20.83	24.95	30
3	N2	arrd-16 RNAi	240	17.98	0.31	17.38 ~ 18.58	14	17	20	24	28	17.0 ~ 17.0	13.36	16.89	19.81	23.92	28
3	N2	arrd-18 RNAi	240	18.89	0.33	18.25 ~ 19.54	14	18	22	26	32	17.0 ~ 18.0	13.89	17.94	21.14	25.05	32
3	daf-2(e1370)	control RNAi	210	44.89	0.85	43.23 ~ 46.55	38	47	53	58	71	44.0 ~ 47.0	37.86	45.39	52.85	57.13	71
3	daf-2(e1370)	daf-16 RNAi	240	23.45	0.33	22.80 ~ 24.11	20	24	26	28	38	24.0 ~ 24.0	18.94	23.2	25.67	27.83	38
3	daf-2(e1370)	arrd-13 RNAi	210	45.69	0.9	43.92 ~ 47.46	40	47	53	59	70	44.0 ~ 47.0	38.05	46.07	52.35	57.43	70
3	daf-2(e1370)	arrd-14 RNAi	240	42.94	0.84	41.30 ~ 44.58	37	44	50	56	67	44.0 ~ 44.0	36.09	43.51	49.94	54.3	67
3	daf-2(e1370)	arrd-15 RNAi	240	45.79	0.67	44.47 ~ 47.11	41	47	51	56	67	- ~ -	40.15	45.44	50.73	55.53	67
3	daf-2(e1370)	arrd-16 RNAi	240	43.29	0.82	41.68 ~ 44.89	37	47	51	56	64	44.0 ~ 44.0	36.65	44.82	50.49	54.23	64
3	daf-2(e1370)	arrd-18 RNAi	180	45.1	0.95	43.24 ~ 46.96	38	47	53	59	64	44.0 ~ 47.0	37.63	45.44	52.26	58.54	64
4	N2	control RNAi	240	18.04	0.28	17.49 ~ 18.59	15	18	21	23	29	17.0 ~ 18.0	14.13	17.43	20.28	22.45	29
4	N2	daf-16 RNAi	240	14.85	0.21	14.44 ~ 15.26	12	15	17	18	23	14.0 ~ 14.0	11.73	14.24	16.38	17.98	23
4	N2	arrd-23 RNAi	240	16.78	0.3	16.19 ~ 17.38	14	17	19	23	27	15.0 ~ 17.0	13.03	16.15	18.99	21.81	27
4	N2	arrd-24 RNAi	240	16.05	0.22	15.61 ~ 16.49	14	15	18	21	26	15.0 ~ 15.0	12.82	14.91	17.16	19.85	26
4	N2	arrd-25 RNAi	240	16.86	0.27	16.32 ~ 17.39	15	17	19	21	26	- ~ -	12.94	15.94	18.56	20.74	26
4	N2	arrd-26 RNAi	240	17.22	0.28	16.66 ~ 17.78	15	17	19	23	29	15.0 ~ 15.0	12.86	15.37	18.84	22.7	29
4	N2	arrd-28 RNAi	240	17.53	0.27	17.00 ~ 18.07	15	17	21	23	29	17.0 ~ 17.0	13.4	16.79	19.31	21.92	29
4	daf-2(e1370)	control RNAi	210	45.93	0.75	44.45 ~ 47.40	39	50	53	56	61	49.0 ~ 49.0	38.88	49.03	52.68	55.15	61
4	daf-2(e1370)	daf-16 RNAi	240	20.29	0.35	19.60 ~ 20.97	18	21	24	27	29	18.0 ~ 18.0	15.32	18.75	23.43	26.7	29
4	daf-2(e1370)	arrd-23 RNAi	240	44.15	0.7	42.78 ~ 45.51	37	47	52	56	60	46.0 ~ 47.0	36.8	46.71	51.15	53.44	60
4	daf-2(e1370)	arrd-24 RNAi	211	43	0.75	41.52 ~ 44.47	39	46	50	53	59	46.0 ~ 46.0	38.29	44.82	49.36	52.81	59
4	daf-2(e1370)	arrd-25 RNAi	240	43.74	0.64	42.47 ~ 45.00	40	46	50	53	59	46.0 ~ 46.0	39.53	45.97	49.47	52.41	59
4	daf-2(e1370)	arrd-26 RNAi	241	44.67	0.64	43.42 ~ 45.93	39	47	52	53	59	46.0 ~ 47.0	38.39	46.61	50.32	52.94	59
4	daf-2(e1370)	arrd-28 RNAi	150	43.52	0.85	41.85 ~ 45.19	37	47	50	53	56	46.0 ~ 47.0	36.86	46.13	49.65	52.53	56
5	N2	control RNAi	240	20.31	0.43	19.47 ~ 21.16	15	21	24	28	38	19.0 ~ 19.0	15	19.76	23.86	27.2	38
5	N2	daf-16 RNAi	240	15.63	0.35	14.95 ~ 16.31	12	15	18	22	27	14.0 ~ 15.0	10.87	14.45	17.88	21.66	27
5	N2	arrd-11 RNAi	240	20.17	0.42	19.35 ~ 20.99	17	21	25	27	40	19.0 ~ 19.0	15.29	19.65	24.05	26.39	40
5	N2	arrd-17 RNAi	240	20.52	0.41	19.72 ~ 21.33	17	21	24	27	40	19.0 ~ 19.0	16.22	19.65	23.16	26.51	40
5	N2	arrd-19 RNAi	240	19.56	0.41	18.75 ~ 20.36	15	19	24	27	36	18.0 ~ 19.0	14.6	18.7	23.23	25.9	36
5	N2	ttm-2 RNAi	240	19.11	0.44	18.24 ~ 19.97	15	19	24	27	36	18.0 ~ 19.0	14.28	18.07	22.39	26.62	36
5	daf-2(e1370)	control RNAi	240	42.97	0.7	41.60 ~ 44.33	38	46	50	54	61	42.0 ~ 42.0	36.55	42.74	49.38	53.12	61
5	daf-2(e1370)	daf-16 RNAi	243	21.54	0.37	20.82 ~ 22.27	17	21	25	28	38	21.0 ~ 21.0	16.88	20.73	24.2	27.62	38
5	daf-2(e1370)	arrd-11 RNAi	210	42.18	0.72	40.77 ~ 43.59	38	42	50	54	58	42.0 ~ 42.0	35.87	41.78	49.22	52.53	58
5	daf-2(e1370)	arrd-17 RNAi	240	41.32	0.62	40.09 ~ 42.54	38	42	46	54	59	- ~ -	35.57	40.07	45.46	52.16	59
5	daf-2(e1370)	arrd-19 RNAi	240	41.1	0.59	39.94 ~ 42.26	38	42	46	50	59	- ~ -	35.72	40.72	44.68	49.62	59

Dataset 2.Mean lifespan and mortality rates of RNAi experiment results.

Mean lifespan, ages at different percent mortality, linear interpolation of mortality curve at specific mortality rate were calculated from pooled lifespan data of two independent lifespan experiments. Data were obtained using OASIS2 (https://sbi.postech.ac.kr/oasis2/).

Set	Condition 1 strain	Condition 1 treatment	Condition 2 strain	Condition 2 treatment	Chi square	P value	Bonferroni P value
1	N2	control RNAi	N2	daf-16 RNAi	60.04	0	0
1	N2	control RNAi	N2	arrd-1 RNAi	0.69	0.4068	1
1	N2	control RNAi	N2	arrd-2 RNAi	0.08	0.7799	1
1	N2	control RNAi	N2	arrd-3 RNAi	8.09	0.0045	0.0268
1	N2	control RNAi	N2	arrd-4 RNAi	3.07	0.0798	0.4786
1	N2	control RNAi	N2	arrd-5 RNAi	0.47	0.4941	1
1	daf-2(e1370)	control RNAi	daf-2(e1370)	daf-16 RNAi	342.23	0	0
1	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-1 RNAi	4.87	0.0274	0.1642
1	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-2 RNAi	6.39	0.0115	0.0344
1	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-3 RNAi	0	0.9633	1
1	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-4 RNAi	2.54	0.1107	0.6644
1	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-5 RNAi	9.05	0.0026	0.0157
2	N2	control RNAi	N2	daf-16 RNAi	60.55	0	0
2	N2	control RNAi	N2	arrd-6 RNAi	1.41	0.2356	1
2	N2	control RNAi	N2	arrd-7 RNAi	0.71	0.3993	1
2	N2	control RNAi	N2	arrd-8 RNAi	0.94	0.3324	1
2	N2	control RNAi	N2	arrd-9 RNAi	1.23	0.2675	1
2	N2	control RNAi	N2	arrd-10 RNAi	3.24	0.072	0.432
2	daf-2(e1370)	control RNAi	daf-2(e1370)	daf-16 RNAi	311.97	0	0
2	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-6 RNAi	0.57	0.4514	1
2	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-7 RNAi	0.59	0.443	1
2	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-8 RNAi	3.79	0.0516	0.3099
2	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-9 RNAi	3.22	0.0729	0.4376
2	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-10 RNAi	5.82	0.0158	0.0948
3	N2	control RNAi	N2	daf-16 RNAi	158.43	0	0
3	N2	control RNAi	N2	arrd-13	8.81	0.003	0.018
3	N2	control RNAi	N2	arrd-14	3.81	0.0509	0.3053
3	N2	control RNAi	N2	arrd-15	3	0.0831	0.4983
3	N2	control RNAi	N2	arrd-16	15.5	0.0001	0.0005
3	N2	control RNAi	N2	arrd-18	2.2	0.1381	0.8287
3	daf-2(e1370)	control RNAi	daf-2(e1370)	daf-16 RNAi	309.08	0	0
3	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-13	0.54	0.4607	1
3	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-14	4.09	0.0433	0.2596
3	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-15	0.02	0.8875	1
3	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-16	2.45	0.1174	0.7042
3	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-18	0.49	0.486	1
4	N2	control RNAi	N2	daf-16 RNAi	85.14	0	0
4	N2	control RNAi	N2	arrd-23 RNAi	7.03	0.008	0.0481
4	N2	control RNAi	N2	arrd-24 RNAi	33.81	0.000000006	0.000000036
4	N2	control RNAi	N2	arrd-25 RNAi	9.4	0.0022	0.013
4	N2	control RNAi	N2	arrd-26 RNAi	2.24	0.1342	0.8053
4	N2	control RNAi	N2	arrd-28 RNAi	1.51	0.2188	1
4	daf-2(e1370)	control RNAi	daf-2(e1370)	daf-16 RNAi	392.76	0	0
4	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-23 RNAi	8.34	0.0039	0.0233
4	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-24 RNAi	20.6	0.0000056	0.000034
4	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-25 RNAi	24.9	0.00000061	0.0000036
4	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-26 RNAi	11.75	0.0006	0.0037
4	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-28 RNAi	17.71	0.000026	0.0002
5	N2	control RNAi	N2	daf-16 RNAi	67.47	0	0
5	N2	control RNAi	N2	arrd-11 RNAi	0.08	0.7707	1
5	N2	control RNAi	N2	arrd-17 RNAi	0	0.9914	1
5	N2	control RNAi	N2	arrd-19 RNAi	1.72	0.1891	0.9455
5	N2	control RNAi	N2	ttm-2 RNAi	3.22	0.0729	0.3644
5	daf-2(e1370)	control RNAi	daf-2(e1370)	daf-16 RNAi	368.1	0	0
5	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-11 RNAi	1.01	0.3156	1
5	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-17 RNAi	5.85	0.0156	0.078
5	daf-2(e1370)	control RNAi	daf-2(e1370)	arrd-19 RNAi	11.49	0.0007	0.0035

Dataset 3.Statistical analysis of lifespan data.

Statistical test results (Chi square, p value, Bonferroni p value) were calculated between ‘condition 1’ and ‘condition 2’. Test results were obtained using OASIS2 (https://sbi.postech.ac.kr/oasis2/).

Discussion

The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. As knocking down each of several α-arrestins resulted in minor changes in lifespan, these C. elegans α-arrestins may play modulatory roles in longevity regulation. RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes for various reasons. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons^7,9,23–25, which are refractory to RNAi^26–28. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals^28–30, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. Another possibility is that C. elegans α-arrestins may have functional redundancy, considering the large number of the α-arrestin family members in C. elegans and their sequence similarity^1,7. In addition, some α-arrestins may mostly function by modulating the action of their interacting proteins⁶. In this case, genetic inhibition of α-arrestins may rather subtly affect the functions of their interacting partners that directly regulate physiology, such as aging and longevity, causing weak or no phenotypes. Thus, it will be interesting to test the effects of simultaneous inhibition of α-arrestins, and to identify and to functionally characterize proteins that bind C. elegans α-arrestins.

In mammals, several α-arrestins are implicated in metabolic regulation⁵. TXNIP (thioredoxin-interacting protein), an inhibitor of thioredoxin in mammals^31–33, is a crucial negative regulator of glucose uptake^34,35. ARRDC4 inhibits glucose uptake in cultured mammalian cells as well³⁵, and ARRDC3 deficiency protects against obesity in male mice through increasing energy expenditure³⁶. Because metabolism is closely associated with aging³⁷, it will be interesting to test whether α-arrestins in complex metazoans play roles in aging via regulating metabolism.

Data availability

Dataset 1. Kaplan-Meier estimator of RNAi lifespan experiments. Kaplan-Meier estimate values were calculated from pooled lifespan data of two independent lifespan experiments using OASIS2 (https://sbi.postech.ac.kr/oasis2/). ‘At risk’ indicates the number of individuals at risk just prior to a specific time point. ‘S_hat’ indicates Kaplan-Meier estimate of survival function, ‘Var S_hat’ indicates variance of ‘S_hat’, and ‘SE(S)’ indicates standard error of ‘S_hat’. doi, 10.5256/f1000research.12337.d173158³⁸

Dataset 2. Mean lifespan and mortality rates of RNAi experiment results. Mean lifespan, ages at different percent mortality, linear interpolation of mortality curve at specific mortality rate were calculated from pooled lifespan data of two independent lifespan experiments. Data were obtained using OASIS2 (https://sbi.postech.ac.kr/oasis2/). doi, 10.5256/f1000research.12337.d173159³⁹

Dataset 3. Statistical analysis of lifespan data. Statistical test results (Chi square, p value, Bonferroni p value) were calculated between ‘condition 1’ and ‘condition 2’. Test results were obtained using OASIS2 (https://sbi.postech.ac.kr/oasis2/). doi, 10.5256/f1000research.12337.d173160⁴⁰

Competing interests

No competing interests were disclosed.

Grant information

This work was supported by the Korean Government (MSIP) through the National Research Foundation of Korea (NRF) [NRF-2016R1E1A1A01941152] funded to S-J.V.L.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgments

We thank the Lee laboratory members and Dr. Young Kwon for critical comments on the manuscript.

Supplementary material

Figure S1. RNAi knockdown of each Caenorhabditis elegans α-arrestin had minor effects on the lifespan of wild-type or daf-2(-) mutants. (A–U) Lifespan curves of wild-type (WT) and daf-2(e1370) (daf-2(-)) mutants treated with RNAi targeting daf-16 (A), which was used as a positive control, arrd-2 (B), arrd-4 (C), arrd-5 (D), arrd-6 (E), arrd-7 (F), arrd-8 (G), arrd-9 (H), arrd-10 (I), arrd-11 (J), arrd-13 (K), arrd-14 (L), arrd-15 (M), arrd-17 (N), arrd-18 (O), arrd-19 (P), arrd-23 (Q), arrd-25 (R), arrd-26 (S), arrd-28 (T) and ttm-2 (U). See Table S1 for statistical analysis and for additional repeats of lifespan data using daf-16 RNAi.

Click here to access the data.

Table S1. Lifespan data of Caenorhabditis elegans α-arrestin RNAi clones. Lifespan data within the solid lines indicate the pooled experimental datasets that are performed at the same time by two independent researchers. P values were calculated within the sets using log-rank (Mantel-Cox) method²². Percent (%) changes and p values for the lifespan data of specific α-arrestin RNAi-treated wild-type (WT) and daf-2(e1370) worms were calculated against those of control RNAi-treated WT and daf-2(e1370) worms within the sets, respectively. Percent (%) changes and p values for the lifespan data of control RNAi-treated daf-2(e1370) mutants were calculated against those of control RNAi-treated WT worms. SEM represents standard error of the mean, and 75th percentile indicates the age (days) at 75% mortality.

Click here to access the data.

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Version 4

VERSION 4 PUBLISHED 18 Aug 2017

Author details Author details

Sangsoon Park
Roles: Conceptualization, Formal Analysis, Investigation, Project Administration, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Yoonji Jung
Roles: Investigation, Validation, Writing – Review & Editing

Seon Woo A. An
Roles: Investigation

Heehwa G. Son
Roles: Investigation

Wooseon Hwang
Roles: Investigation

Dongyeop Lee
Roles: Investigation

Murat Artan
Roles: Investigation

Hae-Eun H. Park
Roles: Investigation

Dae-Eun Jeong
Roles: Investigation

Yujin Lee
Roles: Investigation

Seung-Jae V. Lee
Roles: Conceptualization, Funding Acquisition, Project Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This work was supported by the Korean Government (MSIP) through the National Research Foundation of Korea (NRF) [NRF-2016R1E1A1A01941152] funded to S-J.V.L.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Park S, Jung Y, An SWA et al. RNAi targeting Caenorhabditis elegans α-arrestins marginally affects lifespan [version 1; peer review: 2 approved with reservations]. F1000Research 2017, 6:1515 (https://doi.org/10.12688/f1000research.12337.1)

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Reviewer Report 06 Sep 2017

Roger Pocock, Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University, Melbourne, Vic, Australia

Approved with Reservations

https://doi.org/10.5256/f1000research.13357.r25722

In this manuscript Park et al. performed RNAi knockdown of 24 of the 29 alpha-arrestin genes in C. elegans to ask whether they are important for lifespan regulation in wild type and daf-2 mutant animals. Their data shows that, in their experimental conditions, most alpha-arrestin genes are individually not important for regulating lifespan.

Concerns:
One major issue with the study is whether RNAi knockdown of the alpha-arrestin genes is working. As some alpha-arrestins are expressed in the nervous system, which is refractory to RNAi, this is a concern. To determine whether the RNAi knockdown is working then Q-RTPCR should be performed. However, this is not a small task for 24 genes, therefore, further experiments using sensitized genetic backgrounds for RNAi or elevated temperatures may tease out phenotypes for some alpha-arrestin genes. This could particularly be performed for the alpha-arrestin genes that when knocked down engender a slight effect on lifespan.

In parallel to this, the authors could align the alpha-arrestin gene sequences to ask if there is a common sequence that could be used by RNAi targeting to knockdown multiple alpha-arrestin genes simultaneously.

The authors knockdown of 24 of the 29 alpha-arrestin genes. I wonder why they didn’t generate RNAi clones for the remaining 5, especially since they did generate 3 other constructs for their analysis. Potentially, the important alpha-arrestin genes is amongst those not examined.

The authors did not report any other behavioural or morphological phenotypes observed by alpha-arrestin gene knockdown. Any information on observed phenotypes would be of interest to the research community.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response (F1000Research Advisory Board Member) 14 Nov 2017

Seung-Jae V Lee, Korea Advanced Institute of Science and Technology, South Korea

14 Nov 2017

Author Response F1000Research Advisory Board Member

Dear Dr. Roger Pocock,
We sincerely appreciate your valuable comments on our manuscript. We agree with all your points and revised our manuscript to describe the limitations of this study. ... Continue reading Dear Dr. Roger Pocock,
We sincerely appreciate your valuable comments on our manuscript. We agree with all your points and revised our manuscript to describe the limitations of this study. We would like to mention that the main motivation for publishing this manuscript is to provide our negative results to researchers who are interested in characterizing α-arrestins in C. elegans. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research.

In this manuscript Park et al. performed RNAi knockdown of 24 of the 29 alpha-arrestin genes in C. elegans to ask whether they are important for lifespan regulation in wild type and daf-2 mutant animals. Their data shows that, in their experimental conditions, most alpha-arrestin genes are individually not important for regulating lifespan.

Concerns:
One major issue with the study is whether RNAi knockdown of the alpha-arrestin genes is working. As some alpha-arrestins are expressed in the nervous system, which is refractory to RNAi, this is a concern. To determine whether the RNAi knockdown is working then Q-RTPCR should be performed. However, this is not a small task for 24 genes, therefore, further experiments using sensitized genetic backgrounds for RNAi or elevated temperatures may tease out phenotypes for some alpha-arrestin genes. This could particularly be performed for the alpha-arrestin genes that when knocked down engender a slight effect on lifespan.

> We agree with the reviewer’s comment that the RNAi knockdown efficiency is a major concern in interpreting our screen results. It would have been great if we had performed qRT-PCR analysis. However, as the reviewers pointed out, this is not a small task. We currently do not have any plan for following up this project and want to inform other researchers of our screen data. Therefore, we further emphasized the pitfall of our results in the Discussion as follows,
In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons^{7, 9, 24-26}, which are refractory to RNAi^27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals^29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.”

In parallel to this, the authors could align the alpha-arrestin gene sequences to ask if there is a common sequence that could be used by RNAi targeting to knockdown multiple alpha-arrestin genes simultaneously.

> We appreciate the reviewer’s comment. The α-arrestin family members share arrestin-domains (O. Hobert, Wormbook, 2013). Thus, it will be intriguing if we could examine whether targeting arrestin domain for RNAi to knock down multiple arrd genes simultaneously and if multiple arrd gene knock down has effects on lifespan. We modified our manuscript to address this issue in Discussion as follows,
In Discussion: “Second, C. elegans α-arrestins may have functional redundancy considering the large number of the α-arrestin family members in C. elegans and their sequence similarity^{1, 7}, which may obscure examining the functional importance of each α-arrestin. In addition, some α-arrestins may mostly function by modulating their interacting proteins⁶. In this case, genetic inhibition of α-arrestins may rather subtly affect the functions of their interacting partners that directly regulate physiology, such as aging and longevity, causing weak or no phenotypes. Thus, it will be interesting to test the effects of simultaneous inhibition of α-arrestins possibly through targeting the arrestin domain, and to identify and to functionally characterize proteins that bind C. elegans α-arrestins.”

The authors knockdown of 24 of the 29 alpha-arrestin genes. I wonder why they didn’t generate RNAi clones for the remaining 5, especially since they did generate 3 other constructs for their analysis. Potentially, the important alpha-arrestin genes is amongst those not examined.

> We appreciate the reviewers’ comment. We aimed at obtaining all the α arrestin RNAi clones that are not commercially available, except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones, targeting arrd-12, arrd-22 and arrd-27. We addressed this issue in Methods for RNAi clones and in Discussion as follows,
In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.”
In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.”

The authors did not report any other behavioural or morphological phenotypes observed by alpha-arrestin gene knockdown. Any information on observed phenotypes would be of interest to the research community.

> We thank the reviewer for the valuable comment. One noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantify the results. We described this issue in Methods as follows,
In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”

Dear Dr. Roger Pocock,
We sincerely appreciate your valuable comments on our manuscript. We agree with all your points and revised our manuscript to describe the limitations of this study. We would like to mention that the main motivation for publishing this manuscript is to provide our negative results to researchers who are interested in characterizing α-arrestins in C. elegans. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research.

In this manuscript Park et al. performed RNAi knockdown of 24 of the 29 alpha-arrestin genes in C. elegans to ask whether they are important for lifespan regulation in wild type and daf-2 mutant animals. Their data shows that, in their experimental conditions, most alpha-arrestin genes are individually not important for regulating lifespan.

Concerns:
One major issue with the study is whether RNAi knockdown of the alpha-arrestin genes is working. As some alpha-arrestins are expressed in the nervous system, which is refractory to RNAi, this is a concern. To determine whether the RNAi knockdown is working then Q-RTPCR should be performed. However, this is not a small task for 24 genes, therefore, further experiments using sensitized genetic backgrounds for RNAi or elevated temperatures may tease out phenotypes for some alpha-arrestin genes. This could particularly be performed for the alpha-arrestin genes that when knocked down engender a slight effect on lifespan.

> We agree with the reviewer’s comment that the RNAi knockdown efficiency is a major concern in interpreting our screen results. It would have been great if we had performed qRT-PCR analysis. However, as the reviewers pointed out, this is not a small task. We currently do not have any plan for following up this project and want to inform other researchers of our screen data. Therefore, we further emphasized the pitfall of our results in the Discussion as follows,
In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons^{7, 9, 24-26}, which are refractory to RNAi^27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals^29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.”

In parallel to this, the authors could align the alpha-arrestin gene sequences to ask if there is a common sequence that could be used by RNAi targeting to knockdown multiple alpha-arrestin genes simultaneously.

> We appreciate the reviewer’s comment. The α-arrestin family members share arrestin-domains (O. Hobert, Wormbook, 2013). Thus, it will be intriguing if we could examine whether targeting arrestin domain for RNAi to knock down multiple arrd genes simultaneously and if multiple arrd gene knock down has effects on lifespan. We modified our manuscript to address this issue in Discussion as follows,
In Discussion: “Second, C. elegans α-arrestins may have functional redundancy considering the large number of the α-arrestin family members in C. elegans and their sequence similarity^{1, 7}, which may obscure examining the functional importance of each α-arrestin. In addition, some α-arrestins may mostly function by modulating their interacting proteins⁶. In this case, genetic inhibition of α-arrestins may rather subtly affect the functions of their interacting partners that directly regulate physiology, such as aging and longevity, causing weak or no phenotypes. Thus, it will be interesting to test the effects of simultaneous inhibition of α-arrestins possibly through targeting the arrestin domain, and to identify and to functionally characterize proteins that bind C. elegans α-arrestins.”

The authors knockdown of 24 of the 29 alpha-arrestin genes. I wonder why they didn’t generate RNAi clones for the remaining 5, especially since they did generate 3 other constructs for their analysis. Potentially, the important alpha-arrestin genes is amongst those not examined.

> We appreciate the reviewers’ comment. We aimed at obtaining all the α arrestin RNAi clones that are not commercially available, except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones, targeting arrd-12, arrd-22 and arrd-27. We addressed this issue in Methods for RNAi clones and in Discussion as follows,
In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.”
In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.”

The authors did not report any other behavioural or morphological phenotypes observed by alpha-arrestin gene knockdown. Any information on observed phenotypes would be of interest to the research community.

> We thank the reviewer for the valuable comment. One noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantify the results. We described this issue in Methods as follows,
In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response (F1000Research Advisory Board Member) 14 Nov 2017

Seung-Jae V Lee, Korea Advanced Institute of Science and Technology, South Korea

14 Nov 2017

Author Response F1000Research Advisory Board Member

Dear Dr. Roger Pocock,
We sincerely appreciate your valuable comments on our manuscript. We agree with all your points and revised our manuscript to describe the limitations of this study. ... Continue reading Dear Dr. Roger Pocock,
We sincerely appreciate your valuable comments on our manuscript. We agree with all your points and revised our manuscript to describe the limitations of this study. We would like to mention that the main motivation for publishing this manuscript is to provide our negative results to researchers who are interested in characterizing α-arrestins in C. elegans. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research.

In this manuscript Park et al. performed RNAi knockdown of 24 of the 29 alpha-arrestin genes in C. elegans to ask whether they are important for lifespan regulation in wild type and daf-2 mutant animals. Their data shows that, in their experimental conditions, most alpha-arrestin genes are individually not important for regulating lifespan.

Concerns:
One major issue with the study is whether RNAi knockdown of the alpha-arrestin genes is working. As some alpha-arrestins are expressed in the nervous system, which is refractory to RNAi, this is a concern. To determine whether the RNAi knockdown is working then Q-RTPCR should be performed. However, this is not a small task for 24 genes, therefore, further experiments using sensitized genetic backgrounds for RNAi or elevated temperatures may tease out phenotypes for some alpha-arrestin genes. This could particularly be performed for the alpha-arrestin genes that when knocked down engender a slight effect on lifespan.

> We agree with the reviewer’s comment that the RNAi knockdown efficiency is a major concern in interpreting our screen results. It would have been great if we had performed qRT-PCR analysis. However, as the reviewers pointed out, this is not a small task. We currently do not have any plan for following up this project and want to inform other researchers of our screen data. Therefore, we further emphasized the pitfall of our results in the Discussion as follows,
In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons^{7, 9, 24-26}, which are refractory to RNAi^27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals^29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.”

In parallel to this, the authors could align the alpha-arrestin gene sequences to ask if there is a common sequence that could be used by RNAi targeting to knockdown multiple alpha-arrestin genes simultaneously.

> We appreciate the reviewer’s comment. The α-arrestin family members share arrestin-domains (O. Hobert, Wormbook, 2013). Thus, it will be intriguing if we could examine whether targeting arrestin domain for RNAi to knock down multiple arrd genes simultaneously and if multiple arrd gene knock down has effects on lifespan. We modified our manuscript to address this issue in Discussion as follows,
In Discussion: “Second, C. elegans α-arrestins may have functional redundancy considering the large number of the α-arrestin family members in C. elegans and their sequence similarity^{1, 7}, which may obscure examining the functional importance of each α-arrestin. In addition, some α-arrestins may mostly function by modulating their interacting proteins⁶. In this case, genetic inhibition of α-arrestins may rather subtly affect the functions of their interacting partners that directly regulate physiology, such as aging and longevity, causing weak or no phenotypes. Thus, it will be interesting to test the effects of simultaneous inhibition of α-arrestins possibly through targeting the arrestin domain, and to identify and to functionally characterize proteins that bind C. elegans α-arrestins.”

The authors knockdown of 24 of the 29 alpha-arrestin genes. I wonder why they didn’t generate RNAi clones for the remaining 5, especially since they did generate 3 other constructs for their analysis. Potentially, the important alpha-arrestin genes is amongst those not examined.

> We appreciate the reviewers’ comment. We aimed at obtaining all the α arrestin RNAi clones that are not commercially available, except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones, targeting arrd-12, arrd-22 and arrd-27. We addressed this issue in Methods for RNAi clones and in Discussion as follows,
In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.”
In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.”

The authors did not report any other behavioural or morphological phenotypes observed by alpha-arrestin gene knockdown. Any information on observed phenotypes would be of interest to the research community.

> We thank the reviewer for the valuable comment. One noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantify the results. We described this issue in Methods as follows,
In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”

Dear Dr. Roger Pocock,
We sincerely appreciate your valuable comments on our manuscript. We agree with all your points and revised our manuscript to describe the limitations of this study. We would like to mention that the main motivation for publishing this manuscript is to provide our negative results to researchers who are interested in characterizing α-arrestins in C. elegans. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research.

In this manuscript Park et al. performed RNAi knockdown of 24 of the 29 alpha-arrestin genes in C. elegans to ask whether they are important for lifespan regulation in wild type and daf-2 mutant animals. Their data shows that, in their experimental conditions, most alpha-arrestin genes are individually not important for regulating lifespan.

Concerns:
One major issue with the study is whether RNAi knockdown of the alpha-arrestin genes is working. As some alpha-arrestins are expressed in the nervous system, which is refractory to RNAi, this is a concern. To determine whether the RNAi knockdown is working then Q-RTPCR should be performed. However, this is not a small task for 24 genes, therefore, further experiments using sensitized genetic backgrounds for RNAi or elevated temperatures may tease out phenotypes for some alpha-arrestin genes. This could particularly be performed for the alpha-arrestin genes that when knocked down engender a slight effect on lifespan.

> We agree with the reviewer’s comment that the RNAi knockdown efficiency is a major concern in interpreting our screen results. It would have been great if we had performed qRT-PCR analysis. However, as the reviewers pointed out, this is not a small task. We currently do not have any plan for following up this project and want to inform other researchers of our screen data. Therefore, we further emphasized the pitfall of our results in the Discussion as follows,
In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons^{7, 9, 24-26}, which are refractory to RNAi^27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals^29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.”

In parallel to this, the authors could align the alpha-arrestin gene sequences to ask if there is a common sequence that could be used by RNAi targeting to knockdown multiple alpha-arrestin genes simultaneously.

> We appreciate the reviewer’s comment. The α-arrestin family members share arrestin-domains (O. Hobert, Wormbook, 2013). Thus, it will be intriguing if we could examine whether targeting arrestin domain for RNAi to knock down multiple arrd genes simultaneously and if multiple arrd gene knock down has effects on lifespan. We modified our manuscript to address this issue in Discussion as follows,
In Discussion: “Second, C. elegans α-arrestins may have functional redundancy considering the large number of the α-arrestin family members in C. elegans and their sequence similarity^{1, 7}, which may obscure examining the functional importance of each α-arrestin. In addition, some α-arrestins may mostly function by modulating their interacting proteins⁶. In this case, genetic inhibition of α-arrestins may rather subtly affect the functions of their interacting partners that directly regulate physiology, such as aging and longevity, causing weak or no phenotypes. Thus, it will be interesting to test the effects of simultaneous inhibition of α-arrestins possibly through targeting the arrestin domain, and to identify and to functionally characterize proteins that bind C. elegans α-arrestins.”

The authors knockdown of 24 of the 29 alpha-arrestin genes. I wonder why they didn’t generate RNAi clones for the remaining 5, especially since they did generate 3 other constructs for their analysis. Potentially, the important alpha-arrestin genes is amongst those not examined.

> We appreciate the reviewers’ comment. We aimed at obtaining all the α arrestin RNAi clones that are not commercially available, except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones, targeting arrd-12, arrd-22 and arrd-27. We addressed this issue in Methods for RNAi clones and in Discussion as follows,
In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.”
In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.”

The authors did not report any other behavioural or morphological phenotypes observed by alpha-arrestin gene knockdown. Any information on observed phenotypes would be of interest to the research community.

> We thank the reviewer for the valuable comment. One noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantify the results. We described this issue in Methods as follows,
In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”
Competing Interests: No competing interests were disclosed. Close
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Reviewer Report 04 Sep 2017

Alessandro Bitto, Department of Pathology, University of Washington, Seattle, Seattle, WA, USA

Jason Pitt, Department of Pathology, University of Washington, Seattle, WA, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.13357.r25290

In the manuscript RNAi targeting Caenorhabditis elegans alpha-arrestins marginally affects lifespan Park and co-workers provide largely negative results from lifespan experiments following RNAi knockdown of most of the worm alpha-arrestins. While we welcome the effort to publish negative results, there is one major concern with the experimental approach and several minor concerns with the interpretation of the results that should be addressed before the manuscript is fully accepted for indexing in F1000Research.

Major concern:
The authors data is almost entirely negative: there were only 4 clones which had a statistically significant effect. The authors attempt to address this concern by providing RNAi data for daf-16, which is known to strongly suppress the lifespan extension of daf-2(e1370) as well as decreasing N2 lifespan. However, the authors must demonstrate that alpha-arrestin RNAi knockdown is functioning. As suggested by the authors, there is some evidence that alpha-arrestins are expressed in neurons which are notoriously refractory to RNAi. The authors must show that they can use RNAi to knockdown alpha-arrestins by QRTPCR analysis. The authors could also address this concern by performing the RNAi in one of the mutant strains in which RNAi works in neurons and report if the lifespan still does not change, as they suggest in the discussion. We believe that providing these data is necessary to correctly interpret the results from the lifespan analyses.

Minor concerns:

1. In the text the authors speak broadly of individual arrestins having no role in longevity however the authors only tested 24 of the 29 arrestins in the worm. If the authors had limited themselves to only those clones available in commercial RNAi libraries this would be understandable, however the authors did construct 3 new RNAi feeding constructs. This brings up the question to why the authors did not prepare the remaining 5 constructs. If the authors have reason to exclude those genes from their analysis (ie. they are pseudogenes, not expressed, etc) they should discuss these reasons in the text. Besides, there is the possibility that one of the missing constructs may indeed have major effects on lifespan in C. elegans.

2. The title of the manuscript implies there are more than one result than was observed. The simpler conclusion from the results provided is that individual alpha-arrestin knockdown has no effect of lifespan. This is the fundamental observation of the paper considering that most of them (20 out of 24) had no effect and that 4 of them had such an extremely small effect that it is arguable that the sample size is insufficient to support any conclusion¹. We understand the authors' motivation to make the manuscript sound more appealing, however, their interpretation is disingenuous, given their results.

3. Because alpha-arrestins share significant sequence homology the authors should make some mention as to the likelihood that RNAi of any one arrestin would have off target effects on other arrestins. The authors could easily address this question experimentally with QRTPCR analysis. This is particularly relevant as one of the main conclusions of the manuscript is that future alpha-arrestin studies should knockdown multiple alpha-arrestins simultaneously.

4. Given that the authors observed the RNAi treated animals for weeks or months (in daf-2) it is surprising that no other behavioral or physical phenotypes from the alpha-arrestin RNAi were observed during the course of the analysis. If the authors truly did not observe any other phenotypes they should state it briefly in the text.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

References

1. Petrascheck M, Miller DL: Computational Analysis of Lifespan Experiment Reproducibility.Front Genet. 2017; 8: 92 PubMed Abstract | Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Biology of Aging

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

CITE

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Author Response (F1000Research Advisory Board Member) 14 Nov 2017

Seung-Jae V Lee, Korea Advanced Institute of Science and Technology, South Korea

14 Nov 2017

Author Response F1000Research Advisory Board Member

Dear Dr. Alessandro Bitto and Dr. Jason Pitt,
We greatly appreciate the reviewers’ valuable comments on our manuscript. We agree with all the reviewers’ comments and modified our manuscript to ... Continue reading Dear Dr. Alessandro Bitto and Dr. Jason Pitt,
We greatly appreciate the reviewers’ valuable comments on our manuscript. We agree with all the reviewers’ comments and modified our manuscript to describe the limitations of our studies raised by the reviewers. We would like to mention that the main purpose of publishing our current manuscript is to provide our data to other researchers who are interested in the characterization of C. elegans arrd genes, instead of keeping our current data unpublished. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research.

Reviewers’ comments:

In the manuscript RNAi targeting Caenorhabditis elegans alpha-arrestins marginally affects lifespan Park and co-workers provide largely negative results from lifespan experiments following RNAi knockdown of most of the worm alpha-arrestins. While we welcome the effort to publish negative results, there is one major concern with the experimental approach and several minor concerns with the interpretation of the results that should be addressed before the manuscript is fully accepted for indexing in F1000Research.

Major concern:
The authors data is almost entirely negative: there were only 4 clones which had a statistically significant effect. The authors attempt to address this concern by providing RNAi data for daf-16, which is known to strongly suppress the lifespan extension of daf-2(e1370) as well as decreasing N2 lifespan. However, the authors must demonstrate that alpha-arrestin RNAi knockdown is functioning. As suggested by the authors, there is some evidence that alpha-arrestins are expressed in neurons which are notoriously refractory to RNAi. The authors must show that they can use RNAi to knockdown alpha-arrestins by QRTPCR analysis. The authors could also address this concern by performing the RNAi in one of the mutant strains in which RNAi works in neurons and report if the lifespan still does not change, as they suggest in the discussion. We believe that providing these data is necessary to correctly interpret the results from the lifespan analyses.

> We completely agree with the reviewers for the comment. It would have been great if we had performed qRTPCR for validating the RNAi efficiency, while we were doing lifespan assays. As our experiments were done as an RNAi screen, our initial plan was validating RNAi efficiency and using RNAi-sensitive mutants if we had identified some positive RNAi clones. However, as the reviewers pointed out, our screen results were negative. We currently do not have any plan for following up this project and just want to inform other researchers of our screen data. Therefore, instead of putting efforts on this project retrospectively, we emphasized the limitations of our data raised by the reviewers to inform the readers of pitfalls by revising the manuscript as follows,
In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons^{7, 9, 24-26}, which are refractory to RNAi ^27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals ^29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.”

Minor concerns:
1. In the text the authors speak broadly of individual arrestins having no role in longevity however the authors only tested 24 of the 29 arrestins in the worm. If the authors had limited themselves to only those clones available in commercial RNAi libraries this would be understandable, however the authors did construct 3 new RNAi feeding constructs. This brings up the question to why the authors did not prepare the remaining 5 constructs. If the authors have reason to exclude those genes from their analysis (ie. they are pseudogenes, not expressed, etc) they should discuss these reasons in the text. Besides, there is the possibility that one of the missing constructs may indeed have major effects on lifespan in C. elegans.

> We appreciate the reviewers’ comment. We initially aimed at obtaining all the α arrestin RNAi clones that are not commercially available except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones for arrd-12, arrd-22 and arrd-27 because of unknown technical problems. We agree with the reviewers’ point that these three genes that were not tested may have major effects on lifespan. We addressed this issue in Methods for RNAi clones as well as in Discussion as follows,
In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.”
In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.”

2. The title of the manuscript implies there are more than one result than was observed. The simpler conclusion from the results provided is that individual alpha-arrestin knockdown has no effect of lifespan. This is the fundamental observation of the paper considering that most of them (20 out of 24) had no effect and that 4 of them had such an extremely small effect that it is arguable that the sample size is insufficient to support any conclusion¹. We understand the authors' motivation to make the manuscript sound more appealing, however, their interpretation is disingenuous, given their results.

> We thank the reviewers’ comment on the title and changed the title as follows,
Title: “RNAi targeting Caenorhabditis elegans α-arrestins has small or no effects on lifespan”

3. Because alpha-arrestins share significant sequence homology the authors should make some mention as to the likelihood that RNAi of any one arrestin would have off target effects on other arrestins. The authors could easily address this question experimentally with QRTPCR analysis. This is particularly relevant as one of the main conclusions of the manuscript is that future alpha-arrestin studies should knockdown multiple alpha-arrestins simultaneously.

> We agree with the reviewers that the RNAi targeting each of α-arrestin genes may have caused off-target effects on other α-arrestin genes because of sequence similarities. We performed off-target prediction using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap), and at least with the prediction, the RNAi clones used in our screen do not appear to have major off-target effects on any other genes including α-arrestin genes, except arrd-19 RNAi which may also target arrd-21 which is a predicted pseudogene. Although qRT-PCR will be a straightforward way as the reviewers suggested, again currently we do not have any plan on following up this project and we therefore instead acknowledged the limitation. We added the prediction results in the Supplementary Table S1 and described this issue in the Methods, RNAi clones part as follows,
In Methods, RNAi clones: “All RNAi clones used in our screen were examined for their potential off-target effects by using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)²², and no significant off-target was predicted except arrd-19 RNAi, which may additionally target a predicted pseudogene arrd-21 (Table S1). Experimental validation by qRT-PCR will be necessary to completely exclude the possible off-target effects by RNAi clones.”
In Supplementary material: “Table S1. Possible RNAi target prediction. RNAi clones used in the screen were examined for their possible off-targets by Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)²². For RNAi clone names, ‘mv’ indicates a RNAi clone from Vidal library and ‘sjj’ indicates a RNAi clone from Ahringer library. RNAi clones generated by us for this study are labeled as ‘Lee lab’. Scores for potential target transcripts were calculated as described previously²² and were rounded off to the second digit after the decimal point.”
In Reference: 22. Thakur, N., et al., Clone Mapper: An Online Suite of Tools for RNAi Experiments in Caenorhabditis elegans. G3 (Bethesda), 2014. 4(11): p. 2137-2145.23.

4. Given that the authors observed the RNAi treated animals for weeks or months (in daf-2) it is surprising that no other behavioral or physical phenotypes from the alpha-arrestin RNAi were observed during the course of the analysis. If the authors truly did not observe any other phenotypes they should state it briefly in the text.

> We appreciate the reviewers’ comment. As our main interests were lifespan changes from a large scale screen, we did not pay attention to other phenotypes. Nevertheless, one noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantitate the results. We described this issue in Methods as follows,
In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”

Dear Dr. Alessandro Bitto and Dr. Jason Pitt,
We greatly appreciate the reviewers’ valuable comments on our manuscript. We agree with all the reviewers’ comments and modified our manuscript to describe the limitations of our studies raised by the reviewers. We would like to mention that the main purpose of publishing our current manuscript is to provide our data to other researchers who are interested in the characterization of C. elegans arrd genes, instead of keeping our current data unpublished. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research.

Reviewers’ comments:

In the manuscript RNAi targeting Caenorhabditis elegans alpha-arrestins marginally affects lifespan Park and co-workers provide largely negative results from lifespan experiments following RNAi knockdown of most of the worm alpha-arrestins. While we welcome the effort to publish negative results, there is one major concern with the experimental approach and several minor concerns with the interpretation of the results that should be addressed before the manuscript is fully accepted for indexing in F1000Research.

Major concern:
The authors data is almost entirely negative: there were only 4 clones which had a statistically significant effect. The authors attempt to address this concern by providing RNAi data for daf-16, which is known to strongly suppress the lifespan extension of daf-2(e1370) as well as decreasing N2 lifespan. However, the authors must demonstrate that alpha-arrestin RNAi knockdown is functioning. As suggested by the authors, there is some evidence that alpha-arrestins are expressed in neurons which are notoriously refractory to RNAi. The authors must show that they can use RNAi to knockdown alpha-arrestins by QRTPCR analysis. The authors could also address this concern by performing the RNAi in one of the mutant strains in which RNAi works in neurons and report if the lifespan still does not change, as they suggest in the discussion. We believe that providing these data is necessary to correctly interpret the results from the lifespan analyses.

> We completely agree with the reviewers for the comment. It would have been great if we had performed qRTPCR for validating the RNAi efficiency, while we were doing lifespan assays. As our experiments were done as an RNAi screen, our initial plan was validating RNAi efficiency and using RNAi-sensitive mutants if we had identified some positive RNAi clones. However, as the reviewers pointed out, our screen results were negative. We currently do not have any plan for following up this project and just want to inform other researchers of our screen data. Therefore, instead of putting efforts on this project retrospectively, we emphasized the limitations of our data raised by the reviewers to inform the readers of pitfalls by revising the manuscript as follows,
In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons^{7, 9, 24-26}, which are refractory to RNAi ^27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals ^29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.”

Minor concerns:
1. In the text the authors speak broadly of individual arrestins having no role in longevity however the authors only tested 24 of the 29 arrestins in the worm. If the authors had limited themselves to only those clones available in commercial RNAi libraries this would be understandable, however the authors did construct 3 new RNAi feeding constructs. This brings up the question to why the authors did not prepare the remaining 5 constructs. If the authors have reason to exclude those genes from their analysis (ie. they are pseudogenes, not expressed, etc) they should discuss these reasons in the text. Besides, there is the possibility that one of the missing constructs may indeed have major effects on lifespan in C. elegans.

> We appreciate the reviewers’ comment. We initially aimed at obtaining all the α arrestin RNAi clones that are not commercially available except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones for arrd-12, arrd-22 and arrd-27 because of unknown technical problems. We agree with the reviewers’ point that these three genes that were not tested may have major effects on lifespan. We addressed this issue in Methods for RNAi clones as well as in Discussion as follows,
In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.”
In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.”

2. The title of the manuscript implies there are more than one result than was observed. The simpler conclusion from the results provided is that individual alpha-arrestin knockdown has no effect of lifespan. This is the fundamental observation of the paper considering that most of them (20 out of 24) had no effect and that 4 of them had such an extremely small effect that it is arguable that the sample size is insufficient to support any conclusion¹. We understand the authors' motivation to make the manuscript sound more appealing, however, their interpretation is disingenuous, given their results.

> We thank the reviewers’ comment on the title and changed the title as follows,
Title: “RNAi targeting Caenorhabditis elegans α-arrestins has small or no effects on lifespan”

3. Because alpha-arrestins share significant sequence homology the authors should make some mention as to the likelihood that RNAi of any one arrestin would have off target effects on other arrestins. The authors could easily address this question experimentally with QRTPCR analysis. This is particularly relevant as one of the main conclusions of the manuscript is that future alpha-arrestin studies should knockdown multiple alpha-arrestins simultaneously.

> We agree with the reviewers that the RNAi targeting each of α-arrestin genes may have caused off-target effects on other α-arrestin genes because of sequence similarities. We performed off-target prediction using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap), and at least with the prediction, the RNAi clones used in our screen do not appear to have major off-target effects on any other genes including α-arrestin genes, except arrd-19 RNAi which may also target arrd-21 which is a predicted pseudogene. Although qRT-PCR will be a straightforward way as the reviewers suggested, again currently we do not have any plan on following up this project and we therefore instead acknowledged the limitation. We added the prediction results in the Supplementary Table S1 and described this issue in the Methods, RNAi clones part as follows,
In Methods, RNAi clones: “All RNAi clones used in our screen were examined for their potential off-target effects by using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)²², and no significant off-target was predicted except arrd-19 RNAi, which may additionally target a predicted pseudogene arrd-21 (Table S1). Experimental validation by qRT-PCR will be necessary to completely exclude the possible off-target effects by RNAi clones.”
In Supplementary material: “Table S1. Possible RNAi target prediction. RNAi clones used in the screen were examined for their possible off-targets by Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)²². For RNAi clone names, ‘mv’ indicates a RNAi clone from Vidal library and ‘sjj’ indicates a RNAi clone from Ahringer library. RNAi clones generated by us for this study are labeled as ‘Lee lab’. Scores for potential target transcripts were calculated as described previously²² and were rounded off to the second digit after the decimal point.”
In Reference: 22. Thakur, N., et al., Clone Mapper: An Online Suite of Tools for RNAi Experiments in Caenorhabditis elegans. G3 (Bethesda), 2014. 4(11): p. 2137-2145.23.

4. Given that the authors observed the RNAi treated animals for weeks or months (in daf-2) it is surprising that no other behavioral or physical phenotypes from the alpha-arrestin RNAi were observed during the course of the analysis. If the authors truly did not observe any other phenotypes they should state it briefly in the text.

> We appreciate the reviewers’ comment. As our main interests were lifespan changes from a large scale screen, we did not pay attention to other phenotypes. Nevertheless, one noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantitate the results. We described this issue in Methods as follows,
In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”
Competing Interests: No competing interests were disclosed. Close
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Author Response (F1000Research Advisory Board Member) 14 Nov 2017

Seung-Jae V Lee, Korea Advanced Institute of Science and Technology, South Korea

14 Nov 2017

Author Response F1000Research Advisory Board Member

Dear Dr. Alessandro Bitto and Dr. Jason Pitt,
We greatly appreciate the reviewers’ valuable comments on our manuscript. We agree with all the reviewers’ comments and modified our manuscript to ... Continue reading Dear Dr. Alessandro Bitto and Dr. Jason Pitt,
We greatly appreciate the reviewers’ valuable comments on our manuscript. We agree with all the reviewers’ comments and modified our manuscript to describe the limitations of our studies raised by the reviewers. We would like to mention that the main purpose of publishing our current manuscript is to provide our data to other researchers who are interested in the characterization of C. elegans arrd genes, instead of keeping our current data unpublished. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research.

Reviewers’ comments:

In the manuscript RNAi targeting Caenorhabditis elegans alpha-arrestins marginally affects lifespan Park and co-workers provide largely negative results from lifespan experiments following RNAi knockdown of most of the worm alpha-arrestins. While we welcome the effort to publish negative results, there is one major concern with the experimental approach and several minor concerns with the interpretation of the results that should be addressed before the manuscript is fully accepted for indexing in F1000Research.

Major concern:
The authors data is almost entirely negative: there were only 4 clones which had a statistically significant effect. The authors attempt to address this concern by providing RNAi data for daf-16, which is known to strongly suppress the lifespan extension of daf-2(e1370) as well as decreasing N2 lifespan. However, the authors must demonstrate that alpha-arrestin RNAi knockdown is functioning. As suggested by the authors, there is some evidence that alpha-arrestins are expressed in neurons which are notoriously refractory to RNAi. The authors must show that they can use RNAi to knockdown alpha-arrestins by QRTPCR analysis. The authors could also address this concern by performing the RNAi in one of the mutant strains in which RNAi works in neurons and report if the lifespan still does not change, as they suggest in the discussion. We believe that providing these data is necessary to correctly interpret the results from the lifespan analyses.

> We completely agree with the reviewers for the comment. It would have been great if we had performed qRTPCR for validating the RNAi efficiency, while we were doing lifespan assays. As our experiments were done as an RNAi screen, our initial plan was validating RNAi efficiency and using RNAi-sensitive mutants if we had identified some positive RNAi clones. However, as the reviewers pointed out, our screen results were negative. We currently do not have any plan for following up this project and just want to inform other researchers of our screen data. Therefore, instead of putting efforts on this project retrospectively, we emphasized the limitations of our data raised by the reviewers to inform the readers of pitfalls by revising the manuscript as follows,
In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons^{7, 9, 24-26}, which are refractory to RNAi ^27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals ^29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.”

Minor concerns:
1. In the text the authors speak broadly of individual arrestins having no role in longevity however the authors only tested 24 of the 29 arrestins in the worm. If the authors had limited themselves to only those clones available in commercial RNAi libraries this would be understandable, however the authors did construct 3 new RNAi feeding constructs. This brings up the question to why the authors did not prepare the remaining 5 constructs. If the authors have reason to exclude those genes from their analysis (ie. they are pseudogenes, not expressed, etc) they should discuss these reasons in the text. Besides, there is the possibility that one of the missing constructs may indeed have major effects on lifespan in C. elegans.

> We appreciate the reviewers’ comment. We initially aimed at obtaining all the α arrestin RNAi clones that are not commercially available except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones for arrd-12, arrd-22 and arrd-27 because of unknown technical problems. We agree with the reviewers’ point that these three genes that were not tested may have major effects on lifespan. We addressed this issue in Methods for RNAi clones as well as in Discussion as follows,
In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.”
In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.”

2. The title of the manuscript implies there are more than one result than was observed. The simpler conclusion from the results provided is that individual alpha-arrestin knockdown has no effect of lifespan. This is the fundamental observation of the paper considering that most of them (20 out of 24) had no effect and that 4 of them had such an extremely small effect that it is arguable that the sample size is insufficient to support any conclusion¹. We understand the authors' motivation to make the manuscript sound more appealing, however, their interpretation is disingenuous, given their results.

> We thank the reviewers’ comment on the title and changed the title as follows,
Title: “RNAi targeting Caenorhabditis elegans α-arrestins has small or no effects on lifespan”

3. Because alpha-arrestins share significant sequence homology the authors should make some mention as to the likelihood that RNAi of any one arrestin would have off target effects on other arrestins. The authors could easily address this question experimentally with QRTPCR analysis. This is particularly relevant as one of the main conclusions of the manuscript is that future alpha-arrestin studies should knockdown multiple alpha-arrestins simultaneously.

> We agree with the reviewers that the RNAi targeting each of α-arrestin genes may have caused off-target effects on other α-arrestin genes because of sequence similarities. We performed off-target prediction using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap), and at least with the prediction, the RNAi clones used in our screen do not appear to have major off-target effects on any other genes including α-arrestin genes, except arrd-19 RNAi which may also target arrd-21 which is a predicted pseudogene. Although qRT-PCR will be a straightforward way as the reviewers suggested, again currently we do not have any plan on following up this project and we therefore instead acknowledged the limitation. We added the prediction results in the Supplementary Table S1 and described this issue in the Methods, RNAi clones part as follows,
In Methods, RNAi clones: “All RNAi clones used in our screen were examined for their potential off-target effects by using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)²², and no significant off-target was predicted except arrd-19 RNAi, which may additionally target a predicted pseudogene arrd-21 (Table S1). Experimental validation by qRT-PCR will be necessary to completely exclude the possible off-target effects by RNAi clones.”
In Supplementary material: “Table S1. Possible RNAi target prediction. RNAi clones used in the screen were examined for their possible off-targets by Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)²². For RNAi clone names, ‘mv’ indicates a RNAi clone from Vidal library and ‘sjj’ indicates a RNAi clone from Ahringer library. RNAi clones generated by us for this study are labeled as ‘Lee lab’. Scores for potential target transcripts were calculated as described previously²² and were rounded off to the second digit after the decimal point.”
In Reference: 22. Thakur, N., et al., Clone Mapper: An Online Suite of Tools for RNAi Experiments in Caenorhabditis elegans. G3 (Bethesda), 2014. 4(11): p. 2137-2145.23.

4. Given that the authors observed the RNAi treated animals for weeks or months (in daf-2) it is surprising that no other behavioral or physical phenotypes from the alpha-arrestin RNAi were observed during the course of the analysis. If the authors truly did not observe any other phenotypes they should state it briefly in the text.

> We appreciate the reviewers’ comment. As our main interests were lifespan changes from a large scale screen, we did not pay attention to other phenotypes. Nevertheless, one noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantitate the results. We described this issue in Methods as follows,
In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”

Dear Dr. Alessandro Bitto and Dr. Jason Pitt,
We greatly appreciate the reviewers’ valuable comments on our manuscript. We agree with all the reviewers’ comments and modified our manuscript to describe the limitations of our studies raised by the reviewers. We would like to mention that the main purpose of publishing our current manuscript is to provide our data to other researchers who are interested in the characterization of C. elegans arrd genes, instead of keeping our current data unpublished. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research.

Reviewers’ comments:

In the manuscript RNAi targeting Caenorhabditis elegans alpha-arrestins marginally affects lifespan Park and co-workers provide largely negative results from lifespan experiments following RNAi knockdown of most of the worm alpha-arrestins. While we welcome the effort to publish negative results, there is one major concern with the experimental approach and several minor concerns with the interpretation of the results that should be addressed before the manuscript is fully accepted for indexing in F1000Research.

Major concern:
The authors data is almost entirely negative: there were only 4 clones which had a statistically significant effect. The authors attempt to address this concern by providing RNAi data for daf-16, which is known to strongly suppress the lifespan extension of daf-2(e1370) as well as decreasing N2 lifespan. However, the authors must demonstrate that alpha-arrestin RNAi knockdown is functioning. As suggested by the authors, there is some evidence that alpha-arrestins are expressed in neurons which are notoriously refractory to RNAi. The authors must show that they can use RNAi to knockdown alpha-arrestins by QRTPCR analysis. The authors could also address this concern by performing the RNAi in one of the mutant strains in which RNAi works in neurons and report if the lifespan still does not change, as they suggest in the discussion. We believe that providing these data is necessary to correctly interpret the results from the lifespan analyses.

> We completely agree with the reviewers for the comment. It would have been great if we had performed qRTPCR for validating the RNAi efficiency, while we were doing lifespan assays. As our experiments were done as an RNAi screen, our initial plan was validating RNAi efficiency and using RNAi-sensitive mutants if we had identified some positive RNAi clones. However, as the reviewers pointed out, our screen results were negative. We currently do not have any plan for following up this project and just want to inform other researchers of our screen data. Therefore, instead of putting efforts on this project retrospectively, we emphasized the limitations of our data raised by the reviewers to inform the readers of pitfalls by revising the manuscript as follows,
In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons^{7, 9, 24-26}, which are refractory to RNAi ^27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals ^29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.”

Minor concerns:
1. In the text the authors speak broadly of individual arrestins having no role in longevity however the authors only tested 24 of the 29 arrestins in the worm. If the authors had limited themselves to only those clones available in commercial RNAi libraries this would be understandable, however the authors did construct 3 new RNAi feeding constructs. This brings up the question to why the authors did not prepare the remaining 5 constructs. If the authors have reason to exclude those genes from their analysis (ie. they are pseudogenes, not expressed, etc) they should discuss these reasons in the text. Besides, there is the possibility that one of the missing constructs may indeed have major effects on lifespan in C. elegans.

> We appreciate the reviewers’ comment. We initially aimed at obtaining all the α arrestin RNAi clones that are not commercially available except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones for arrd-12, arrd-22 and arrd-27 because of unknown technical problems. We agree with the reviewers’ point that these three genes that were not tested may have major effects on lifespan. We addressed this issue in Methods for RNAi clones as well as in Discussion as follows,
In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.”
In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.”

2. The title of the manuscript implies there are more than one result than was observed. The simpler conclusion from the results provided is that individual alpha-arrestin knockdown has no effect of lifespan. This is the fundamental observation of the paper considering that most of them (20 out of 24) had no effect and that 4 of them had such an extremely small effect that it is arguable that the sample size is insufficient to support any conclusion¹. We understand the authors' motivation to make the manuscript sound more appealing, however, their interpretation is disingenuous, given their results.

> We thank the reviewers’ comment on the title and changed the title as follows,
Title: “RNAi targeting Caenorhabditis elegans α-arrestins has small or no effects on lifespan”

3. Because alpha-arrestins share significant sequence homology the authors should make some mention as to the likelihood that RNAi of any one arrestin would have off target effects on other arrestins. The authors could easily address this question experimentally with QRTPCR analysis. This is particularly relevant as one of the main conclusions of the manuscript is that future alpha-arrestin studies should knockdown multiple alpha-arrestins simultaneously.

> We agree with the reviewers that the RNAi targeting each of α-arrestin genes may have caused off-target effects on other α-arrestin genes because of sequence similarities. We performed off-target prediction using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap), and at least with the prediction, the RNAi clones used in our screen do not appear to have major off-target effects on any other genes including α-arrestin genes, except arrd-19 RNAi which may also target arrd-21 which is a predicted pseudogene. Although qRT-PCR will be a straightforward way as the reviewers suggested, again currently we do not have any plan on following up this project and we therefore instead acknowledged the limitation. We added the prediction results in the Supplementary Table S1 and described this issue in the Methods, RNAi clones part as follows,
In Methods, RNAi clones: “All RNAi clones used in our screen were examined for their potential off-target effects by using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)²², and no significant off-target was predicted except arrd-19 RNAi, which may additionally target a predicted pseudogene arrd-21 (Table S1). Experimental validation by qRT-PCR will be necessary to completely exclude the possible off-target effects by RNAi clones.”
In Supplementary material: “Table S1. Possible RNAi target prediction. RNAi clones used in the screen were examined for their possible off-targets by Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)²². For RNAi clone names, ‘mv’ indicates a RNAi clone from Vidal library and ‘sjj’ indicates a RNAi clone from Ahringer library. RNAi clones generated by us for this study are labeled as ‘Lee lab’. Scores for potential target transcripts were calculated as described previously²² and were rounded off to the second digit after the decimal point.”
In Reference: 22. Thakur, N., et al., Clone Mapper: An Online Suite of Tools for RNAi Experiments in Caenorhabditis elegans. G3 (Bethesda), 2014. 4(11): p. 2137-2145.23.

4. Given that the authors observed the RNAi treated animals for weeks or months (in daf-2) it is surprising that no other behavioral or physical phenotypes from the alpha-arrestin RNAi were observed during the course of the analysis. If the authors truly did not observe any other phenotypes they should state it briefly in the text.

> We appreciate the reviewers’ comment. As our main interests were lifespan changes from a large scale screen, we did not pay attention to other phenotypes. Nevertheless, one noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantitate the results. We described this issue in Methods as follows,
In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”
Competing Interests: No competing interests were disclosed. Close
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Alessandro Bitto, University of Washington, Seattle, Seattle, USA

Jason Pitt, University of Washington, Seattle, USA
Roger Pocock, Monash University, Melbourne, Australia
Meng-Qiu Dong, National Institute of Biological Sciences (NCBS), Beijing, China

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Alessandro Bitto, Department of Pathology, University of Washington, Seattle, Seattle, WA, USA

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Alessandro Bitto, Department of Pathology, University of Washington, Seattle, Seattle, WA, USA

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Approved With Reservations

I appreciate the wish for the authors to move past this set of experiments and communicate their results to the field. Nevertheless, I must reiterate that, without testing the efficacy of their RNAi, their results cannot be interpreted, rather than be "interpreted with caution" as the authors suggest in the discussion.

Indeed, the authors themselves agree in their response to my previous review that "providing evidence for RNAi targeting arrd genes is a key for concluding that knockdown of arrd genes does not affect lifespan."

The authors suggest that performing qPCR on a new set of RNAi experiments would not demonstrate whether the knockdown worked on the worms followed for lifespan curves. While technically correct, I think it is fair to assume that their RNAi results are reproducible enough to warrant the assumption that whatever happens in a new set of RNAi experiments has happened in a previous one.

The authors bring forth a third reason for not testing the efficiency of their RNAi by claiming that their results are still useful to the field as they are. In fact, as I have suggested in my previous review, without qPCR testing of the efficacy of their RNAi, their results cannot be distinguished from the natural variation in lifespan of a control population of N2 worms, and are thus potentially meaningless.

In all honesty, I do not think that performing 24 qPCR reactions is an unreasonable request, especially when it is agreed that it would be "key for concluding that knockdown of arrd genes does not affect lifespan", which is the main point of the manuscript.

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Reviewer Expertise

Biology of Aging

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Author Response F1000Research Advisory Board Member

08 Dec 2017

Seung-Jae V Lee, Korea Advanced Institute of Science and Technology, South Korea

Dear Dr. Alessandro Bitto,

We greatly appreciate your time and efforts for further valuable comments. We totally agree with your concern that we do not know the degree of RNAi knockdown efficiency in worms except daf-16 RNAi, which was used as a positive control and showed expected lifespan phenotypes. Therefore, to precisely describe what we did, we changed “RNAi knockdown” to “feeding bacteria expressing dsRNA” throughout the manuscript. We sincerely thank you for fixing our misleading interpretation.

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19 Views

26 Oct 2017 | for Version 2

Alessandro Bitto, Department of Pathology, University of Washington, Seattle, Seattle, WA, USA

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Approved With Reservations

The authors took effort to address most of the concerns we raised in our initial review, but did not address at all our major concern, apart from a note in the discussion about the limitations of their results.

Thus our major concern stands: the authors must provide evidence that RNAi knockdown is functioning for each of the genes tested before their lifespan results can be interpreted correctly. Until such evidence is provided, there is no way of determining whether knockdown of individual arrestins really has limited effect on lifespan.

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No competing interests were disclosed.

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Biology of Aging

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Author Response F1000Research Advisory Board Member

14 Nov 2017

Seung-Jae V Lee, Korea Advanced Institute of Science and Technology, South Korea

Dear Dr. Alessandro Bitto,
We greatly appreciate your comments and respect your concerns on our paper. We totally agree with your point that providing evidence for RNAi targeting arrd genes is a key for concluding that knockdown of arrd genes does not affect lifespan. Nevertheless, we think it is more beneficial for the research field and for our lab to publish our data as they are, instead of not publishing a paper or performing all the qRT-PCR experiments retrospectively because of the following reasons.

First, we emphasized the limitation and the pitfall of our data thoroughly and therefore the readers will be fully aware of that.
Second, even if we perform the arrd RNAi experiments again and measure mRNA levels now, we will not be able to know whether the RNAi worked for the ones that we used for our lifespan screen. In addition, we do not have a plan on working on arrd genes any more.
Third, with all these issues and limitations in mind, we think our negative data are still useful for the field. If some researchers want to do lifespan experiments for arrd genes in the future, they would rather use RNAi-sensitive strains and/or mutants by knowing our negative results. We think publishing these results is better for the field than keeping the data in our lab unpublished.

In any case, we sincerely appreciate your efforts for evaluating our manuscript.

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Reviewer Report

14 Views

24 Oct 2017 | for Version 2

Meng-Qiu Dong, National Institute of Biological Sciences (NCBS), Beijing, China

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Approved

In this paper, Park et al. knocked down 24 out of 29 alpha-arrestin genes in C. elegans and found that none of them produced a sizable effect on the lifespans of the wild type or the long-lived daf-2 worms. The methods used in this study were sound, and the data quality were up to standard with hundreds of worms assayed for RNAi of each alpha-arrestin gene. I think the work is honest, and the negative results in this paper are reliable.

Concerns:

(1) I agree with the previous 2 reviewers that it is strange to leave out 5 members of the C. elegans alpha-arrestin genes.
(2) I think it is fair to say that none of the 24 genes extended lifespan when knocked down. The statistically significant 10% increase of wild-type lifespan by arrd-3 RNAi is negligible, given the variation of the lifespan assay. The relatively small p-value (0.0045) is blessed by the large sample size (n = 240). I wonder what the p-values would be if the two independent lifespan experiments had not been pooled.
(3) I would like the authors to report how many worms were censored.
(4) I wonder whether the authors had observed any noticeable phenotypes from their RNAi experiments, in particular, egg-laying defects. Strong egg-laying defects are easy to detect. G-protein signaling and beta-arrestin regulate the egg-laying behavior.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

C. elegans aging

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Author Response F1000Research Advisory Board Member

14 Nov 2017

Seung-Jae V Lee, Korea Advanced Institute of Science and Technology, South Korea

Dear Dr. Meng-Qiu Dong,
We sincerely thank you for your valuable comments on our manuscript. We admit the limitations of our research and revised our manuscript to downplay our results so as not to confuse the readers. We addressed your concerns and revised our manuscript as described below.

In this paper, Park et al. knocked down 24 out of 29 alpha-arrestin genes in C. elegans and found that none of them produced a sizable effect on the lifespans of the wild type or the long-lived daf-2 worms. The methods used in this study were sound, and the data quality were up to standard with hundreds of worms assayed for RNAi of each alpha-arrestin gene. I think the work is honest, and the negative results in this paper are reliable.

Concerns:
(1) I agree with the previous 2 reviewers that it is strange to leave out 5 members of the C. elegans alpha-arrestin genes.

> We appreciate your comments. We leave out arrd-20 and arrd-21 because they are predicted to be pseudogenes (Wormbase, version WS259) that are expected not to encode functional proteins. In addition, we did not test arrd-12, arrd-22 or arrd-27 because we were not able to obtain the corresponding RNAi clones during our cloning. We addressed this issue in Methods and discussed potential lifespan-regulatory roles of those genes in Discussion as follows,
In Methods, RNAi clones: “RNAi clone for arrd-12, arrd-22, or arrd-27 was not generated because we were not able to obtain transformed E. coli (HT115) colonies using arrd-12 or arrd-22 infusion reaction products, or the PCR product for arrd-27 genomic or complementary DNA.”
In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.”

(2) I think it is fair to say that none of the 24 genes extended lifespan when knocked down. The statistically significant 10% increase of wild-type lifespan by arrd-3 RNAi is negligible, given the variation of the lifespan assay. The relatively small p-value (0.0045) is blessed by the large sample size (n = 240). I wonder what the p-values would be if the two independent lifespan experiments had not been pooled.

> We appreciate your valuable comments. We agree with you that the extent of lifespan changes by several arrd RNAi clones was small but statistically significant because of the large sample size. Even if the lifespan data are not pooled, many independent lifespan results for arrd RNAi clones were again statistically significant. In either case, pooled or not pooled, we think the main point of our data indicates little effects of arrd RNAi on lifespan.

(3) I would like the authors to report how many worms were censored.

> We appreciate your comments. We provided raw lifespan data that contain the number of censored worms on each day of counting in Dataset1. One can also easily know the total number of censored worms by the difference between the number of animals that died and the total number of worms in Supplementary Table S2.

(4) I wonder whether the authors had observed any noticeable phenotypes from their RNAi experiments, in particular, egg-laying defects. Strong egg-laying defects are easy to detect. G-protein signaling and beta-arrestin regulate the egg-laying behavior.

> We appreciate your comments. Because we focused on the lifespan phenotype of arrd RNAi, unfortunately we did not pay attention to other physiological phenotypes and might have missed egg-laying phenotypes caused by some arrd RNAi clones. It will be an important point for future research.

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Reviewer Report

11 Views

20 Oct 2017 | for Version 2

Roger Pocock, Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University, Melbourne, Vic, Australia

11 Views Cite this report Responses(1)

Approved

The authors have partially addressed my concerns. Below I suggest further changes.

Change title from 'RNAi targeting Caenorhabditis elegans α-arrestins has small or no effects on lifespan' to 'RNAi targeting Caenorhabditis elegans α-arrestins has little effect on lifespan'
Also change from 'small or no effects' to 'little effect' throughout the text.
The authors state that they did not study arrd-12, 22 and 27 because of 'unknown technical problems'. It would be helpful for the field to be more specific here. Could they not be amplified?
In the results paragraph the authors state that 'knockdown of each of individual α-arrestin genes tended to slightly reduce lifespan in wild-type or in daf-2(-) mutants'
This is a little misleading. Change to 'knockdown of specific α-arrestin genes caused a minor reduction of lifespan in wild-type or in daf-2(-) mutants'
Change from 'In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR' to 'In addition, as we did not test whether RNAi targeting of each α-arrestin gene was effective by using quantitative RT-PCR'

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Author Response F1000Research Advisory Board Member

14 Nov 2017

Seung-Jae V Lee, Korea Advanced Institute of Science and Technology, South Korea

Dear Dr. Roger Pocock,
We greatly appreciate your comments on our manuscript. We further addressed your concerns and revised our manuscript as described below.

The authors have partially addressed my concerns. Below I suggest further changes.
1. Change title from 'RNAi targeting Caenorhabditis elegans α-arrestins has small or no effects on lifespan' to 'RNAi targeting Caenorhabditis elegans α-arrestins has little effect on lifespan'

> We appreciate your comment on the title. We changed the title as follows,
Title: RNAi targeting Caenorhabditis elegans α-arrestins has little effect on lifespan

2. Also change from 'small or no effects' to 'little effect' throughout the text.

> We also changed the ‘small or no effects’ to ‘little effect’ in the main text as follows,
In Abstract: “We found that knocking down each of 24 out of total 29 C. elegans α-arrestins had little effect on lifespan.”
In Introduction: “We found that knocking down each of the α-arrestins had little effect on the lifespan of wild-type and daf-2 mutants.”
In Discussion: “Here we showed that RNAi knockdown of individual C. elegans α-arrestins had little effect on lifespan in wild-type or daf-2 mutants.”
In Figure legend: “Figure 1. RNAi targeting each of Caenorhabditis elegans α-arrestins had little effect on lifespan.”

3. The authors state that they did not study arrd-12, 22 and 27 because of 'unknown technical problems'. It would be helpful for the field to be more specific here. Could they not be amplified?

> We appreciate your comments. We were not able to obtain transformed E. coli HT115 with infusion reaction products for arrd-12 or arrd-22 RNAi clones, or the PCR product for arrd-27 genomic or complementary DNA. We specified the ‘unknown technical problems’ by mentioning these details in the main text as follows,
In Methods, RNAi clones: “RNAi clone for arrd-12, arrd-22, or arrd-27 was not generated because we were not able to obtain transformed E. coli (HT115) colonies using arrd-12 or arrd-22 infusion reaction products, or the PCR product for arrd-27 genomic or complementary DNA.”

4. In the results paragraph the authors state that 'knockdown of each of individual α-arrestin genes tended to slightly reduce lifespan in wild-type or in daf-2(-) mutants'
This is a little misleading. Change to 'knockdown of specific α-arrestin genes caused a minor reduction of lifespan in wild-type or in daf-2(-) mutants'

> We appreciate your comments. We changed the sentence as follows,
In Results: “We found that knockdown of specific α-arrestin genes caused a minor reduction of lifespan in wild-type or in daf-2(-) mutants (Figure 1B, Figure S1B-U, Table S2).”

5. Change from 'In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR' to 'In addition, as we did not test whether RNAi targeting of each α-arrestin gene was effective by using quantitative RT-PCR'

> We appreciate your comments. We changed the sentence as suggested.
In Discussion: “In addition, as we did not test whether RNAi targeting each α-arrestin gene was effective by using quantitative RT-PCR”

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22 Views

06 Sep 2017 | for Version 1

Roger Pocock, Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University, Melbourne, Vic, Australia

22 Views Cite this report Responses(1)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

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Author Response F1000Research Advisory Board Member

14 Nov 2017

Seung-Jae V Lee, Korea Advanced Institute of Science and Technology, South Korea

Dear Dr. Roger Pocock,
We sincerely appreciate your valuable comments on our manuscript. We agree with all your points and revised our manuscript to describe the limitations of this study. We would like to mention that the main motivation for publishing this manuscript is to provide our negative results to researchers who are interested in characterizing α-arrestins in C. elegans. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research.

In this manuscript Park et al. performed RNAi knockdown of 24 of the 29 alpha-arrestin genes in C. elegans to ask whether they are important for lifespan regulation in wild type and daf-2 mutant animals. Their data shows that, in their experimental conditions, most alpha-arrestin genes are individually not important for regulating lifespan.

Concerns:
One major issue with the study is whether RNAi knockdown of the alpha-arrestin genes is working. As some alpha-arrestins are expressed in the nervous system, which is refractory to RNAi, this is a concern. To determine whether the RNAi knockdown is working then Q-RTPCR should be performed. However, this is not a small task for 24 genes, therefore, further experiments using sensitized genetic backgrounds for RNAi or elevated temperatures may tease out phenotypes for some alpha-arrestin genes. This could particularly be performed for the alpha-arrestin genes that when knocked down engender a slight effect on lifespan.

> We agree with the reviewer’s comment that the RNAi knockdown efficiency is a major concern in interpreting our screen results. It would have been great if we had performed qRT-PCR analysis. However, as the reviewers pointed out, this is not a small task. We currently do not have any plan for following up this project and want to inform other researchers of our screen data. Therefore, we further emphasized the pitfall of our results in the Discussion as follows,
In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons^{7, 9, 24-26}, which are refractory to RNAi^27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals^29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.”

In parallel to this, the authors could align the alpha-arrestin gene sequences to ask if there is a common sequence that could be used by RNAi targeting to knockdown multiple alpha-arrestin genes simultaneously.

> We appreciate the reviewer’s comment. The α-arrestin family members share arrestin-domains (O. Hobert, Wormbook, 2013). Thus, it will be intriguing if we could examine whether targeting arrestin domain for RNAi to knock down multiple arrd genes simultaneously and if multiple arrd gene knock down has effects on lifespan. We modified our manuscript to address this issue in Discussion as follows,
In Discussion: “Second, C. elegans α-arrestins may have functional redundancy considering the large number of the α-arrestin family members in C. elegans and their sequence similarity^{1, 7}, which may obscure examining the functional importance of each α-arrestin. In addition, some α-arrestins may mostly function by modulating their interacting proteins⁶. In this case, genetic inhibition of α-arrestins may rather subtly affect the functions of their interacting partners that directly regulate physiology, such as aging and longevity, causing weak or no phenotypes. Thus, it will be interesting to test the effects of simultaneous inhibition of α-arrestins possibly through targeting the arrestin domain, and to identify and to functionally characterize proteins that bind C. elegans α-arrestins.”

The authors knockdown of 24 of the 29 alpha-arrestin genes. I wonder why they didn’t generate RNAi clones for the remaining 5, especially since they did generate 3 other constructs for their analysis. Potentially, the important alpha-arrestin genes is amongst those not examined.

> We appreciate the reviewers’ comment. We aimed at obtaining all the α arrestin RNAi clones that are not commercially available, except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones, targeting arrd-12, arrd-22 and arrd-27. We addressed this issue in Methods for RNAi clones and in Discussion as follows,
In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.”
In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.”

The authors did not report any other behavioural or morphological phenotypes observed by alpha-arrestin gene knockdown. Any information on observed phenotypes would be of interest to the research community.

> We thank the reviewer for the valuable comment. One noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantify the results. We described this issue in Methods as follows,
In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”

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Reviewer Report

30 Views

04 Sep 2017 | for Version 1

Alessandro Bitto, Department of Pathology, University of Washington, Seattle, Seattle, WA, USA

Jason Pitt, Department of Pathology, University of Washington, Seattle, WA, USA

30 Views Cite this report Responses(1)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

References

1. Petrascheck M, Miller DL: Computational Analysis of Lifespan Experiment Reproducibility.Front Genet. 2017; 8: 92 PubMed Abstract | Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biology of Aging

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Author Response F1000Research Advisory Board Member

14 Nov 2017

Seung-Jae V Lee, Korea Advanced Institute of Science and Technology, South Korea

Dear Dr. Alessandro Bitto and Dr. Jason Pitt,
We greatly appreciate the reviewers’ valuable comments on our manuscript. We agree with all the reviewers’ comments and modified our manuscript to describe the limitations of our studies raised by the reviewers. We would like to mention that the main purpose of publishing our current manuscript is to provide our data to other researchers who are interested in the characterization of C. elegans arrd genes, instead of keeping our current data unpublished. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research.

Reviewers’ comments:

In the manuscript RNAi targeting Caenorhabditis elegans alpha-arrestins marginally affects lifespan Park and co-workers provide largely negative results from lifespan experiments following RNAi knockdown of most of the worm alpha-arrestins. While we welcome the effort to publish negative results, there is one major concern with the experimental approach and several minor concerns with the interpretation of the results that should be addressed before the manuscript is fully accepted for indexing in F1000Research.

Major concern:
The authors data is almost entirely negative: there were only 4 clones which had a statistically significant effect. The authors attempt to address this concern by providing RNAi data for daf-16, which is known to strongly suppress the lifespan extension of daf-2(e1370) as well as decreasing N2 lifespan. However, the authors must demonstrate that alpha-arrestin RNAi knockdown is functioning. As suggested by the authors, there is some evidence that alpha-arrestins are expressed in neurons which are notoriously refractory to RNAi. The authors must show that they can use RNAi to knockdown alpha-arrestins by QRTPCR analysis. The authors could also address this concern by performing the RNAi in one of the mutant strains in which RNAi works in neurons and report if the lifespan still does not change, as they suggest in the discussion. We believe that providing these data is necessary to correctly interpret the results from the lifespan analyses.

> We completely agree with the reviewers for the comment. It would have been great if we had performed qRTPCR for validating the RNAi efficiency, while we were doing lifespan assays. As our experiments were done as an RNAi screen, our initial plan was validating RNAi efficiency and using RNAi-sensitive mutants if we had identified some positive RNAi clones. However, as the reviewers pointed out, our screen results were negative. We currently do not have any plan for following up this project and just want to inform other researchers of our screen data. Therefore, instead of putting efforts on this project retrospectively, we emphasized the limitations of our data raised by the reviewers to inform the readers of pitfalls by revising the manuscript as follows,
In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons^{7, 9, 24-26}, which are refractory to RNAi ^27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals ^29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.”

Minor concerns:
1. In the text the authors speak broadly of individual arrestins having no role in longevity however the authors only tested 24 of the 29 arrestins in the worm. If the authors had limited themselves to only those clones available in commercial RNAi libraries this would be understandable, however the authors did construct 3 new RNAi feeding constructs. This brings up the question to why the authors did not prepare the remaining 5 constructs. If the authors have reason to exclude those genes from their analysis (ie. they are pseudogenes, not expressed, etc) they should discuss these reasons in the text. Besides, there is the possibility that one of the missing constructs may indeed have major effects on lifespan in C. elegans.

> We appreciate the reviewers’ comment. We initially aimed at obtaining all the α arrestin RNAi clones that are not commercially available except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones for arrd-12, arrd-22 and arrd-27 because of unknown technical problems. We agree with the reviewers’ point that these three genes that were not tested may have major effects on lifespan. We addressed this issue in Methods for RNAi clones as well as in Discussion as follows,
In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.”
In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.”

2. The title of the manuscript implies there are more than one result than was observed. The simpler conclusion from the results provided is that individual alpha-arrestin knockdown has no effect of lifespan. This is the fundamental observation of the paper considering that most of them (20 out of 24) had no effect and that 4 of them had such an extremely small effect that it is arguable that the sample size is insufficient to support any conclusion¹. We understand the authors' motivation to make the manuscript sound more appealing, however, their interpretation is disingenuous, given their results.

> We thank the reviewers’ comment on the title and changed the title as follows,
Title: “RNAi targeting Caenorhabditis elegans α-arrestins has small or no effects on lifespan”

3. Because alpha-arrestins share significant sequence homology the authors should make some mention as to the likelihood that RNAi of any one arrestin would have off target effects on other arrestins. The authors could easily address this question experimentally with QRTPCR analysis. This is particularly relevant as one of the main conclusions of the manuscript is that future alpha-arrestin studies should knockdown multiple alpha-arrestins simultaneously.

> We agree with the reviewers that the RNAi targeting each of α-arrestin genes may have caused off-target effects on other α-arrestin genes because of sequence similarities. We performed off-target prediction using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap), and at least with the prediction, the RNAi clones used in our screen do not appear to have major off-target effects on any other genes including α-arrestin genes, except arrd-19 RNAi which may also target arrd-21 which is a predicted pseudogene. Although qRT-PCR will be a straightforward way as the reviewers suggested, again currently we do not have any plan on following up this project and we therefore instead acknowledged the limitation. We added the prediction results in the Supplementary Table S1 and described this issue in the Methods, RNAi clones part as follows,
In Methods, RNAi clones: “All RNAi clones used in our screen were examined for their potential off-target effects by using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)²², and no significant off-target was predicted except arrd-19 RNAi, which may additionally target a predicted pseudogene arrd-21 (Table S1). Experimental validation by qRT-PCR will be necessary to completely exclude the possible off-target effects by RNAi clones.”
In Supplementary material: “Table S1. Possible RNAi target prediction. RNAi clones used in the screen were examined for their possible off-targets by Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)²². For RNAi clone names, ‘mv’ indicates a RNAi clone from Vidal library and ‘sjj’ indicates a RNAi clone from Ahringer library. RNAi clones generated by us for this study are labeled as ‘Lee lab’. Scores for potential target transcripts were calculated as described previously²² and were rounded off to the second digit after the decimal point.”
In Reference: 22. Thakur, N., et al., Clone Mapper: An Online Suite of Tools for RNAi Experiments in Caenorhabditis elegans. G3 (Bethesda), 2014. 4(11): p. 2137-2145.23.

4. Given that the authors observed the RNAi treated animals for weeks or months (in daf-2) it is surprising that no other behavioral or physical phenotypes from the alpha-arrestin RNAi were observed during the course of the analysis. If the authors truly did not observe any other phenotypes they should state it briefly in the text.

> We appreciate the reviewers’ comment. As our main interests were lifespan changes from a large scale screen, we did not pay attention to other phenotypes. Nevertheless, one noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantitate the results. We described this issue in Methods as follows,
In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”

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Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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RNAi targeting Caenorhabditis elegans α-arrestins marginally affects lifespan

Abstract

Keywords

Introduction

Methods

Caenorhabditis elegans strains

Phylogenetic analysis

RNAi clones

RNAi screen using lifespan assay

Results

Knocking down each of several C. elegans α-arrestins marginally influenced lifespan

Figure 1. RNAi targeting each of Caenorhabditis elegans α-arrestins had small or no effects on lifespan.

Discussion

Data availability

Competing interests

Grant information

Acknowledgments

Supplementary material

References

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