Analysis of the<i> Arabidopsis</i> organellar rhomboid At1g74140 transcript population uncovered splicing patterns different from its close relative At1g74130

Kenton Ko; Jeremy Guenther; Nicholas Ostan; Joshua Powles

doi:10.12688/f1000research.21219.1

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Research Article

Analysis of the Arabidopsis organellar rhomboid At1g74140 transcript population uncovered splicing patterns different from its close relative At1g74130

[version 1; peer review: 2 not approved]

Kenton Ko ¹, Jeremy Guenther¹, Nicholas Ostan¹, Joshua Powles¹

PUBLISHED 18 Nov 2019

Author details Author details

¹ Department of Biology, Queen's University, Kingston, ON, K7L 3N6, Canada

Kenton Ko
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Jeremy Guenther
Roles: Data Curation, Formal Analysis, Investigation, Methodology

Nicholas Ostan
Roles: Data Curation, Formal Analysis, Investigation, Methodology

Joshua Powles
Roles: Formal Analysis, Methodology, Project Administration, Supervision

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: Four distinct rhomboid genes appear to function in Arabidopsis plastids, two “active” types from the secretases and presenilin-like associated rhomboid-like (PARL) categories (At1g25290 and At5g25752) and two “inactive” rhomboid forms (At1g74130 and At1g74140). The number of working rhomboids is further increased by alternative splicing, two reported for At1g25290 and three for At1g74130. Since At1g25290 and At1g74130 exist as alternative splice variants, it would be necessary to assess the splicing patterns of the other two plastid rhomboid genes, At5g25752 and At1g74140, before studying the Arabidopsis plastid rhomboid system as a whole.
Methods: This study thus specifically focused on an analysis of the At1g74140 transcript population using various RT-PCR strategies.
Results: The exon mapping results indicate splicing patterns different from the close relative At1g74130, despite similarity between the exonic sequences. The splicing patterns indicate a high level of sequence “discontinuity” in the At1g74140 transcript population with a significant portion of the discontinuity being generated by two regions of the gene.
Conclusion: The overall discontinuous splicing pattern of At1g74140 may be reflective of its mode of involvement in activities like controlling gene expression.

Keywords

Arabidopsis, plastid rhomboid proteins, alternative splicing, short RNA transcript production

Corresponding author: Kenton Ko

Competing interests: No competing interests were disclosed.

Grant information: The research was supported by funds (grant number 43698) from the Natural Sciences and Engineering Research Council of Canada and Queen’s University. Joshua Powles was supported by graduate awards from Queen’s University. Jeremy Guenther and Nicholas Ostan were supported partially by SWEP funds from Queen’s University.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2019 Ko K 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.

How to cite: Ko K, Guenther J, Ostan N and Powles J. Analysis of the Arabidopsis organellar rhomboid At1g74140 transcript population uncovered splicing patterns different from its close relative At1g74130 [version 1; peer review: 2 not approved]. F1000Research 2019, 8:1925 (https://doi.org/10.12688/f1000research.21219.1) First published: 18 Nov 2019, 8:1925 (https://doi.org/10.12688/f1000research.21219.1) Latest published: 18 Nov 2019, 8:1925 (https://doi.org/10.12688/f1000research.21219.1)

Introduction

The rhomboid protein gene system in Arabidopsis is complex with 13 transcribed genes encoding both “active” and “inactive” rhomboid protein types (Knopf & Adam, 2012; Koonin et al., 2003; Tripathi & Sowdhamini, 2006). A recent survey of the databases indicates at least 22 entries for Arabidopsis (Powles & Ko, 2018). These entries encompass the same range of rhomboid types as that observed in other organisms (Lemberg & Freeman, 2007). One intriguing aspect about these different rhomboids is that they appear to operate simultaneously, such as in the Arabidopsis plastid compartment. In Arabidopsis, there are four distinct plastid rhomboid genes predicted - two encoding “active” types (At1g25290 and At5g25752) and two encoding “inactive” forms (At1g74130 and At1g74140). This number is further increased by alternative splicing, as reported for At1g25290 and At1g74130 (Powles et al., 2013; Sedivy-Haley et al., 2012). Alternative splicing of At1g25290 transcripts introduced variant forms with and without a putative cyclin-binding RVL motif (Sedivy-Haley et al., 2012). For At1g74130, alternative splicing generated three variants with different carboxyl segments, changing from a predicted seven to a six transmembrane structure (Powles et al., 2013). These three At1g74130 variants exhibited different functionalities, such as their interactions with the Tic40 substrate and the yeast mitochondrial rhomboid protease Rbd1 and Mgm1 in live cell contexts, and their abilities in sensitizing cells to antimicrobial drugs (Powles et al., 2013; Powles & Ko, 2018). These different functionalities are especially interesting since one potential role of At1g74130 appears to be in the early stages of tissue development and plastid biogenesis (Sedivy-Haley et al., 2011).

The picture emerging for the Arabidopsis plastid rhomboids suggests that alternative splice variants are involved in regulatory type activities. Such a phenomenon is observed in other organisms, such as for the human rhomboid gene RHBDD2, as an example (Abba et al., 2009). The levels of the two alternatively spliced RHBDD2 mRNAs were elevated in breast cancer cell lines (Abba et al., 2009). In terms of other regulatory roles, rhomboid proteins themselves are often associated with regulatory activities in different organisms and in a range of cellular pathways, such as development, mitochondrial membrane remodeling, protein transport, quorum sensing, and stress response (see examples in Adrian & Freeman, 2012; Jeyaraju et al., 2013; Lemberg, 2013; McQuibban et al., 2003; Sedivy-Haley et al., 2011; Sekine et al., 2012; Stevenson et al., 2007; Thompson et al., 2012; Urban, 2006; Yan et al., 2008). The possibility of alternative splicing contributing further to these different types of regulatory activities is thus intriguing and warrants investigation.

It is now clear that to understand how the plastid rhomboid system works, it is necessary to identify all potential rhomboid variants in play. This study was thus designed to examine the splicing activities of At1g74140, a close relative to At1g74130 and located tandemly next to At1g74130. An analysis of the At1g74140 transcript population would help uncover potential modes of operation for this gene and its possible role in Arabidopsis plastids.

Methods

Arabidopsis and the propagation regime used

Growth chambers were used to propagate the plants employed in this study. The cultivation conditions for the Arabidopsis line CS60000 (“wild-type”) were derived from information posted on the Arabidopsis Biological Resource Center (Ohio State University) website (http://abrc.osu.edu) (Arabidopsis Biological Resource Center, RRID:SCR_008136) (Alonso et al., 2003). The growth chamber parameters used were: 21°C; 70% humidity; and a 16:8 hour light:dark photoperiod using 150–200 μmol·m^-2·s^-1 of fluorescent and incandescent lighting. Seeds were cold stratified for 3 days before transfer to growth chambers. All plants were subjected to a regime of daily watering and weekly fertilization.

Database sequences utilized

The genomic DNA entries used for At1g74130 and At1g74140 are of the tandem genes located on chromosome 1 (Chromosome 1 NC_003070.9). The accession numbers used for the At1g74140 cDNA work were: NM_001124127.1, NM_001084350.2, NM_106074.2, NM_001036203.1, and NM_001084351.2. The accession numbers used for the At1g74130 cDNA entries were NM_106073.5 and NM_202412.1 (NCBI, RRID:SCR_006472; NCBI BLAST, RRID:SCR_004870).

PCR-based procedures for the analysis of transcript populations

Procedures and optimization details related to the analysis of transcript structure were the same as those described by Sedivy-Haley et al. (2012). Briefly, all plant tissues destined for RNA extraction were collected between 11:00 and 13:00 h during the light period to equalize the circadian effects. Total RNA was isolated and treated with DNase using Qiagen kits (RNeasy kit and RNase-free DNase set, Qiagen, Hilden, Germany) (QIAGEN, RRID:SCR_008539). Transcript structure was studied using Qiagen PCR kits (One-Step RT-PCR kit, Qiagen, Hilden, Germany) (QIAGEN, RRID:SCR_008539) and a set of exon-exon specific DNA primers. A summary of the DNA primers used is provided in the Extended data: Table S1. The RT-PCR steps and cycle timings used were as recommended by the manufacturer, Qiagen, without modifications. The RT-PCR assays were all conducted with 20 ng of total RNA with cycles between 35 and 40 to visualize lower level products. The temperature used was 55°C. Prior optimization RT-PCR assays were conducted with varying temperatures, cycles, and total RNA amounts. All assays were conducted using an Eppendorf Mastercycler for microplates. PCR products were resolved by running samples in standard 4% (w/v) Tris-borate – EDTA polyacrylamide gels. The DNA markers used were purchased from Frogabbio (Toronto, Ontario, Canada).

Results

Comparison of At1g74140 and the nearby related At1g74130

There are currently four genes predicted for plastidial rhomboid proteins in Arabidopsis (Kanaoka et al., 2005; Knopf & Adam, 2012; Koonin et al., 2003; Tripathi & Sowdhamini, 2006). Three are located in chromosome 1 and one in chromosome 5. Our previous work on the transcript structures of At1g25290 and At1g74130 revealed alternatively spliced variants, two for At1g25290 and three for At1g74130 (Powles et al., 2013; Sedivy-Haley et al., 2012). The other rhomboid gene in chromosome 1, At1g74140, is highly similar to At1g74130 and located downstream from At1g74130. At1g74130 is located between nucleotide positions 27873611 and 27876033, and At1g74140 occupies 27876905 to 27879780. The overall exon-intron structures of At1g74130 and At1g74140 are similar, each with 8 exons and 7 introns (Figure 1A). Although the exon lengths are similar, there appear to be substantial differences in the lengths of the middle introns 3, 4, and 5 (Figure 1A). A comparison of the translated products from the available At1g74130 and At1g74140 cDNA entries or sequences (five and three, respectively) also indicated a high degree of similarity with a few gaps (Figure 1B). The differences in intronic composition may thus be reflective of the roles of the two genes and this notion continues to be the case when examining the outcomes of the At1g74140 transcript structure analysis below.

Figure 1. A comparison of the related genes At1g74130 and At1g74140.

(A) A schematic comparison of the related gene structures (using information derived from the entry Chromosome 1 NC_003070.9). Exons are in depicted black and numbered sequentially. Introns are depicted in gray. (B) A comparison of the amino acid sequences derived from five At1g74140 variants and three At1g74130 variants (The accession numbers for the At1g74140 variants were: NM_001124127.1, NM_001084350.2, NM_106074.2, NM_001036203.1, and NM_001084351.2. The accession numbers for the At1g74130 variants were NM_106073.5 and NM_202412.1, and from Powles et al., 2013). Gaps are denoted by hyphens. The yellow highlighted amino acids indicate the residue locations for part of the active site.

Analysis of At1g74140 transcript structure and its splicing activities

For the analysis of At1g74140 transcripts, the overall RT-PCR strategy and primer pairs used were designed to map alternative splicing activities in the context of transcript populations (Table S1 summarizes the details of the exon-exon specific primers used). These primers should allow detection of the many differently spliced exonic structures in the transcript population, along with changes to levels of neighboring exons represented in the resulting products. Such modulations in RT-PCR product levels should be indicative of different transcript splicing activities. This approach was employed to characterize alternatively spliced variants in our previous studies on At1g25290 and At1g74130 (Powles et al., 2013; Sedivy-Haley et al., 2012).

A combined RT-PCR strategy, stepped and laddered primer pairs, was used to characterize the different structures that existed in the transcript population. The outcomes were sorted and summarized schematically in Figure 2–Figure 4 along with the corresponding primer strategies. The various RT-PCR results used for the construction of Figure 2–Figure 4 are presented in Figure 5–Figure 10. The presence or absence of each exon and their relative levels were surveyed and compiled. These exon specific RT-PCR outcomes are sorted and organized schematically as black lines in Figure 2 (below the gene map). The weight of the black lines, for example, thick versus thin, represents relative RT-PCR product levels. From this summation map of the stepped RT-PCR strategy, we were able to observe missing exon-exon products as well as unbalances in the relative levels of neighboring exon-exon products. Products primed from the start region of exon 1 and forward region of exon 8 were not detected. Products primed from the latter end of exon 1 to exon 7 were present, but exhibited changes to their relative levels, even if the neighboring RT-PCR products were expected to be comparable with respect to size and RT-PCR assay parameters.

Figure 2. RT-PCR analysis of the At1g74140 transcript population.

The results summarized in this figure are presented in Figure 5 through Figure 10 and Figures S1 to S3. Primer pairs focused on exon-exon borders are summarized as black solid lines. Primer pairs focused on 5’UTR and START are summarized separately as red lines. The weight of the lines reflects relative levels of resulting RT-PCR products. A hatched red line represents the absence of a predicted RT-PCR product. The At1g74140 gene structure and map of priming sites are provided schematically.

Figure 3. RT-PCR analysis of the At1g74140 transcript population with a focus on the exon 2 region.

The results summarized are presented in Figure 5 through Figure 10 and Figures S1 to S3. The primer pair focused on exons 3 to 5 is represented as a black solid line. Primer pairs focused on the exon 2 region are summarized separately as red lines. The weight of the lines reflects relative levels of resulting RT-PCR products. A hatched red line represents the absence of a predicted RT-PCR product.

Figure 4. RT-PCR analysis of the At1g74140 transcript population with a focus on the exon 7 region.

The results summarized are presented in Figure 5 through Figure 10 and Figures S1 to S3. Primer pairs focused on the exon 7 region are summarized as red lines. The weight of the lines reflects relative levels of resulting RT-PCR products. A hatched red line represents the absence of a predicted RT-PCR product.

Figure 5. RT-PCR results for the forward primer 1F set.

The forward 1F primer used is depicted above the reverse primers. Changes to splicing predictions are outlined elliptically. The relative size markers used are labelled M for the Froggabio 100 base pair ladder.