Time-resolved small RNA transcriptomics of the ichthyosporean Sphaeroforma arctica

Ichthyosporea, a clade of holozoans, represent a clade closely related to animals, and thus hold a key phylogenetic position for understanding the origin of animals. We have previously discovered that an ichthyosporean, Sphaeroforma arctica, contains microRNAs (miRNAs) as well as the miRNA processing machinery. This was the first discovery of miRNAs among the closest single-celled relatives of animals and raised intriguing questions about the roles of regulatory small RNAs in cell development and differentiation in unicellular eukaryotes. Like many ichthyosporeans, S. arctica also undergoes a transient multicellular developmental life cycle. As miRNAs are, among other roles, key regulators of gene expression during development in animals, we wanted to investigate the dynamics of miRNAs during the developmental cycle in S. arctica. Here we have therefore collected a comprehensive time-resolved small RNA transcriptome linked to specific life stages with a substantially higher sequencing depth than before, which can enable further discovery of functionally relevant small RNAs. The data consists of Illumina-sequenced small RNA libraries from two independent biological replicates of the entire life cycle of S. arctica with high temporal resolution. The dataset is directly linked and comes from the same samples as a previously published mRNA-seq dataset, thus enabling direct cross-functional analyses.


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Introduction
Ichthyosporeans hold a key position in the evolutionary tree for understanding the origin of animals and animal multicellularity.Ichthyosporea is a clade of holozoans, of which many characterized representatives undergo multinucleate (coenocytic) life cycles and exhibit a transient multicellular stage during cellularization of the coenocytes. 1,2mong ichthyosporeans, the life cycle of Sphaeroforma arctica has been best characterized.Multiple nuclear division cycles in a single cell occur with highly regular timing, forming multinuclear coenocytes, 3 which is followed by actomyosin-dependent cellularization. 4The whole life cycle has been characterized through mRNA transcriptomics, showing dynamic transcriptional regulation during the life cycle, including transcriptional regulation of the putative key regulators of cellularization. 4croRNAs (miRNAs) are short RNA molecules that, among many roles, regulate the activity of genes important for multicellular development in both animals and plants (e.g., Refs.5, 6).Although miRNAs have been reported from other eukaryote groups spanning the tree of life, such as brown and green algae (e.g., Refs.7, 8) Amoebozoa (e.g., Ref. 9) excavates (e.g., Ref. 10) and unicellular Holozoa, 11 their presence and function in many cases remain controversial. 12evertheless, these discoveries raise the intriguing question of whether small regulatory RNAs also play a role during development in unicellular and facultatively multicellular organisms.To understand whether miRNAs play a role in its development, we collected a high-quality small RNA dataset in S. arctica at both high depth and high temporal resolution throughout its entire developmental cycle.

Methods
The purpose of this study was to generate a dataset in order to (1) investigate the temporal dynamics of miRNA expression in S. arctica and to provide potential functional insights into the miRNAs, (2) discover novel miRNA genes in S. arctica, and ( 3) discover other potentially functionally relevant small RNAs, such as piRNAs (e.g., Refs.13, 14).
We have acquired high throughput sequencing datasets of the fraction of RNA molecules smaller than approximately 200 nucleotides (small RNAs).We have isolated and sequenced the small RNA content from synchronized cultures every six hours over a period of 72 hours, spanning the entire cell cycle.The sequencing was done in two biological replicates.
The experiment was performed in parallel with the previously published mRNA transcriptome dataset 4 and the small RNA libraries were prepared from the same total RNA samples; thus, the present dataset can be analyzed simultaneously with the mRNA data.

Experimental design and culturing conditions and RNA extraction
The cultures were prepared according to the protocol originally described in Ref. 4. In detail, S. arctica cells were cultured in Marine Broth media (Marine Broth, Difco BD, NJ, USA; 37.4 g/L) in sterile culture flasks at 12°C in dark conditions.The cultures were grown to a stationary phase, which has been shown to synchronize the coenocytic cycles (see Ref. 3).
At the start of the experiment, the saturated cultures were diluted 1:300 into fresh marine broth media.Aliquots were collected every six hours for 72 hours, spanning an entire coenocytic life cycle.RNA was extracted using the miRNeasy Mini Kit (QIAGEN, Venlo, Netherlands) from approximately 50 mL of culture for each culture aliquot, and RNA integrity was evaluated using a Bioanalyzer 2100 (Agilent, CA, US).

Library preparation and sequencing details
The small RNA libraries were prepared using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs, MA, US).A total of 50 bp single-end reads were obtained by sequencing the libraries on the Illumina HiSeq 2500 platform with the v4 chemistry and high output mode.Library preparation and sequencing was carried out by the CRG Genomics core unit, Barcelona, Spain.The data presented here is not processed in any way.The sequencing libraries contained on average 22.7 million reads (SD = 5.3 mill reads).Together, this data represents a more than 40-fold higher sequencing depth than the previous study, 11 where small RNA reads were obtained from only two samples.

Limitations
The small RNA extractions were sequenced as is, without any external, or spiked-in, controls.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: My area of expertise is in microbiology, bioinformatics, and evolutionary biology.I also have a background in molecular biology (including DNA and RNA).
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.The methodological section of the manuscript provides detailed procedures for culturing, RNA extraction, library preparation, and sequencing, thereby enabling the replication of all experiments.Data files are well-organized, summarized in a table, and deposited in the RNA database under a unique accession number.The references are thoughtfully selected, offering opportunities for deeper exploration of the topic.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Phylogeny, evolution and genomics of protists 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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Reviewer Report 01
April 2024 https://doi.org/10.5256/f1000research.146953.r252697© 2024 Milanowski R.This is an open access peer review report 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.Rafał Milanowski University of Warsaw, Warsaw, PolandIchthyosporea is a group of Opisthokonta that plays a crucial role in discussions about the origins of animals and animal multicellularity.The Data Note article by Ondracka et al. describes the previously unpublished small RNA transcriptome associated with specific life stages of the ichthyosporean species Sphaeroforma arctica.This dataset holds the potential to facilitate further discovery of functionally relevant small RNAs in this species.

Table 1
details the individual files under accession number PRJEB55646.Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).