A Tudor domain protein, SIMR-1, promotes siRNA production at piRNA-targeted mRNAs in C. elegans
- University of Southern California, United States
- Colorado State University, United States
- Institute of Molecular Biology, Germany
Abstract
piRNAs play a critical role in the regulation of transposons and other germline genes. In Caenorhabditis elegans, regulation of piRNA target genes is mediated by the mutator complex, which synthesizes high levels of siRNAs through the activity of an RNA-dependent RNA polymerase. However, the steps between mRNA recognition by the piRNA pathway and siRNA amplification by the mutator complex are unknown. Here, we identify the Tudor domain protein, SIMR-1, as acting downstream of piRNA production and upstream of mutator complex-dependent siRNA biogenesis. Interestingly, SIMR-1 also localizes to distinct subcellular foci adjacent to P granules and Mutator foci, two phase-separated condensates that are the sites of piRNA-dependent mRNA recognition and mutator complex-dependent siRNA amplification, respectively. Thus, our data suggests a role for multiple perinuclear condensates in organizing the piRNA pathway and promoting mRNA regulation by the mutator complex.
Data availability
High-throughput sequencing data for RNA-sequencing libraries generated during this study are available through Gene Expression Omnibus (GSE138220 for preliminary simr-1 small RNA, and simr-1 mRNA sequencing data, GSE134573 for mut-16 small RNA and mRNA sequencing data, and GSE145217 for prg-1 and ergo-1 small RNA sequencing data).
-
A Tudor domain protein, SIMR-1, promotes siRNA production at piRNA-targeted mRNAs in C. elegansNCBI Gene Expression Omnibus, GSE138220.
-
RNAi pathways repress reprogramming of C. elegans germ cells during heat stressNCBI Gene Expression Omnibus, GSE134573.
Article and author information
Author details
Bruno F M de Albuquerque
Carolyn Marie Phillips
Funding
National Cancer Institute (K22 CA177897)
- Carolyn Marie Phillips
National Institute of General Medical Sciences (R35 GM119656)
- Carolyn Marie Phillips
National Institute of General Medical Sciences (T32 GM118289)
- Dieu An H Nguyen
National Institute of General Medical Sciences (R35 GM119775)
- Taiowa A Montgomery
Deutsche Forschungsgemeinschaft (KE1888/1-1)
- Rene F Ketting
Deutsche Forschungsgemeinschaft (KE1888/1-2)
- Rene F Ketting
Pew Charitable Trusts (Pew Scholar in the Biomedical Sciences)
- Carolyn Marie Phillips
National Science Foundation (DGE 1418060)
- Celja J Uebel
University of Southern California (Dornsife College Chemistry-Biology Interface trainee)
- Celja J Uebel
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Manage et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,914
- views
-
- 450
- downloads
-
- 61
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A Tudor domain protein, SIMR-1, promotes siRNA production at piRNA-targeted mRNAs in C. elegans
eLife 9:e56731.
https://doi.org/10.7554/eLife.56731
Further reading
-
- Chromosomes and Gene Expression
- Genetics and Genomics
Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA structure and nucleotide modifications can each interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We show that OTTR successfully captures both full-length tRNAs and tRNA fragments in budding yeast and in mouse reproductive tissues without any prior enzymatic treatment, and that tRNA cloning efficiency can be further enhanced via AlkB-mediated demethylation of modified nucleotides. As with other recent tRNA cloning protocols, we find that a subset of nucleotide modifications leave misincorporation signatures in OTTR datasets, enabling their detection without any additional protocol steps. Focusing on tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRNA fragments - including both 5′ and 3′ tRNA halves derived from the majority of tRNAs – than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.
-
- Chromosomes and Gene Expression
O-GlcNAcylation is the reversible post-translational addition of β-N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins. It plays an important role in several cellular processes through the modification of thousands of protein substrates. O-GlcNAcylation in humans is mediated by a single essential enzyme, O-GlcNAc transferase (OGT). OGT, together with the sole O-GlcNAcase OGA, form an intricate feedback loop to maintain O-GlcNAc homeostasis in response to changes in cellular O-GlcNAc using a dynamic mechanism involving nuclear retention of its fourth intron. However, the molecular mechanism of this dynamic regulation remains unclear. Using an O-GlcNAc responsive GFP reporter cell line, we identify SFSWAP, a poorly characterized splicing factor, as a trans-acting factor regulating OGT intron detention. We show that SFSWAP is a global regulator of retained intron splicing and exon skipping that primarily acts as a negative regulator of splicing. In contrast, knockdown of SFSWAP leads to reduced inclusion of a ‘decoy exon’ present in the OGT retained intron which may mediate its role in OGT intron detention. Global analysis of decoy exon inclusion in SFSWAP and UPF1 double knockdown cells indicate altered patterns of decoy exon usage. Together, these data indicate a role for SFSWAP as a global negative regulator of pre-mRNA splicing and positive regulator of intron retention.
