Caenorhabditis elegans
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ABSTRACT:
PROVIDER: PRJNA118225 | ENA |
REPOSITORIES: ENA
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Project description:Short interfering RNAs (siRNAs) are a class of regulatory effectors that enforce gene silencing though formation of RNA duplexes. While progress has been made in identifying the capabilities of siRNAs in silencing of foreign RNA and transposable elements, siRNA functions in endogenous gene regulation have remained mysterious. In certain organisms, siRNA biosynthesis involves novel enzymes that act as RNA-directed RNA polymerases (RdRPs). Here we analyze the function of a C. elegans RdRP, RRF-3, during spermatogenesis. We found that loss of RRF-3 function resulted in pleiotropic defects in sperm development, and that sperm defects led to embryonic lethality. Notably, sperm nuclei in mutants of either rrf-3 or another component of the siRNA pathway, eri-1, were frequently surrounded by ectopic microtubule structures, with spindle abnormalities in a subset of the resulting embryos. Through high-throughput small RNA sequencing, we identified a population of cellular mRNAs from spermatogenic cells that appear to serve as templates for antisense siRNA synthesis. This set of genes includes the majority of genes known to have enriched expression during spermatogenesis, as well as many genes not previously known to be expressed during spermatogenesis. For a subset of these genes, we found that RRF-3 was required for effective siRNA accumulation. These and other data suggest a working model in which a major role for the RRF-3/ERI pathway is to generate siRNAs that set patterns of gene expression through feedback repression of a set of critical targets during spermatogenesis Sequencing of small RNAs from a population of C. elegans isolated spermatogenic cells and from two paired sets of rrf-3 mutant and control whole males
2010-01-13 | E-GEOD-18429 | biostudies-arrayexpress
Project description:Short interfering RNAs (siRNAs) are a class of regulatory effectors that enforce gene silencing though formation of RNA duplexes. While progress has been made in identifying the capabilities of siRNAs in silencing of foreign RNA and transposable elements, siRNA functions in endogenous gene regulation have remained mysterious. In certain organisms, siRNA biosynthesis involves novel enzymes that act as RNA-directed RNA polymerases (RdRPs). Here we analyze the function of a C. elegans RdRP, RRF-3, during spermatogenesis. We found that loss of RRF-3 function resulted in pleiotropic defects in sperm development, and that sperm defects led to embryonic lethality. Notably, sperm nuclei in mutants of either rrf-3 or another component of the siRNA pathway, eri-1, were frequently surrounded by ectopic microtubule structures, with spindle abnormalities in a subset of the resulting embryos. Through high-throughput small RNA sequencing, we identified a population of cellular mRNAs from spermatogenic cells that appear to serve as templates for antisense siRNA synthesis. This set of genes includes the majority of genes known to have enriched expression during spermatogenesis, as well as many genes not previously known to be expressed during spermatogenesis. For a subset of these genes, we found that RRF-3 was required for effective siRNA accumulation. These and other data suggest a working model in which a major role for the RRF-3/ERI pathway is to generate siRNAs that set patterns of gene expression through feedback repression of a set of critical targets during spermatogenesis
2010-01-13 | GSE18429 | GEO
Project description:Endogenous RNA-directed RNA polymerases (RdRPs) are cellular components capable of synthesizing new complementary RNAs from existing RNA templates. We present evidence for successive engagement of two different RdRPs in an endogenous siRNA-based mechanism targeting specific mRNAs in C. elegans soma. In the initiation stage of this process, a group of mRNA species are chosen as targets for downregulation, leading to accumulation of rare 26-nt 5'-phosphorylated antisense RNAs that depend on the RdRP homolog RRF-3, the argonaute ERGO-1, DICER, and a series of associated (ERI) factors. This primary process leads to production of a much more abundant class of 22-nt antisense RNAs, dependent on a secondary RdRP (RRF-1) and associating with at least one distinct Argonaute (NRDE-3). The requirement for two RdRP/Argonaute combinations and initiation by a rare class of uniquely-structured siRNAs in this pathway illustrate the caution and flexibility used as biological systems exploit the physiological copying of RNA. 24 small RNA and 2 polyA RNA samples
2010-02-01 | E-GEOD-19414 | biostudies-arrayexpress
Project description:Endogenous RNA-directed RNA polymerases (RdRPs) are cellular components capable of synthesizing new complementary RNAs from existing RNA templates. We present evidence for successive engagement of two different RdRPs in an endogenous siRNA-based mechanism targeting specific mRNAs in C. elegans soma. In the initiation stage of this process, a group of mRNA species are chosen as targets for downregulation, leading to accumulation of rare 26-nt 5'-phosphorylated antisense RNAs that depend on the RdRP homolog RRF-3, the argonaute ERGO-1, DICER, and a series of associated (ERI) factors. This primary process leads to production of a much more abundant class of 22-nt antisense RNAs, dependent on a secondary RdRP (RRF-1) and associating with at least one distinct Argonaute (NRDE-3). The requirement for two RdRP/Argonaute combinations and initiation by a rare class of uniquely-structured siRNAs in this pathway illustrate the caution and flexibility used as biological systems exploit the physiological copying of RNA.
2010-02-01 | GSE19414 | GEO
Project description:The nematode C. elegans contains several types of endogenous small interfering RNAs (endo-siRNAs) produced by RNA-dependent RNA polymerase (RdRP) complexes. Both “silencing” siRNAs bound by Worm-specific Argonautes (WAGO) and “activating” siRNAs bound by the CSR-1 Argonaute require the DRH-3 helicase, an RdRP component. Here we show that, in the drh-3(ne4253) mutant deficient in RdRP-produced secondary endo-siRNAs, the silencing histone mark H3K9me3 is largely depleted, whereas in the csr-1 partial loss-of-function mutant this mark is ectopically deposited on CSR-1 target genes. Moreover, we observe ectopic H3K9me3 at enhancer elements in both drh-3 and csr-1 partial loss-of-function mutants and describe small RNAs matching enhancers. Finally, we detect accumulation of H3K27me3 at highly expressed genes in the drh-3(ne4253) mutant, which correlates with their reduced transcription. Our study shows that when abundant RdRP-produced siRNAs are depleted, there is ectopic elevation of noncoding RNAs linked to increase in silencing chromatin marks. Moreover, our results suggest that enhancer small RNAs may guide local H3K9 methylation.
2019-04-04 | GSE115629 | GEO
Project description:How lifespan and the rate of aging are set is a key problem in biology. Small RNAs are conserved molecules that impact diverse biological processes through the control of gene expression. However, in contrast to miRNAs, the role of endo-siRNAs in aging remains unexplored. Here, by combining deep sequencing and genomic and genetic approaches in C.CaenorhabditisC. elegans elegans, we reveal an unprecedented role for endo-siRNA molecules in the maintenance of proteostasis and lifespan extension in germline-less animals. Furthermore, we identify an endo-siRNA-regulated tyrosine phosphatase, which limits the longevity of germline-less animals by restricting the activity of the heat shock transcription factor HSF-1. Altogether, our findings point to endo-siRNAs as a link between germline removal and the HSF-1 proteostasis and longevity-promoting somatic pathway. This establishes a role for endo siRNAs in the aging process and identifies downstream genes and physiological processes that are regulated by the endo siRNAs to affect longevity.
2020-04-03 | GSE128935 | GEO
Project description:RRF-3 and ERI-1 are first identified proteins required for accumulation of at least some endogenous secondary siRNAs in C.elegans. Genome wide gene expression analysis was performed on L4 stage rrf-3 and eri-1 mutant C. elegans to study effects caused by loss of these proteins. Mutant rrf-3 and eri-1 strains exhibited similar expression patterns when compared to N2 wild type, while 72 transcripts were found to be co-overexpressed and 4 transcripts co-underexpressed (> 2-fold, p< 0.05). Ontology analysis indicated many of the gene products were associated with protein phosphorylation and sperm function. These results provide additional support for the hypothesis that RRF-3 and ERI-1 act together in a siRNA pathway and may indicate biological processes that are related to endo-siRNAs. Keywords: Mutant vs. wild type -comparison
2007-10-19 | GSE8659 | GEO
Project description:RRF-3 and ERI-1 are first identified proteins required for accumulation of at least some endogenous secondary siRNAs in C.elegans. Genome wide gene expression analysis was performed on L4 stage rrf-3 and eri-1 mutant C. elegans to study effects caused by loss of these proteins. Mutant rrf-3 and eri-1 strains exhibited similar expression patterns when compared to N2 wild type, while 72 transcripts were found to be co-overexpressed and 4 transcripts co-underexpressed (> 2-fold, p< 0.05). Ontology analysis indicated many of the gene products were associated with protein phosphorylation and sperm function. These results provide additional support for the hypothesis that RRF-3 and ERI-1 act together in a siRNA pathway and may indicate biological processes that are related to endo-siRNAs. Experiment Overall Design: rrf-3(pk1426) mutant worms (NL2099), eri-1 (mg366) mutant worms (GR1373), and wild type worms (N2) were grown on culturing plates containing NGM agar media with OP50 Escherichia coli (E. coli). Worms were grown at 20°C and harvested at L4 stage. RNA isolation was made using Ribopure Total RNA isolation -kit (Ambion). Animals were grown and RNA isolated separately for each biological replicate. Total RNA was labeled for chip experiments using the cRNA protocol (Affymetrix, Palo Alto, CA). DNA from each mutant was hybridized to Gene Chip C. elegans whole genome arrays containing 22,500 transcripts (Affymetrix, Palo Alto, CA) as three biological replicates (3 chips each for N2, rrf-3, eri-1). Microarray hybridization and scanning was performed at the Biomedicum Biochip Center (Helsinki, Finland).
2008-06-16 | E-GEOD-8659 | biostudies-arrayexpress
Project description:Adenosine deaminases that act on RNA (ADARs) catalyze the conversion of adenosine to inosine in dsRNA. C. elegans ADARs, ADR-1 and ADR-2, promote the expression of genes containing dsRNA structures by preventing their processing into siRNAs and silencing by RNAi. The 26G endogenous siRNA (endo-siRNA) pathway generates a subset of siRNAs distinct from those made in adr-1;adr-2 mutants, but using many of the same factors. We found that adr-1;adr-2;rrf-3 mutants, lacking both ADARs and the RNA-dependent RNA polymerase RRF-3 required for the 26G pathway, display a bursting phenotype rescued by the RNAi factors RDE-1 and RDE-4. To determine what gene expression changes underlie the synthetic phenotype of adr-1;adr-2;rrf-3 mutants, we sequenced poly(A)+ RNA from adr-1;adr-2;rrf-3 embryos, their parent strains, and strains rescued with mutations in rde-1 and rde-4. We found that genes associated with edited structures were robustly downregulated in adr-1;adr-2;rrf-3 mutants in a manner partially dependent on rde-1 and rde-4. Additionally, genes induced during Orsay virus infections were induced in rrf-3 mutants and further upregulated in adr-1;adr-2;rrf-3 mutants, again dependent in part on rde-1 and rde-4.
2018-02-23 | GSE106647 | GEO
Project description:Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that in the germ-line, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germ-line nuage structures called P-granules. WAGO-1 silences certain genes, transposons, pseudogenes and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest novel regulatory functions for small RNAs.
2009-10-01 | GSE18230 | GEO