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Distinct phases of siRNA synthesis in an endogenous RNAi pathway in C. elegans soma

ABSTRACT: 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.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE19414 | GEO | 2010/02/01

SECONDARY ACCESSION(S): PRJNA121791

REPOSITORIES: GEO

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Distinct phases of siRNA synthesis in an endogenous RNAi pathway in C. elegans soma

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

Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in ERGO-1/Argonaute pathway

Project description:Argonaute (AGO) proteins interact with distinct classes of small RNAs to direct multiple regulatory outcomes. In many organisms, including plants, fungi and nematodes, cellular RNAdependent RNA polymerases (RdRPs) utilize AGO targets as templates for amplification of silencing signals. Here, we show that distinct RdRPs function sequentially to produce small RNAs that target endogenous loci in C. elegans. We show that DCR-1, the RdRP RRF-3, and the dsRNA-binding protein RDE-4, are required for the biogenesis of 26nt RNAs, termed 26GRNAs, and that 26G-RNAs engage the Piwi-clade AGO, ERGO-1. Our findings support a model in which targeting by ERGO-1 recruits a second RdRP (RRF-1 or EGO-1), which in turn transcribes 22G-RNAs that interact with worm-specific AGOs (WAGOs) to direct gene silencing. ERGO-1 targets exhibit a non-random distribution in the genome and appear to include many gene duplications, suggesting that this pathway may control over-expression resulting from gene expansion. 8 samples examined. Small RNA libraries generated from: C. elegans animals.

2010-02-25 | E-GEOD-18714 | biostudies-arrayexpress

Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in ERGO-1/Argonaute pathway

2010-02-25 | GSE18714 | GEO

RppH can faithfully replace TAP to allow cloning of 5’-triphosphate carrying small RNAs

Project description:RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans, maintain the 5’ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5’PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5’ pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation.

2018-03-20 | GSE111995 | GEO

Endo-siRNAs in C. elegans spermatogenesis and their requirement for the RdRP RRF-3

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

RNA-dependent RNA polymerases in the black-legged tick produce Argonaute-dependent small RNAs and regulate genes

Project description:Small regulatory RNAs (sRNAs) are involved in anti-viral defense and gene regulation. Although RNA-dependent RNA Polymerases (RdRPs) play essential roles in sRNA biogenesis in nematodes, plants and fungi, their involvement in other animals remains controversial. We identify abundant classes of ~22nt sRNAs that require specific combinations of RdRPs and sRNA effector proteins (Argonautes or AGOs) expressed in the tick ISE6 cell line. The RdRP-dependent sRNAs are mainly derived from sense and antisense strands of RNA polymerase III-transcribed genes and repetitive elements. Knockdown of AGO/RdRP causes miregulation of protein-coding genes.These results demonstrate that arachnid RdRPs are important sRNA biogenesis factors, and the discovery of novel pathways underscores the importance of characterizing sRNA biogenesis in various organisms to understand virus-vector interactions and exploit RNAi for pest control.

2021-09-11 | GSE183810 | GEO

Endo-siRNAs in C. elegans spermatogenesis and their requirement for the RdRP RRF-3

2010-01-13 | GSE18429 | GEO

mRNA sequencing from ego-1(om84) and control L3, L4, and adult C. elegans hermaphrodites

Project description:Cellular RdRPs play a critical role in the development of many organisms. Using high-throughput small RNA and messenger RNA sequencing we found that the Caenorhabditis elegans RdRP, EGO-1, is required to produce small RNAs antisense to a number of germline-expressed genes through several developmental stages. We found that these genes fall into distinct classes including genes required for kinetochore and nuclear pore assembly, as well as the production of histone-modifying and centromeric proteins. We also found several RNAi-related genes to be targets of EGO-1. Finally, we show a strong correlation between the loss of small RNAs and the rise of mRNA levels in ego-1 mutant animals. mRNA sequencing from ego-1(om84) and control L3, L4, and adult C. elegans hermaphrodites

2011-06-01 | E-GEOD-26579 | biostudies-arrayexpress

5'-monophosphate-independent small RNA sequencing from ego-1(om84) and control L3, L4, and adult C. elegans hermaphrodites

Project description:Cellular RdRPs play a critical role in the development of many organisms. Using high-throughput small RNA and messenger RNA sequencing we found that the Caenorhabditis elegans RdRP, EGO-1, is required to produce small RNAs antisense to a number of germline-expressed genes through several developmental stages. We found that these genes fall into distinct classes including genes required for kinetochore and nuclear pore assembly, as well as the production of histone-modifying and centromeric proteins. We also found several RNAi-related genes to be targets of EGO-1. Finally, we show a strong correlation between the loss of small RNAs and the rise of mRNA levels in ego-1 mutant animals. 5'-monophosphate-independent small RNA sequencing from ego-1(om84) and control L3, L4, and adult C. elegans hermaphrodites

2011-06-01 | E-GEOD-26581 | biostudies-arrayexpress

5'-monophosphate-dependent small RNA sequencing from ego-1(om84) and control adult C. elegans hermaphrodites

Project description:Cellular RdRPs play a critical role in the development of many organisms. Using high-throughput small RNA and messenger RNA sequencing we found that the Caenorhabditis elegans RdRP, EGO-1, is required to produce small RNAs antisense to a number of germline-expressed genes through several developmental stages. We found that these genes fall into distinct classes including genes required for kinetochore and nuclear pore assembly, as well as the production of histone-modifying and centromeric proteins. We also found several RNAi-related genes to be targets of EGO-1. Finally, we show a strong correlation between the loss of small RNAs and the rise of mRNA levels in ego-1 mutant animals. 5'-monophosphate-dependent small RNA sequencing from ego-1(om84) and control adult C. elegans hermaphrodites

2011-06-01 | E-GEOD-26580 | biostudies-arrayexpress

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