The Vasa homolog RDE-12 engages target mRNA and a secondary-Argonaute WAGO-1 to promote RNAi in C. elegans
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ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
SUBMITTER: Craig Mello
PROVIDER: E-GEOD-54396 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Diverse naturally-occurring small RNA species interact with Argonaute proteins to mediate sequence-specific regulation in animals. In addition to micro-RNAs (miRNAs), which collectively regulate thousands of target mRNAs, other endogenous small RNA species include the Piwi-associated piRNAs that are important for fertility and a less well-characterized class of small RNAs often referred to simply as endo-siRNAs. Here we have utilized deep-sequencing technology and C. elegans genetics to explore the biogenesis and function of endo-siRNAs. We describe conditional alleles of the dicer-related helicase, drh-3, that implicate DRH-3 in both the response to foreign dsRNA as well as the RNA-dependent RNA Polymerase (RdRP)-dependent biogenesis of a diverse class of endogenous small RNAs, termed 22G-RNAs. We show that 22G-RNAs are abundantly expressed in the germline and maternally inherited and are the products of at least two distinct 22G-RNA systems. One system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage-related structures termed P-granules. The WAGO 22G-RNA system silences transposons, pseudogenes and cryptic loci as well as a number of genes. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one of the multiple, distinct WAGO surveillance pathways. These findings broaden our understanding of the biogenesis and diversity of 22G-RNA species and suggest potential novel regulatory functions for these small RNAs. 18 samples examined. Small RNA libraries generated from: C. elegans animals with mutations in the WAGO pathway and a WAGO-1 immunopercipitate.
2009-10-01 | E-GEOD-18215 | biostudies-arrayexpress
Project description:Organisms exhibit a fascinating array of gene-silencing pathways, which have evolved in part, to confront invasive nucleic acids such as transposons and viruses. A key question raised by the existence of these pathways is how do they distinguish M-bM-^@M-^\selfM-bM-^@M-^] from M-bM-^@M-^\non-selfM-bM-^@M-^] nucleic acids? Evidence exists for a number of mechanisms that might facilitate detection of foreign sequences including mechanisms that sense copy-number, unpaired DNA, or aberrant RNA (e.g.dsRNA). Here we describe an RNA-induced epigenetic silencing pathway, RNAe, that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate RNAe, while maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences, while two other Argonaute pathways serve as epigenetic memories of "self" and "non-self" RNAs. These findings suggest how organisms may utilize RNAi-related mechanisms not only to recognize and silence foreign genes, but also to keep inventory of all genes expressed in the germ-line. Examine small RNA population changes in different transgene lines. FLAG::WAGO-9 was immunoprecipitated from 20 mg of lysate essentially as described (Gu et al., 2009). Small RNAs were extracted from WAGO-9 immune complexes as well as a portion of the input lysate, gel-purified, pre-treated with TAP, cloned and sequenced as described (Gu et al., 2009).
2012-07-17 | E-GEOD-38724 | biostudies-arrayexpress
Project description:Argonaute proteins (AGOs) are key nuclease effectors of RNA interference (RNAi) [1]. Although purified AGOs can mediate a single round of target-RNA cleavage in vitro, accessory factors are required for siRNA loading and to achieve multiple-target turnover [2, 3]. To identify AGO co-factors we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi [4]. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 co-localizes with WAGO-1 in germline P-granules and to peri-nuclear cytoplasmic foci in somatic cells. These findings and our genetic studies suggest that (i) RDE-12 is first recruited to target mRNAs by upstream AGOs (RDE-1 and ERGO-1) where it promotes small-RNA amplification and/or WAGO-1 loading, and that (ii) downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 that may scan for additional target mRNAs.
2014-06-24 | GSE54396 | GEO
Project description:Epigenetic inheritance is the transmission of information independently of the nucleotide sequence of the genome. A fundamental question in biology is to what extent does epigenetics contribute to trans-generational inheritance. Here we investigate the role of Argonaute-small-RNA pathways in epigenetic inheritance in the nematode C. elegans. Argonautes present their guide RNAs for base-pairing with target sequences and, upon binding, can cleave the target RNA and/or recruit cofactors that mediate post-transcriptional or transcriptional silencing 1,2. Previous studies have shown that Argonaute small-RNA pathways reinforce and maintain epigenetic silencing in C. elegans 3-5. For example, the conserved PIWI-related Argonaute PRG-1 initiates a remarkably stable mode of epigenetic silencing, termed RNA-induced epigenetic silencing (RNAe) 3. Alleles that are silenced by RNAe send trans-acting Argonaute-small-RNA signals that can act in a sequence-specific manner to induce the permanent Epigenetic inheritance is the transmission of information independently of the nucleotide sequence of the genome. A fundamental question in biology is to what extent does epigenetics contribute to trans-generational inheritance. Here we investigate the role of Argonaute-small-RNA pathways in epigenetic inheritance in the nematode C. elegans. Argonautes present their guide RNAs for base-pairing with target sequences and, upon binding, can cleave the target RNA and/or recruit cofactors that mediate post-transcriptional or transcriptional silencing 1,2. Previous studies have shown that Argonaute small-RNA pathways reinforce and maintain epigenetic silencing in C. elegans 3-5. For example, the conserved PIWI-related Argonaute PRG-1 initiates a remarkably stable mode of epigenetic silencing, termed RNA-induced epigenetic silencing (RNAe) 3. Alleles that are silenced by RNAe send trans-acting Argonaute-small-RNA signals that can act in a sequence-specific manner to induce the permanent silencing of homologous genes 3. Here we explore an opposite phenomenon, RNA-induced gene activation (RNAa), in which an expressed gene provides a sequence-specific signal that can activate a silent homologous gene. We provide evidence that the CSR-1 Argonaute is required for this trans-activating signal. CSR-1 engages antisense small RNAs complementary to most, if not all, germline-expressed mRNAs 6,7. Moreover, we show that the ability of a foreign sequence to mediate RNAa is correlated with acquisition of CSR-1-associated small RNAs targeting the foreign sequence. Thus CSR-1 small RNAs constitute a memory of previous germline-gene expression that protects endogenous genes from epigenetic silencing. These findings reveal a remarkably sophisticated epigenetic surveillance mechanism that monitors the flow of transgenerational information ensuring that progeny express only those genes also expressed in their parents. Examine small RNA population changes in different transgene lines
2013-12-23 | E-GEOD-49532 | biostudies-arrayexpress
Project description:During each life cycle germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute (AGO) proteins and their small-RNA cofactors are expressed during male gametogenesis and promote male fertility. Here we show that the CSR-1 AGO functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal that represents an epigenetic memory of male-specific gene expression, while CSR-1, which is abundant in mature sperm, transmits this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small-RNA complexes that constitute a memory of which genes were active in preceding generations. Examine small RNA changes in WT and alg-3/4 mutant males cultured at 20M-BM-0C and 25M-BM-0C, as well as determine the small RNAs enriched in a FLAG::CSR-1 IP from male worms grown at 25M-BM-0C. mRNA sequencing was also performed to determine how transcripts targeted by small RNAs change in mutant background at 20M-BM-0C and 25M-BM-0C.
2013-12-19 | E-GEOD-49672 | biostudies-arrayexpress
Project description:High-throughput pyrosequencing of endogenous small RNAs from CSR-1 IP complexes and csr-1(tm892) and ego-1(om97) mutants with corresponding controls. RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1 interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci, but does not down-regulate target mRNA or protein levels. Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans. 5 samples examined. Small RNAs that co-immunopercipitate with CSR-1 protein and input sample. Small RNAs from csr-1(tm892) and ego-1(om97) mutants and corresponding congenic wild type strain.
2009-10-02 | E-GEOD-18165 | biostudies-arrayexpress
Project description:The endogenous RNA substrates of Translin-TRAX complexes (also known as C3POs) and how they regulate diverse biological processes remain unknown. Here we show that Translin and TRAX do not play a significant role in RNAi in the filamentous fungus Neurospora crassa. Instead, the Neurospora C3PO complex functions as a ribonuclease that removes the 5M-bM-^@M-^Y pre-tRNA fragments after the processing of pre-tRNAs by RNase P. In translin and trax mutants, 5M-bM-^@M-^Y pre-tRNA fragments accumulate to very high levels that can be degraded specifically by both recombinant and endogenous Neurospora C3PO and recombinant Drosophila C3PO. In addition, the mutants have elevated tRNA levels and increased levels of protein translation and are more resistant to a programmed cell-death inducing agent. Together, this study identified the endogenous RNA substrates of C3PO and provides a potential explanation for its roles in seemingly diverse biological processes. Examine small RNA population changes in two different strain background
2012-07-30 | E-GEOD-38334 | biostudies-arrayexpress
Project description:Piwi Argonautes and Piwi-interacting RNAs (piRNAs) mediate genome defense by targeting transposons. However, many piRNA species lack obvious sequence complementarity to transposons or other loci. For example, only one C. elegans transposon is a known piRNA target. Here we show that, in mutants lacking the Piwi Argonaute PRG-1 and associated piRNAs (21U-RNAs), many silent loci in the germline exhibit increased levels of mRNA expression and depletion of an amplified RNAdependent RNA polymerase (RdRP)-derived species of small RNA termed 22G-RNAs. Sequences depleted of 22G-RNAs are enriched nearby potential target sites that base pair imperfectly but extensively to 21U-RNAs. We show that PRG-1 is required to initiate, but not to maintain, silencing of transgenes engineered to contain complementarity to endogenous 21U-RNAs. Our findings support a model in which C. elegans piRNAs utilize their enormous repertoire of targeting capacity to scan the germline transcriptome for foreign sequences, while endogenous germline-expressed genes are actively protected from piRNA-induced silencing. Examine small RNA population changes in prg-1 and rescued strains
2012-09-01 | E-GEOD-38723 | biostudies-arrayexpress
Project description:High-throughput pyrosequencing of endogenous small RNAs from >95% male enriched populations of alg-3(tm1155);alg-4(ok1041);fog-2(q71) and fog-2(q71) worms as well as purified spermatids from fem-3(q20) adult worms. Gametogenesis is thermosensitive in numerous metazoa ranging from worms to man. In C. elegans a variety of germ-line nuage- (P-granule) -associated RNA-binding proteins including the Piwi-clade Argonaute, PRG-1, have been implicated in temperature-dependent fertility. Here, we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3) in promoting male fertility at elevated temperatures. A rescuing GFP::alg-3 transgene is localized in P-granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, named 26G-RNAs, which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for temperature-dependent fertility in C. elegans and support a model in which AGOs and their small RNA co-factors function to promote robustness in gene-expression networks. 3 samples examined. Small RNAs from alg-3(tm1155);alg-4(ok1041);fog-2(q71) males and fog-2(q71) males. Small RNAs from spermatids isolated from ferm-3(q20) worms.
2010-02-24 | E-GEOD-18729 | biostudies-arrayexpress
Project description:These datasets profile the expression of small RNAs with 5p monophosphates and 3p hydroxyls (including miRNAs, 21U-RNAs) in young adult worms from the wt, prg-1 mutant, and fog-2 mutant backgrounds. These datasets were prepared in the Craig C. Mello laboratory using T4 RNA ligase 1. Keywords: miRNA and 21U-RNA discovery and mutant expression profiling CE-mutProfile-5pDependent-Illumina 1 flowcell each for the wt, prg-1 mutant, and fog-2 mutant
2008-06-15 | E-GEOD-11735 | biostudies-arrayexpress