C. elegans piRNAs mediate the genome-wide surveillance of germline transcripts
ABSTRACT: 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
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; only one C. elegans transposon is a known piRNA target. Here, we show that, in mutants lacking the Piwi Argonaute PRG-1 (and consequently its associated piRNAs/21U-RNAs), many silent loci in the germline exhibit increased levels of mRNA expression with a concomitant depletion of RNA-dependent RNA polymerase (RdRP)-derived secondary small RNAs termed 22G-RNAs. Sequences depleted of 22G-RNAs are proximal to 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.
Project description:The Piwi-piRNA pathway represents a small RNA-based mechanism responsible for the recognition and silencing of invading DNA. Biogenesis of piRNAs (21U-RNAs) is poorly understood. In Caenorhabditis elegans, the piRNA-binding Argonaute protein PRG-1 is the only known player acting downstream from precursor transcription. From a screen aimed at the isolation of piRNA-induced silencing-defective (Pid) mutations, we identified, among known Piwi pathway components, PID-1 as a novel player. PID-1 is a mostly cytoplasmic, germline-specific factor essential for 21U-RNA biogenesis, affecting an early step in the processing or transport of 21U precursor transcripts. We also show that maternal 21U-RNAs are essential to initiate silencing.
Project description:In metazoans, Piwi-related Argonaute proteins have been linked to germline maintenance, and to a class of germline-enriched small RNAs termed piRNAs. Here we show that an abundant class of 21 nucleotide small RNAs (21U-RNAs) are expressed in the C. elegans germline, interact with the C. elegans Piwi family member PRG-1, and depend on PRG-1 activity for their accumulation. The PRG-1 protein is expressed throughout development and localizes to nuage-like structures called P granules. Although 21U-RNA loci share a conserved upstream sequence motif, the mature 21U-RNAs are not conserved and, with few exceptions, fail to exhibit complementarity or evidence for direct regulation of other expressed sequences. Our findings demonstrate that 21U-RNAs are the piRNAs of C. elegans and link this class of small RNAs and their associated Piwi Argonaute to the maintenance of temperature-dependent fertility.
Project description:Epigenetic regulation by diverse classes of small RNAs is mediated by the highly conserved Argonaute/Piwi family of proteins. Although Argonautes are broadly expressed, the Piwi subfamily primarily functions in the germ line. Piwi proteins are associated with germline-specific ribonucleoprotein (RNP) granules in Drosophila, zebrafish, and mouse. Depending on the species and on the specific family member, Piwis play important roles in spermatogenesis and/or in maintaining germ cell and stem cell totipotency. Piwis bind to a newly discovered class of small RNAs, called piRNAs. C. elegans contains a large set of Argonaute/Piwi-related proteins, including two closely related to piwi called prg-1 and prg-2. The function of prg-1 and prg-2 and whether piRNAs exist in C. elegans is unknown.Here, we demonstrate that the Piwi-like protein PRG-1 is localized to P granules in germ cells entering spermatogenesis and is required for successful spermatogenesis. Loss of prg-1 causes a marked reduction in expression of a subset of mRNAs expressed during spermatogenesis, and prg-1 mutant sperm exhibit extensive defects in activation and fertilization. Moreover, prg-1 activity is required for the presence of the small RNAs called 21U-RNAs.Our data suggest that PRG-1 promotes expression, processing, or stability of 21U-RNAs, which, in turn or in concert with PRG-1, promote proper expression of spermatogenesis transcripts.
Project description:The Piwi proteins of the Argonaute superfamily are required for normal germline development in Drosophila, zebrafish, and mice and associate with 24-30 nucleotide RNAs termed piRNAs. We identify a class of 21 nucleotide RNAs, previously named 21U-RNAs, as the piRNAs of C. elegans. Piwi and piRNA expression is restricted to the male and female germline and independent of many proteins in other small-RNA pathways, including DCR-1. We show that Piwi is specifically required to silence Tc3, but not other Tc/mariner DNA transposons. Tc3 excision rates in the germline are increased at least 100-fold in piwi mutants as compared to wild-type. We find no evidence for a Ping-Pong model for piRNA amplification in C. elegans. Instead, we demonstrate that Piwi acts upstream of an endogenous siRNA pathway in Tc3 silencing. These data might suggest a link between piRNA and siRNA function.
Project description:Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and are essential for fertility in diverse organisms. An interesting feature of piRNAs is that, while piRNA lengths are stereotypical within a species, they can differ widely between species. For example, piRNAs are mainly 29 and 30 nucleotides in humans, 24 to 30 nucleotides in D. melanogaster, and uniformly 21 nucleotides in C. elegans. However, how piRNA length is determined and whether length impacts function remains unknown. Here, we show that C. elegans deficient for PARN-1, a conserved RNase, accumulate untrimmed piRNAs with 3' extensions. Surprisingly, these longer piRNAs are stable and associate with the Piwi protein PRG-1 but fail to robustly recruit downstream silencing factors. Our findings identify PARN-1 as a key regulator of piRNA length in C. elegans and suggest that length is regulated to promote efficient transcriptome surveillance.
Project description:In animals, piRNAs and their associated Piwi proteins guard germ cell genomes against mobile genetic elements via an RNAi-like mechanism. In Caenorhabditis elegans, 21U-RNAs comprise the piRNA class, and these collaborate with 22G RNAs via unclear mechanisms to discriminate self from nonself and selectively and heritably silence the latter. Recent work indicates that 21U-RNAs are post-transcriptional processing products of individual transcription units that produce ? 26-nucleotide capped precursors. However, nothing is known of how the expression of precursors is controlled or how primary transcripts give rise to mature small RNAs. We conducted a genome-wide RNAi screen to identify components of the 21U biogenesis machinery. Screening by direct, quantitative PCR (qPCR)-based measurements of mature 21U-RNA levels, we identified 22 genes important for 21U-RNA production, termed TOFUs (Twenty-One-u Fouled Ups). We also identified seven genes that normally repress 21U production. By measuring mature 21U-RNA and precursor levels for the seven strongest hits from the screen, we assigned factors to discrete stages of 21U-RNA production. Our work identifies for the first time factors separately required for the transcription of 21U precursors and the processing of these precursors into mature 21U-RNAs, thereby providing a resource for studying the biogenesis of this important small RNA class.
Project description:Animal germ cells employ small RNA-based mechanisms to recognize and silence DNA that invades their genome. One of these pathways is named the Piwi:piRNA pathway. Biogenesis of piRNAs is poorly understood. In C. elegans, the piRNA (21U-RNA)-binding Argonaute protein PRG-1 is the only known player acting downstream of pre-cursor transcription. From a screen aimed at the isolation of ‘piRNA-induced silencing defective’ mutations we identified, amongst known Piwi-pathway components like MUT-7, RDE-3 and HRDE-1, PID-1 as a novel player. PID-1 is essential for 21U RNA biogenesis and affects an early step in the processing or transport of 21U precursor transcripts. 12 small RNA samples were analyzed as singletons.
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
Project description:Piwi-interacting (pi) RNAs are a class of germline-expressed small RNAs that have been linked to epigenetic programming in metazoa. C. elegans piRNAs known as 21U-RNAs are defined by more than 15,000 genome-encoded species. To explore the origin of 21U-RNAs we employed methods to enrich the 5' ends of Pol II transcripts. We show that a species of capped-short (cs) RNA is frequently expressed bidirectionally at Pol II loci in C. elegans. Interestingly, at annotated 21U-RNA loci, csRNAs originate precisely 2 nt upstream of the mature piRNA species suggesting that csRNAs are piRNA precursors. In addition, we show that csRNAs associated with TS sites genome-wide define a previously overlooked class of 21U-RNA loci, and nearly double the number of piRNA species available for genome surveillance. Our methods should be of general utility in TS site identification and 5' anchored RNA-expression profiling. Identification of capped RNA including capped small RNA and long capped RNA in C. elegans. The mouse data are independent data to test the CapSeq sequencing protocol.