piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs ...tolerate mismatched pairing and in principle could target every transcript. Here we employ in vivo cross-linking to identify transcriptome-wide interactions between piRNAs and target RNAs. We show that piRNAs engage all germline mRNAs and that piRNA binding follows microRNA-like pairing rules. Targeting correlates better with binding energy than with piRNA abundance, suggesting that piRNA concentration does not limit targeting. In mRNAs silenced by piRNAs, secondary small RNAs accumulate at the center and ends of piRNA binding sites. In germline-expressed mRNAs, however, targeting by the CSR-1 Argonaute correlates with reduced piRNA binding density and suppression of piRNA-associated secondary small RNAs. Our findings reveal physiologically important and nuanced regulation of individual piRNA targets and provide evidence for a comprehensive post-transcriptional regulatory step in germline gene expression.
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•PIWI CLASH identifies piRNA binding sites transcriptome-wide•piRNAs engage all germline mRNAs via microRNA-like pairing rules•piRNA target sites have distinct 22G-RNA patterns on silenced and expressed mRNAs•Targeting by CSR-1 Argonaute correlates with reduced piRNA binding density
Transcriptome-wide profiling of piRNA targeting rules provides new insights into the interplay between Argonaute pathways and their physiological roles in C. elegans
Organisms employ a fascinating array of strategies to silence invasive nucleic acids such as transposons and viruses. Although evidence exists for several pathways that detect foreign sequences, ...including pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), in many cases, the mechanisms used to distinguish “self” from “nonself” nucleic acids remain mysterious. Here, we describe an RNA-induced epigenetic silencing pathway that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate permanent silencing, and maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences and two other Argonaute pathways serve as epigenetic memories of “self” and “nonself” RNAs. These findings suggest how organisms can utilize RNAi-related mechanisms to detect foreign sequences not by any molecular signature, but by comparing the foreign sequence to a memory of previous gene expression.
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► Epigenetic silencing triggered by piRNA-mediated recognition of nonself RNA ► piRNAs scan using imperfect base pairing to initiate gene silencing ► Maintenance of silencing requires chromatin factors and RdRP-generated small RNAs ► Activating and silencing signals may compete in self versus nonself discrimination
Evidence for a provocative theory that places piRNAs at the center of a “self” versus “nonself” sensor, with some piRNAs functioning to silence foreign DNA and others as part of a mechanism that protects endogenous germline-expressed genes from aberrant silencing.
Organisms can develop adaptive sequence-specific immunity by reexpressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piwi-interacting RNA ...(piRNA) pathway recruits RNA-dependent RNA polymerase (RdRP) to foreign sequences to amplify a transgenerational small-RNA-induced epigenetic silencing signal (termed RNAe). Here, we provide evidence that, in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self-mRNAs. We refer to this mechanism, which can prevent or reverse RNAe, as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a transgenerational CSR-1 memory that recognizes and protects self-mRNAs, allowing piRNAs to recognize foreign sequences innately, without the need for prior exposure.
•C. elegans develops an adaptive memory of expressed/self-mRNAs•The CSR-1 Argonaute mediates RNA-induced epigenetic gene activation (RNAa)•RNAa counteracts Piwi-Argonaute-dependent epigenetic silencing (RNAe)•Multigenerational exposure to RNAa adapts an RNAe allele for independent expression
Seth et al. show that the C. elegans Argonaute CSR-1 protects cognate genes from Piwi-Argonaute-mediated silencing. Their findings suggest how Argonaute pathways function together in the germline to monitor the flow of transgenerational information, ensuring that progeny will express only those genes that are also expressed in their parents.
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 downregulate 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.
In metazoans, Piwi-related Argonaute proteins engage piRNAs (Piwi-interacting small RNAs) to defend the genome against invasive nucleic acids, such as transposable elements. Yet many ...organisms—including worms and humans—express thousands of piRNAs that do not target transposons, suggesting that piRNA function extends beyond genome defense. Here, we show that the X chromosome-derived piRNA 21ux-1 downregulates XOL-1 (XO Lethal), a master regulator of X chromosome dosage compensation and sex determination in Caenorhabditis elegans. Mutations in 21ux-1 and several Piwi-pathway components sensitize hermaphrodites to dosage compensation and sex determination defects. We show that the piRNA pathway also targets xol-1 in C. briggsae, a nematode species related to C. elegans. Our findings reveal physiologically important piRNA-mRNA interactions, raising the possibility that piRNAs function broadly to ensure robust gene expression and germline development.
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•An X-linked piRNA, 21ux-1, targets xol-1 and represses its expression in C. elegans•21ux-1 acts with sex-1 to promote dosage compensation and sex determination•The piRNA pathway regulates the expression of the xol-1 ortholog in C. briggsae
Although piRNAs are known to defend the genome against transposons, it remains unclear whether they have additional functions beyond genome defense. Tang et al. show that an X chromosome-derived piRNA downregulates XOL-1 expression to promote robust regulation of dosage compensation and sex determination in C. elegans.
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 germline, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline 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 additional regulatory functions for small RNAs.
Gametogenesis is a thermosensitive process in numerous metazoans, ranging from worms to man. In Caenorhabditis elegans, a variety of RNA-binding proteins that associate with germ-line nuage (P ...granules), including the Piwi-clade argonaute PRG-1, have been implicated in maintaining fertility at elevated temperature. Here we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3), in promoting thermotolerant male fertility. A rescuing GFP::alg-3 transgene is localized to P granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, the 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 thermotolerant fertility in C. elegans and support a model in which AGOs and their small RNA cofactors function to promote robustness in gene-expression networks.
Although single-gene loss-of-function analyses can identify components of particular processes, important molecules are missed owing to the robustness of biological systems. Here we show that ...large-scale RNAi screening for suppression interactions with functionally related mutants greatly expands the repertoire of genes known to act in a shared process and reveals a new layer of functional relationships. We performed RNAi screens for 17 Caenorhabditis elegans cell polarity mutants, generating the most comprehensive polarity network in a metazoan, connecting 184 genes. Of these, 72% were not previously linked to cell polarity and 80% have human homologues. We experimentally confirmed functional roles predicted by the network and characterized through biophysical analyses eight myosin regulators. In addition, we discovered functional redundancy between two unknown polarity genes. Similar systematic genetic interaction screens for other biological processes will help uncover the inventory of relevant genes and their patterns of interactions.
Protein-coding genes undergo a wide array of regulatory interactions with factors that engage non-coding regions. Open reading frames (ORFs), in contrast, are thought to be constrained by coding ...function, precluding a major role in gene regulation. Here, we explore Piwi-interacting (pi)RNA-mediated transgene silencing in C. elegans and show that marked differences in the sensitivity to piRNA silencing map to the endogenous sequences within transgene ORFs. Artificially increasing piRNA targeting within the ORF of a resistant transgene can lead to a partial yet stable reduction in expression, revealing that piRNAs not only silence but can also “tune” gene expression. Our findings support a model that involves a temporal element to mRNA regulation by germline Argonautes, likely prior to translation, and suggest that piRNAs afford incremental control of germline mRNA expression by targeting the body of the mRNA, including the coding region.
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•C. elegans germline mRNAs differ in sensitivity to piRNA targeting•piRNA targeting of coding regions provides incremental control of gene expression•Piwi Argonaute surveillance occurs upstream of nonsense-mediated decay•Model, piRNAs scan mRNAs within perinuclear nuage prior to translation initiation
Some C. elegans transgenes resist piRNA silencing. Seth et al. map resistance to endogenous sequences within transgenes and show that artificially increasing piRNA targeting can incrementally reduce expression without silencing. Their findings identify coding regions as part of a rich piRNA regulatory landscape within perinuclear nuage.
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