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.
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.
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.
Neuropilins (NRPs) are transmembrane receptors that bind class 3 semaphorins and VEGF family members to regulate axon guidance and angiogenesis. Although expression of NRP1 by vascular smooth muscle ...cells (SMCs) has been reported, NRP function in smooth muscle (SM) in vivo is unexplored. Using Nrp2 +/LacZ and Nrp2 +/gfp transgenic mice, we observed robust and sustained expression of Nrp2 in the SM compartments of the bladder and gut, but no expression in vascular SM, skeletal muscle, or cardiac muscle. This expression pattern was recapitulated in vitro using primary human SM cell lines. Alterations in cell morphology after treatment of primary visceral SMCs with the NRP2 ligand semaphorin-3F (SEMA3F) were accompanied by inhibition of RhoA activity and myosin light chain phosphorylation, as well as decreased cytoskeletal stiffness. Ex vivo contractility testing of bladder muscle strips exposed to electrical stimulation or soluble agonists revealed enhanced tension generation of tissues from mice with constitutive or SM-specific knockout of Nrp2 , compared with controls. Mice lacking Nrp2 also displayed increased bladder filling pressures, as assessed by cystometry in conscious mice. Together, these findings identify Nrp2 as a mediator of prorelaxant stimuli in SMCs and suggest a novel function for Nrp2 as a regulator of visceral SM contractility.
Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, ...including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.
Animal cells have a remarkable capacity to adopt durable and heritable gene expression programs or epigenetic states that define the physical properties and diversity of somatic cell types. The ...maintenance of epigenetic programs depends on poorly understood pathways that prevent gain or loss of inherited signals. In the germline, epigenetic factors are enriched in liquid-like perinuclear condensates called nuage. Here, we identify the deeply conserved helicase-domain protein, ZNFX-1, as an epigenetic regulator and component of nuage that interacts with Argonaute systems to balance epigenetic inheritance. Our findings suggest that ZNFX-1 promotes the 3′ recruitment of machinery that propagates the small RNA epigenetic signal and thus counteracts a tendency for Argonaute targeting to shift 5′ along the mRNA. These functional insights support the idea that recently identified subdomains of nuage, including ZNFX-1 granules or “Z-granules,” may define spatial and temporal zones of molecular activity during epigenetic regulation.
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•Genetic identification of ZNFX-1, a conserved epigenetic inheritance factor•ZNFX-1 balances the transgenerational inheritance of epigenetic information•ZNFX-1 interacts with RdRP and three distinct Argonaute systems in germline nuage•ZNFX-1 promotes balanced amplification of small RNA signals along germline mRNAs
Ishidate et al. identify ZNFX-1, a highly conserved helicase protein, as a factor required for epigenetic inheritance in C. elegans. Their findings indicate that ZNFX-1 localizes within nuage, where it interacts with Argonaute systems and RNA-dependent RNA polymerase (RdRP) to ensure balanced amplification of small RNA signals along germline mRNAs.
Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and ...vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.
Argonaute (AGO) proteins are key nuclease effectors of RNAi 1. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering ...RNA (siRNA) loading and to achieve multiple-target turnover 2, 3. To identify AGO cofactors, 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 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1), where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing.
•Vasa ATPase-related protein RDE-12 promotes RNAi and virus suppression in C. elegans•RDE-12 is recruited to target mRNA by primary Ago RDE-1 in the RNAi pathway•RDE-12 promotes secondary siRNA biogenesis and associates with secondary Ago WAGO-1•RDE-12 localizes to P granules in germline and novel perinuclear foci in soma
Shirayama et al. identify the Vasa homolog RDE-12 as a protein that promotes RNAi and virus suppression in C. elegans. RDE-12 is recruited to target mRNA by primary AGO RDE-1 to promote small RNA amplification. RDE-12 forms a complex with secondary AGO WAGO-1 in P granules and may have additional functions in target mRNA surveillance and silencing.
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