Project description:Germ granules are liquid-like condensates that regulate small RNA pathways and gene expression, ensuring genome stability and fertility in animals. In C. elegans, several Argonaute proteins, central players of small RNA pathways, localize to germ granules, yet the functional significance of this spatial enrichment remains unclear. Here, we disrupted the localization of WAGO-4 to germ granules by introducing targeted mutations in the FG repeats of Vasa-like GLH proteins. These mutations did not disrupt overall germ granule architecture but significantly reduced WAGO-4 partitioning, leading to its predominant localization in the cytoplasm. Functional analyses revealed that this mislocalization partially compromised WAGO-4 activity, resulting in reduced WAGO-4 binding of small RNAs targeting specific genes, particularly those not co-regulated by CSR-1. This selective effect highlights the importance of WAGO-4’s spatial localization for efficient small RNA loading and gene regulation. Our findings demonstrate that germ granules serve as specialized compartments that fine-tune Argonaute function, emphasizing the role of phase-separated condensates in modulating RNA pathways and gene regulatory networks.
Project description:Germ granules are liquid-like condensates that regulate small RNA pathways and gene expression, ensuring genome stability and fertility in animals. In C. elegans, several Argonaute proteins, central players of small RNA pathways, localize to germ granules, yet the functional significance of this spatial enrichment remains unclear. Here, we disrupted the localization of WAGO-4 to germ granules by introducing targeted mutations in the FG repeats of Vasa-like GLH proteins. These mutations did not disrupt overall germ granule architecture but significantly reduced WAGO-4 partitioning, leading to its predominant localization in the cytoplasm. Functional analyses revealed that this mislocalization partially compromised WAGO-4 activity, resulting in reduced WAGO-4 binding of small RNAs targeting specific genes, particularly those not co-regulated by CSR-1. This selective effect highlights the importance of WAGO-4’s spatial localization for efficient small RNA loading and gene regulation. Our findings demonstrate that germ granules serve as specialized compartments that fine-tune Argonaute function, emphasizing the role of phase-separated condensates in modulating RNA pathways and gene regulatory networks.
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.
Project description:Small RNA pathways defend the germlines of animals against selfish genetic elements and help to maintain genomic integrity. At the same time, their activity needs to be well-controlled to prevent silencing of ‘self’ genes. Here, we reveal a proteolytic mechanism that controls endogenous small interfering (22G) RNA activity in the Caenorhabditis elegans germline to protect genome integrity and maintain fertility. We find that WAGO-1 and WAGO-3 Argonaute proteins are produced as pro-proteins that are matured through proteolytic processing of their unusually proline-rich N-termini. In the absence of DPF-3, a P-granule-localized N-terminal dipeptidase orthologous to mammalian DPP8/9, processing fails, causing a change of identity of 22G RNAs bound to these WAGO proteins. Accumulation of repeat- and transposon-derived transcripts, DNA damage and sterility ensue. We propose that DPF-3 acts as a licensing factor for 22G RNA activity.
Project description:Small RNA pathways defend the germlines of animals against selfish genetic elements and help to maintain genomic integrity. At the same time, their activity needs to be well-controlled to prevent silencing of ‘self’ genes. Here, we reveal a proteolytic mechanism that controls endogenous small interfering (22G) RNA activity in the Caenorhabditis elegans germline to protect genome integrity and maintain fertility. We find that WAGO-1 and WAGO-3 Argonaute proteins are produced as pro-proteins that are matured through proteolytic processing of their unusually proline-rich N-termini. In the absence of DPF-3, a P-granule-localized N-terminal dipeptidase orthologous to mammalian DPP8/9, processing fails, causing a change of identity of 22G RNAs bound to these WAGO proteins. Accumulation of repeat- and transposon-derived transcripts, DNA damage and sterility ensue. We propose that DPF-3 acts as a licensing factor for 22G RNA activity.
Project description:Small RNA pathways defend the germlines of animals against selfish genetic elements and help to maintain genomic integrity. At the same time, their activity needs to be well-controlled to prevent silencing of ‘self’ genes. Here, we reveal a proteolytic mechanism that controls endogenous small interfering (22G) RNA activity in the Caenorhabditis elegans germline to protect genome integrity and maintain fertility. We find that WAGO-1 and WAGO-3 Argonaute proteins are produced as pro-proteins that are matured through proteolytic processing of their unusually proline-rich N-termini. In the absence of DPF-3, a P-granule-localized N-terminal dipeptidase orthologous to mammalian DPP8/9, processing fails, causing a change of identity of 22G RNAs bound to these WAGO proteins. Accumulation of repeat- and transposon-derived transcripts, DNA damage and sterility ensue. We propose that DPF-3 acts as a licensing factor for 22G RNA activity.
Project description:To explore the roles of piRNAs and WAGO-class 22G-RNAs in regulating gene expression and transposon silencing in Caenorhabditis elegans, we used RNA-seq to assess changes in small RNA and mRNA levels in prg-1 and mut-16 mutants, which disable the piRNA and WAGO-class 22G-RNA pathways respectively. We identified numerous roles for piRNAs and WAGO-class 22G-RNAs in regulating germline genes, including transposons, histones, and spermatogenic and oogenic transcripts.
Project description:Small RNA pathways defend the germlines of animals against selfish genetic elements and help to maintain genomic integrity. At the same time, their activity needs to be well-controlled to prevent silencing of ‘self’ genes. Here, we reveal a proteolytic mechanism that controls endogenous small interfering (22G) RNA activity in the Caenorhabditis elegans germline to protect genome integrity and maintain fertility. We find that WAGO-1 and WAGO-3 Argonaute proteins are produced as pro-proteins that are matured through proteolytic processing of their unusually proline-rich N-termini. In the absence of DPF-3, a P-granule-localized N-terminal dipeptidase orthologous to mammalian DPP8/9, processing fails, causing a change of identity of 22G RNAs bound to these WAGO proteins. Accumulation of repeat- and transposon-derived transcripts, DNA damage and sterility ensue. We propose that DPF-3 acts as a licensing factor for 22G RNA activity.