The Piwi-piRNA pathway represents a germline-specific transposon-defense system. C. elegans Piwi, prg-1, is a non-essential gene and triggers a secondary RNAi response that depends on mutator genes, endo-siRNAs (22G-RNAs), and the 22G-RNA-binding Argonaute protein HRDE-1. Interestingly, silencing of PRG-1 targets can become PRG-1 independent. This state, known as RNAe, is heritable and depends on mutator genes and HRDE-1. We studied how the transgenerational memory of RNAe and the piRNA pathway interact. We find that maternally provided PRG-1 is required for de novo establishment of 22G-RNA populations, especially those targeting transposons. Strikingly, attempts to re-establish 22G-RNAs in absence of both PRG-1 and RNAe memory result in severe germline proliferation defects. This is accompanied by a disturbed balance between gene-activating and -repressing 22G-RNA pathways. We propose a model in which CSR-1 prevents the loading of HRDE-1 and in which both PRG-1 and HRDE-1 help to keep mutator activity focused on the proper targets.
In Caenorhabditis elegans, the piRNA (21U RNA) pathway is required to establish proper gene regulation and an immortal germline. To achieve this, PRG‐1‐bound 21U RNAs trigger silencing mechanisms mediated by RNA‐dependent RNA polymerase (RdRP)‐synthetized 22G RNAs. This silencing can become PRG‐1‐independent and heritable over many generations, a state termed RNA‐induced epigenetic gene silencing (RNAe). How and when RNAe is established, and how it is maintained, is not known. We show that maternally provided 21U RNAs can be sufficient for triggering RNAe in embryos. Additionally, we identify PID‐2, a protein containing intrinsically disordered regions (IDRs), as a factor required for establishing and maintaining RNAe. PID‐2 interacts with two newly identified and partially redundant eTudor domain‐containing proteins, PID‐4 and PID‐5. PID‐5 has an additional domain related to the X‐prolyl aminopeptidase APP‐1, and binds APP‐1, implicating potential N‐terminal proteolysis in RNAe. All three proteins are required for germline immortality, localize to perinuclear foci, affect size and appearance of RNA inheritance‐linked Z granules, and are required for balancing of 22G RNA populations. Overall, our study identifies three new proteins with crucial functions in C. elegans small RNA silencing.
In Caenorhabditis elegans, the piRNA (21U RNA) pathway is required to establish proper gene regulation and an immortal germline. To achieve this, PRG-1-bound 21U RNAs trigger silencing mechanisms mediated by RNA-dependent RNA polymerase (RdRP)-synthetized 22G RNAs. This 25 silencing can become PRG-1-independent, and heritable over many generations. This state is named RNAe. It is unknown how and when RNAe is established, and how it is maintained. We show that maternally provided 21U RNAs can be sufficient to trigger RNAe in embryos. Additionally, we identify the IDR-containing protein PID-2, as a factor required to establish and maintain RNAe. PID-2 interacts with two novel, partially redundant, eTudor domain proteins, PID-4 and PID-5. Additionally, PID-5 has 30 a domain related to the X-prolyl aminopeptidase protein APP-1, and binds APP-1, implicating Nterminal proteolysis in RNAe. All three proteins are required for germline immortality, localize to perinuclear foci, affect Z granules, and are required for balancing of 22G RNA populations. Overall, our study identifies three new proteins with crucial functions in the C. elegans small RNA silencing network. 35
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