Piwi-interacting RNAs (piRNAs) are small RNAs required to maintain germline integrity and fertility but their mechanism of action is poorly understood. Here we demonstrate that C. elegans piRNAs silence transcripts in trans through imperfectly complementary sites. Target silencing is independent of Piwi endonuclease activity or "slicing". Instead, piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous proteincoding gene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are de-repressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes and tend to overlap the start and end of transposons in sense and antisense, respectively. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNAi in C. elegans.The Piwi/piRNA pathway has an evolutionarily conserved role in germline transposon silencing in animals. C. elegans encodes two Piwi family proteins, PRG-1 and PRG-2, although PRG-2 has likely little or no function (1, 2). PRG-1 and piRNA expression is restricted to the male and female germline. The piRNAs of C. elegans are 21 nucleotides in length with a 5′ uracil (21U-RNAs) (1-4). In C. elegans piRNAs have a sequence motif, situated ~40 bp upstream of each piRNA locus, that is thought to be required for piRNA biogenesis (2, 3). A challenge in the field is to understand the mechanism(s) by which piRNAs act on their targets. Proposed functions for Piwi/piRNA complexes include the RNAi-like slicing of RNA transcripts (5-7), transcript deadenylation (8) and de novo DNA methylation (9, 10). Here we identify the targeting mechanism and the endogenous targets of C. elegans piRNAs.
The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humansRACK1, a constituent of the ribosomal 40S subunit, is required for the association of miRISC with translating ribosomes. This suggests that RACK1 contributes to recruit miRISC to the site of translation and supports a post-initiation mode of miRNA-mediated gene repression.
microRNAs function in diverse developmental and physiological processes by regulating target gene expression at the post-transcriptional level. ALG-1 is one of two Caenorhabditis elegans Argonautes (ALG-1 and ALG-2) that together are essential for microRNA biogenesis and function. Here, we report the identification of novel antimorphic (anti) alleles of ALG-1 as suppressors of lin-28(lf) precocious developmental phenotypes. The alg-1(anti) mutations broadly impair the function of many microRNAs and cause dosage-dependent phenotypes that are more severe than the complete loss of ALG-1. ALG-1(anti) mutant proteins are competent for promoting Dicer cleavage of microRNA precursors and for associating with and stabilizing microRNAs. However, our results suggest that ALG-1(anti) proteins may sequester microRNAs in immature and functionally deficient microRNA Induced Silencing Complexes (miRISCs), and hence compete with ALG-2 for access to functional microRNAs. Immunoprecipitation experiments show that ALG-1(anti) proteins display an increased association with Dicer and a decreased association with AIN-1/GW182. These findings suggest that alg-1(anti) mutations impair the ability of ALG-1 miRISC to execute a transition from Dicer-associated microRNA processing to AIN-1/GW182 associated effector function, and indicate an active role for ALG/Argonaute in mediating this transition.
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