Roles for the Caenorhabditis elegans RNA-directed RNA polymerase RRF-3 in endogenous RNA interference.

摘要

The pairing of sense and antisense RNA is a key feature of gene regulation in RNA interference (RNAi) and related processes. Short interfering RNAs (siRNAs) are a general class of small RNAs produced during RNAi that enforce gene silencing through guiding Argonautes to specific target transcripts through base pairing between siRNAs and targets. Although RNAi was originally identified as a defense from parasitic and foreign genetic material, it has become evident that RNAi plays an important role in endogenous gene regulation. In certain organisms, siRNA synthesis involves enzymes that act as RNA-directed RNA polymerases (RdRPs). Here we analyze the function of a C. elegans RdRP, RRF-3, in endogenous RNAi.;We found that loss of RRF-3 function resulted in pleiotropic defects in sperm development and that sperm defects led to embryonic lethality. Notably, sperm nuclei in mutants of either rrf-3 or another component of the RRF-3 RNAi pathway, eri-1, were frequently surrounded by ectopic microtubule structures, and a subset of the resulting embryos had spindle abnormalities. Through deep sequencing of small RNAs, we identified a population of cellular mRNAs from spermatogenic cells that appear to serve as templates for siRNA synthesis. This set of genes included the majority of genes with known spermatogenesis-enriched expression, as well as many genes not previously known to be expressed in spermatogenesis. For a subset of spermatogenesis-enriched genes, we observed that RRF-3 was required for siRNA accumulation. These and other data suggest a working model in which a major role for the RRF-3/ERI pathway is to generate siRNAs that set patterns of gene expression through repression of a set of critical targets during spermatogenesis.;Furthermore, we found that RRF-3 regulates certain somatically expressed transcripts, and we discovered that the somatic RRF-3/ERI pathway represses its target genes through a mechanism involving distinct stages of siRNA synthesis. Two populations of siRNAs involved in this process are distinguished by 5' chemical structures, lengths, and by genetic requirements for their accumulation. The RRF-3/ERI pathway was required for production of a rare group of instigators which provoke synthesis of a shorter and more numerous populations of effectors through a second RdRP (RRF-1). These results establish a rough framework for the RRF-3 somatic RNAi pathway.