Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans

Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that function in the nucleus. We previously found that erroneous rRNAs induce the generation of antisense ribosomal siRNAs (risiRNAs) which silence the expression of rRNAs via the nuclear RNAi defective (Nrde) pathway. To further understand the biological roles and mechanisms of this class of small regulatory RNAs, we conducted forward genetic screening to identify factors involved in risiRNA generation in Caenorhabditis elegans. We found that risiRNAs accumulated in the RNA exosome mutants. risiRNAs directed a NRDE-dependent silencing of pre-rRNAs in the nucleolus. In the presence of risiRNAs, NRDE-2 accumulated in the nucleolus and colocalized with RNA polymerase I. risiRNAs inhibited the transcription elongation of RNA polymerase I by decreasing RNAP I occupancy downstream of the RNAi-targeted site. Meanwhile, exosomes mislocalized from the nucleolus to nucleoplasm in suppressor of siRNA (susi) mutants, in which erroneous rRNAs accumulated. These results established a novel model of rRNA surveillance by combining ribonuclease-mediated RNA degradation with small RNA-directed nucleolar RNAi system. In this study, we compared total small RNAs from (1) wild type animal; (2) dis-3(ust56) mutant; (3) exos-1(ust57) mutant. And we compared total small RNAs from (4) eri-1(mg366) animal; (5) eri-1(mg366);dis-3(ust56) mutant; (6) eri-1(mg366);dis-3(ust56);rrf-1(pk1417);rrf-2(ok210) mutant;(7) eri-1(mg366);exos-1(ust57) mutant; (8) eri-1(mg366);exos-10(ok2269) mutant; And we compared the NRDE-3 associated small RNAs in (9) control animals; (10) dis-3(ust56) mutant; and (11) exos-1(ust57) mutant; (12) exos-10(ok2269); (13) dis-3(ust56);rrf-1(pk1417);rrf-2(ok210) mutant. And we also compared total small RNAs from (14) control animals; (15) fib-1 RNAi animals; (16) mtr-4 RNAi animals; (17) nol-56 RNAi animals;