RNA-dependent RNA polymerases-synthesized antisense ribosomal siRNAs silence pre-rRNA via the nuclear RNAi pathway in C. elegans

The biogenesis of rRNA drives cell growth and proliferation, but mechanisms that modulate rRNA production remain poorly understood. We have conducted a genetic screen to look for factors that negatively regulate endo-siRNA generation in C. elegans, and identified the suppressor of siRNA (susi)-1and a new class of triphosphorylated antisense ribosomal siRNAs (risiRNAs). In susi-1 mutant, we observed an accumulation of siRNA sequences complementary to 18S and 26S rRNAs. risiRNAs are similar to 22G-RNA, as they are 22 nt in length and have a triphosphorylated guanidine at their 5''-end. The generation of risiRNAs depends on RNA-dependent RNA polymerases, but not the ERI/Dicer complex. risiRNAs act through the nuclear RNAi pathway and guide the Argonaute protein NRDE-3 to the nucleoli, which leads to a downregulation of pre-rRNA expression. Interestingly, low temperature and ultraviolet irradiation also induces the accumulation of risiRNA, suggesting potential regulatory functions. Cloned SUSI-1 shows homology to Dis3-like exonuclease 2 (Dis3L2) in other organisms, a gene identified as a key exonuclease involved in the 3'' to 5'' degradation of oligouridylated RNAs. We observed an accumulation of 3''-tail oligouridylated 26S rRNA in susi-1 mutant and in low temperature-treated animals. Consistently, the injection of 3''-tail oligouridylated 26S rRNA elicited a nuclear accumulation of NRDE-3. Our findings thus establish SUSI-1(ceDis3L2) as a suppressor of endo-siRNA generation, identify a novel class of risiRNAs that suppress pre-rRNA expression, and suggest a potential disease mechanism. Overall design: In this study, we compared (1) the small RNAs from wild type N2 animals with susi-1(R457) mutant, (2) NRDE-3-associated small RNAs in wild type animals with in eri-1 animals, (3) small RNAs in animals grow at normal temperature 20oC and animals after a 4oC cold shock; (4) small RNAs in eri-1 animals with or without UV irradiation; (5) NRDE-3-associated small RNAs with or without UV irradiation. To understant the mechanism of risiRNA biogenesis, we further investigated the accumulation of oligouridylated 26S rRNA by (6) 26S rRNA tail-seq of wild type N2 with susi-1(ust1) animals; (7) 26S rRNA tail-seq of eri-1 animals with eri-1;susi1 animals; (8) 26S rRNA tail-seq of eri-1 animals with or without 4C cold shock.