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 the association of NRDE proteins with pre-rRNAs and the silencing of pre-rRNAs. 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.

真核细胞可表达多种在细胞核中发挥功能的内源性小调控RNA。我们此前的研究发现，错误的核糖体RNA可诱导反义核糖体小干扰RNA（antisense ribosomal siRNAs, risiRNAs）的生成，后者通过核RNAi缺陷（Nrde）通路沉默核糖体RNA的表达。为进一步解析这类小调控RNA的生物学功能与作用机制，我们以秀丽隐杆线虫为模型开展正向遗传筛选，以鉴定参与risiRNA生成的调控因子。我们发现，RNA外切体突变体中存在risiRNA的积累。risiRNA可介导NRDE蛋白与核糖体前体RNA（pre-rRNAs）结合，并沉默核糖体前体RNA的表达。在risiRNA存在的条件下，NRDE-2会在核仁中聚集，并与RNA聚合酶I（RNAP I）共定位。risiRNA通过降低RNA聚合酶I在RNAi靶向位点下游的占据率，抑制其转录延伸。与此同时，在siRNA抑制（susi）突变体中，错误的核糖体RNA发生积累，RNA外切体也从核仁异位定位至核质。上述研究结果构建了一种全新的rRNA监控模型：该模型将核糖核酸酶介导的RNA降解与小RNA定向的核仁RNAi系统相结合。