Global Regulatory Functions of the <em>Staphylococcus aureus</em> Endoribonuclease III in Gene Expression

RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III.RNA周转在革兰氏阳性人类致病菌金黄色葡萄球菌（Staphylococcus aureus）的毒力与应激适应过程中均发挥关键作用。然而，这些过程中涉及的分子调控因子与作用机制仍未得到充分阐释。本研究针对普遍存在的双链特异性核糖核酸酶（double-strand-specific endoribonucleases）家族成员——金黄色葡萄球菌核糖核酸酶III（RNase III）的功能展开系统性探索。为精准鉴定可被RNase III结合并加工的基因组转录本，我们对体内与野生型RNase III或两种不同切割缺陷突变体共免疫沉淀的RNA所构建的cDNA文库开展了深度测序分析。通过独立实验方法，我们验证了多个新发现的RNase III靶标。研究团队鉴定出多种结构化RNA作为RNase III的底物，并证实该酶参与核糖体RNA（rRNAs）与转运RNA（tRNAs）的成熟过程，调控信使RNA（mRNAs）与非编码RNA的周转，同时通过在自身mRNA的编码区内部进行切割，实现对自身合成的自主调控。此外，基于全新的作用机制，我们发现RNase III对蛋白质合成具有正向调控效应：RNase III介导的5'非翻译区（5′UTR）内切割可增强编码主要冷休克蛋白的cspA mRNA的稳定性与翻译效率。进一步研究显示，RNase III可切割反向转录基因的重叠5'非翻译区，以生成无先导mRNA，这为共调控相邻基因提供了一种全新途径。与近期研究结果一致，通过与RNase III突变体蛋白共免疫沉淀，我们捕获到了覆盖44%注释基因的低丰度反义RNA。由此可见，除经典的基因调控功能外，RNase III还参与普遍转录的RNA质量控制过程。总体而言，本研究清晰阐明了RNase III所介导的转录后调控的复杂性与多面性。