Target recognition by RNase E RNA-binding domain AR2 drives sRNA decay in the absence of PNPase.
Target recognition by RNase E RNA-binding domain AR2 drives sRNA decay in the absence of PNPase.
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DOI：
10.1073/pnas.2208022119
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发表时间：
2022-11-29
影响因子：
11.1
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RNase E在革兰氏阴性细菌中发挥着不可或缺的作用，它启动了细胞RNA的分解，并为主要的RNA衰变机器--RNA降解体提供了支架。此外，RNase E通过切割mRNAs来执行基因调控，这些mRNAs在与通常与RNA伴侣Hfq相关的小调节RNA(SRNAs)结合时被标记为腐烂。然而，对这四个组成部分之间相互作用的详细机制理解仍然难以捉摸。虽然sRNA结合可以促进mRNA的衰退，但我们的工作表明，sRNAs和HfQ是通过结合靶标mRNAs间接招募到RNaseE C-末端结构域的第二个富含精氨酸的RNA结合区(AR2)。因此，sRNAs被降解，这广泛地影响细菌的生理和生存。主要内切核酸酶RNase E的C末端结构域(CTD)不仅是革兰氏阴性细菌中中央RNA衰退机制的支架，而且还介导了RNA伴侣Hfq促进的SRNA-mRNA碱基配对后小调节RNA(SRNAs)及其同源目标转录物的耦合降解。尽管RNase E CTD在sRNA依赖的基因调控中起着至关重要的作用，但该结构域中的特定残基在碱基配对时招募sRNA和mRNAs的作用仍不清楚。我们先前已经证明，在大肠杆菌中，高度保守的3‘-5’-外切核糖核酸酶多核苷酸磷酸化酶(PNPase)通过限制RNase E对Hfq结合的sRNA的访问和降解本来会促进sRNA衰退的靶mRNA片段来稳定sRNAs。在这里，我们报道在没有PNPase的情况下，CTD中的RNA结合区AR2是RNase E启动降解依赖于Hfq的sRNAs CyaR和RyhB所必需的。此外，我们还发现，在Hfq中引入突变，破坏靶基因与Hfq的结合，或者在RNE的AR2编码区引入突变，都会削弱RNase E与sRNAs的结合。综上所述，我们的数据支持这样一个模型，即在Hfq催化sRNA-mRNA配对后，sRNA通过其AR2结构域与RNase E结合的mRNA靶标而被招募。这些结果也支持我们的结论，在PNPase缺陷菌株中，由于sRNA的积累而加速了与mRNA靶标的配对，sRNA发生了更快的衰退。我们的发现为RNase E调控sRNAs和mRNAs的机制提供了洞察力。
RNase E performs an indispensable function in gram-negative bacteria by initiating breakdown of cellular RNAs and providing the scaffold for the primary RNA decay machine, the RNA degradosome. Additionally, RNase E executes gene regulation by cleaving mRNAs that are flagged for decay upon binding to small regulatory RNAs (sRNAs) often associated with the RNA chaperone Hfq. However, a detailed mechanistic understanding of the interactions between these four components remains elusive. Although sRNA binding is known to promote mRNA decay, our work presented here indicates that sRNAs and Hfq are recruited indirectly through bound target mRNAs to the second arginine-rich RNA-binding region (AR2) within the C-terminal domain of RNase E. Consequently, sRNAs are degraded, which broadly impacts bacterial physiology and survival. The C-terminal domain (CTD) of the major endoribonuclease RNase E not only serves as a scaffold for the central RNA decay machinery in gram-negative bacteria but also mediates coupled degradation of small regulatory RNAs (sRNAs) and their cognate target transcripts following RNA chaperone Hfq–facilitated sRNA–mRNA base pairing. Despite the crucial role of RNase E CTD in sRNA-dependent gene regulation, the contribution of particular residues within this domain in recruiting sRNAs and mRNAs upon base pairing remains unknown. We have previously shown that in Escherichia coli, the highly conserved 3′-5′-exoribonuclease polynucleotide phosphorylase (PNPase) paradoxically stabilizes sRNAs by limiting access of RNase E to Hfq-bound sRNAs and by degrading target mRNA fragments that would otherwise promote sRNA decay. Here, we report that in the absence of PNPase, the RNA-binding region AR2 in the CTD is required for RNase E to initiate degradation of the Hfq-dependent sRNAs CyaR and RyhB. Additionally, we show that introducing mutations in either hfq that disrupts target mRNA binding to Hfq or the AR2 coding region of rne impairs RNase E binding to sRNAs. Altogether, our data support a model where sRNAs are recruited via bound mRNA targets to RNase E by its AR2 domain after Hfq catalyzes sRNA–mRNA pairing. These results also support our conclusion that in a PNPase-deficient strain, more rapid decay of sRNAs occurs due to accelerated pairing with mRNA targets as a consequence of their accumulation. Our findings provide insights into the mechanisms by which sRNAs and mRNAs are regulated by RNase E.