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.

中文翻译：

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RNase E RNA 结合域 AR2 的目标识别在没有 PNPase 的情况下驱动 sRNA 衰变

主要核糖核酸内切酶 RNase E 的 C 端结构域 (CTD) 不仅作为革兰氏阴性菌中央 RNA 衰变机制的支架，而且还介导小调节 RNA (sRNA) 及其在 RNA 后的同源靶转录本的耦合降解伴侣蛋白 Hfq 促进 sRNA-mRNA 碱基配对。尽管 RNase E CTD 在 sRNA 依赖性基因调控中起着至关重要的作用，但该结构域内的特定残基在碱基配对时对募集 sRNA 和 mRNA 的贡献仍然未知。我们之前已经表明，在大肠杆菌，高度保守的 3'-5'-核糖核酸外切酶多核苷酸磷酸化酶 (PNPase) 通过限制 RNase E 进入 Hfq 结合的 sRNA 以及通过降解会促进 sRNA 衰变的目标 mRNA 片段来稳定 sRNA。在这里，我们报告说，在没有 PNPase 的情况下，CTD 中的 RNA 结合区域 AR2 是 RNase E 启动 Hfq 依赖性 sRNA CyaR 和 RyhB 降解所必需的。此外，我们表明在任何一个中引入突变高频破坏目标 mRNA 与 Hfq 或 AR2 编码区的结合rne损害 RNase E 与 sRNA 的结合。总而言之，我们的数据支持一个模型，在该模型中，在 Hfq 催化 sRNA-mRNA 配对后，通过其 AR2 结构域将 mRNA 靶标结合到 RNase E 来募集 sRNA。这些结果也支持我们的结论，即在 PNPase 缺陷菌株中，由于 sRNA 的积累而加速与 mRNA 靶标的配对，因此会发生更快速的 sRNA 衰变。我们的研究结果提供了对 sRNA 和 mRNA 受 RNase E 调控机制的见解。