Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments

In many gram-negative and some gram-positive bacteria small regulatory RNAs (sRNAs) that bind the RNA chaperone Hfq have a pivotal role in modulating virulence, stress responses, metabolism, and biofilm formation. These sRNAs recognize transcripts through base-pairing, and sRNA-mRNA annealing consequently alters the translation and/or stability of transcripts leading to changes in gene expression. We have previously found that the highly conserved 3'-to-5' exoribonuclease polynucleotide phosphorylase (PNPase) has an indispensable role in paradoxically stabilizing Hfq-bound sRNAs and promoting their function in gene regulation in Escherichia coli. Here, we report that PNPase uniquely contributes to the degradation of specific mRNA cleavage products, the majority of which bind Hfq and are derived from targets of sRNAs. Specifically, we found that these mRNA-derived fragments accumulate in the absence of PNPase or its exoribonuclease activity and interact with PNPase. Additionally, we show that mutations in hfq or in the seed pairing region of a sRNA eliminated the requirement of PNPase for sRNA stability. Altogether, our results are consistent with a model that PNPase degrades mRNA-derived fragments that could otherwise deplete cells of Hfq-binding sRNAs through pairing mediated decay. Overall design: Examination of mRNA expression in four samples of strains carrying wild type or mutant genes of rph and/or pnp. Additional examination of four samples from pnp wild type and mutant strains of RNA from Hfq co-immunoprecipitation input and output fractions. All samples were sequenced with two biological replicates.

在诸多革兰氏阴性菌及部分革兰氏阳性菌中，结合RNA伴侣蛋白Hfq的小型调控RNA（small regulatory RNAs, sRNAs）在调控毒力、应激响应、代谢过程与生物被膜形成方面发挥关键作用。这类sRNAs通过碱基互补配对识别靶标转录本，sRNA与mRNA的退火结合会改变转录本的翻译效率及/或稳定性，进而介导基因表达的改变。

我们前期研究发现，高度保守的3'→5' 外切核糖核酸酶（3'-to-5' exoribonuclease）——多核苷酸磷酸化酶（polynucleotide phosphorylase, PNPase）在大肠杆菌（Escherichia coli）中具有不可或缺的功能：它能够反直觉地稳定结合Hfq的sRNAs，并促进其在基因调控中的功能。

本研究中，我们报道PNPase还可特异性参与特定mRNA裂解产物的降解过程，这类产物大多结合Hfq且来源于sRNAs的靶标转录本。具体而言，我们发现，在缺失PNPase或其外切核糖核酸酶活性的菌株中，这些mRNA来源的片段会发生积累，且能够与PNPase发生相互作用。

此外，我们证实，hfq基因突变或sRNA种子配对区域的突变，会消除PNPase对sRNA稳定性的依赖需求。

综上，我们的研究结果支持如下模型：PNPase可降解mRNA来源的片段，这类片段若未被清除，可通过配对介导的降解途径耗尽细胞内结合Hfq的sRNAs。

整体实验设计：对4组携带野生型或突变型rph和/或pnp基因的菌株的mRNA进行表达检测。此外，还对来自pnp野生型与突变型菌株的4组RNA样本进行分析，这些样本取自Hfq免疫共沉淀（co-immunoprecipitation）的输入组分与输出组分。所有样本均设置2次生物学重复，并进行测序。