Chhabra Shivani, Mandell Zachary F, Liu Bo, Babitzke Paul, Bechhofer David H
Icahn School of Medicine at Mount Sinaigrid.59734.3c, Department of Pharmacological Sciences, New York, New York, USA.
The Pennsylvania State University, Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, University Park, Pennsylvania, USA.
mBio. 2022 Apr 26;13(2):e0040022. doi: 10.1128/mbio.00400-22. Epub 2022 Mar 21.
The Bacillus subtilis genome encodes four 3' exoribonucleases: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Previous work showed that PNPase, encoded by the gene, is the major 3' exonuclease involved in mRNA turnover; in a deletion strain, numerous mRNA decay intermediates accumulate. Whether B. subtilis mRNA decay occurs in the context of a degradosome complex is controversial. In this study, global mapping of mRNA decay intermediate 3' ends within coding sequences was performed in strains that were either deleted for or had an inactivating point mutation in the gene. The patterns of 3'-end accumulation in these strains were highly similar, which may have implications for the role of a degradosome in mRNA decay. A comparison with mapped 3' ends in a strain lacking CshA, the major RNA helicase, indicated that many mRNAs require both PNPase and CshA for efficient decay. Transcriptome sequencing (RNA-seq) analysis of strains lacking RNase R suggested that this enzyme did not play a major role in mRNA turnover in the wild-type strain. Strains were constructed that contained only one of the four known 3' exoribonucleases. When RNase R was the only 3' exonuclease present, it was able to degrade a model mRNA efficiently, showing processive decay even through a strong stem-loop structure that inhibits PNPase processivity. Strains containing only RNase PH or only YhaM were also insensitive to this RNA secondary structure, suggesting the existence of another, as-yet-unidentified, 3' exoribonuclease. The ability to rapidly change bacterial gene expression programs in response to environmental conditions is highly dependent on the efficient turnover of mRNA. While much is known about the regulation of gene expression at the transcriptional and translational levels, much less is known about the intermediate step of mRNA decay. Here, we mapped the 3' ends of mRNA decay intermediates in strains that were missing the major 3' exoribonuclease PNPase or the RNA helicase CshA. We also assessed the roles of three other B. subtilis 3' exonucleases in the mRNA decay process. The data confirm the primary role of PNPase in mRNA turnover and suggest the involvement of one or more unknown RNases.
枯草芽孢杆菌基因组编码四种3'外切核糖核酸酶：多核苷酸磷酸化酶（PNPase）、核糖核酸酶R、核糖核酸酶PH和YhaM。先前的研究表明，由 基因编码的PNPase是参与mRNA周转的主要3'外切核糖核酸酶；在 缺失菌株中，大量mRNA衰变中间体积累。枯草芽孢杆菌mRNA衰变是否发生在降解体复合物的背景下存在争议。在本研究中，在 基因缺失或有失活点突变的菌株中，对编码序列内的mRNA衰变中间体3'末端进行了全局定位。这些菌株中3'末端积累的模式高度相似，这可能对降解体在mRNA衰变中的作用有影响。与缺乏主要RNA解旋酶CshA的菌株中定位的3'末端进行比较表明，许多mRNA需要PNPase和CshA两者才能有效衰变。对缺乏核糖核酸酶R的菌株进行转录组测序（RNA-seq）分析表明，该酶在野生型菌株的mRNA周转中不发挥主要作用。构建了仅包含四种已知3'外切核糖核酸酶之一的菌株。当核糖核酸酶R是唯一存在的3'外切核糖核酸酶时，它能够有效降解模型mRNA，即使通过抑制PNPase连续性的强茎环结构也能显示出连续性衰变。仅含有核糖核酸酶PH或仅含有YhaM的菌株对这种RNA二级结构也不敏感，这表明存在另一种尚未鉴定的3'外切核糖核酸酶。细菌响应环境条件快速改变基因表达程序的能力高度依赖于mRNA的有效周转。虽然在转录和翻译水平上对基因表达的调控了解很多，但对mRNA衰变的中间步骤了解较少。在这里，我们绘制了缺失主要3'外切核糖核酸酶PNPase或RNA解旋酶CshA的菌株中mRNA衰变中间体的3'末端图谱。我们还评估了其他三种枯草芽孢杆菌3'外切核酸酶在mRNA衰变过程中的作用。数据证实了PNPase在mRNA周转中的主要作用，并表明涉及一种或多种未知核糖核酸酶。
