Polynucleotide phosphorylase and RNA helicase CshA cooperate in <i>Bacillus subtilis</i> mRNA decay
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Polynucleotide phosphorylase (PNPase), a 3ʹ exoribonuclease that degrades RNA in the 3ʹ-to-5ʹ direction, is the major mRNA decay activity in <i>Bacillus subtilis</i>. PNPase is known to be inhibited <i>in vitro</i> by strong RNA secondary structure, and rapid mRNA turnover <i>in vivo</i> is thought to require an RNA helicase activity working in conjunction with PNPase. The most abundant RNA helicase in <i>B. subtilis</i> is CshA. We found for three small, monocistronic mRNAs that, for some RNA sequences, PNPase processivity was unimpeded even without CshA, whereas others required CshA for efficient degradation. A novel colour screen for decay of mRNA in <i>B. subtilis</i> was created, using mRNA encoded by the <i>slrA</i> gene, which is degraded from its 3ʹ end by PNPase. A significant correlation between the predicted strength of a stem-loop structure, located in the body of the message, and PNPase processivity was observed. Northern blot analysis confirmed that PNPase processivity was greatly hindered by the internal RNA structure, and even more so in the absence of CshA. Three other <i>B. subtilis</i> RNA helicases did not appear to be involved in mRNA decay during vegetative growth. The results confirm the hypothesis that efficient 3ʹ exonucleolytic decay of <i>B. subtilis</i> RNA depends on the combined activity of PNPase and CshA.
多核苷酸磷酸化酶（Polynucleotide phosphorylase，PNPase）是一类沿3'→5'方向降解RNA的3'外切核糖核酸酶，是枯草芽孢杆菌（Bacillus subtilis）体内主要的mRNA降解活性因子。已知PNPase在体外可被强RNA二级结构抑制，而体内mRNA的快速降解周转被认为需要RNA解旋酶与PNPase协同发挥作用。 枯草芽孢杆菌中丰度最高的RNA解旋酶为CshA。针对3种小型单顺反子mRNA，本研究发现：对于部分RNA序列，即便缺乏CshA，PNPase的持续降解活性也不会受到阻碍；而另一些序列则需要CshA才能实现高效降解。 本研究构建了一种用于检测枯草芽孢杆菌mRNA降解的新型显色筛选体系，该体系利用slrA基因编码的mRNA，此类mRNA可被PNPase从3'端起始降解。我们观察到，位于转录本内部的茎环结构的预测稳定性与PNPase的持续降解活性之间存在显著相关性。Northern印迹分析证实，内部RNA结构会极大阻碍PNPase的持续降解活性，而在CshA缺失的情况下，这种阻碍作用更为显著。 另有3种枯草芽孢杆菌RNA解旋酶似乎并不参与营养生长期的mRNA降解过程。本研究结果证实了相关假说：枯草芽孢杆菌RNA的高效3'外切降解依赖于PNPase与CshA的协同活性。
