ABSTRACT

Polynucleotide phosphorylase (PNPase), a 3ʹ exoribonuclease that degrades RNA in the 3ʹ-to-5ʹ direction, is the major mRNA decay activity in Bacillus subtilis. PNPase is known to be inhibited in vitro by strong RNA secondary structure, and rapid mRNA turnover in vivo is thought to require an RNA helicase activity working in conjunction with PNPase. The most abundant RNA helicase in B. subtilis 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 B. subtilis was created, using mRNA encoded by the slrA 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 B. subtilis 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 B. subtilis RNA depends on the combined activity of PNPase and CshA.

摘要

多核苷酸磷酸化酶 (PNPase) 是一种 3' 外切核糖核酸酶，可在 3'-to-5' 方向降解 RNA，是枯草芽孢杆菌中主要的 mRNA 衰变活性。已知 PNPase在体外被强 RNA 二级结构抑制，并且认为体内快速 mRNA 周转需要与 PNPase 一起工作的 RNA 解旋酶活性。枯草芽孢杆菌中最丰富的 RNA 解旋酶是 CshA。我们发现，对于三个小的单顺反子 mRNA，对于某些 RNA 序列，即使没有 CshA，PNPase 的持续合成能力也不受阻碍，而另一些则需要 CshA 才能有效降解。使用由slrA编码的 mRNA，创建了一种用于枯草芽孢杆菌中 mRNA 衰变的新型彩色屏幕PNPase 从其 3ʹ 端降解基因。观察到位于消息主体中的茎环结构的预测强度与 PNPase 合成能力之间的显着相关性。Northern印迹分析证实，内部RNA结构极大地阻碍了PNPase的持续合成能力，在没有CshA的情况下更是如此。其他三种枯草芽孢杆菌RNA 解旋酶似乎不参与营养生长过程中的 mRNA 衰变。结果证实了这样的假设，即枯草芽孢杆菌RNA 的有效 3ʹ 核酸外切衰变取决于 PNPase 和 CshA 的组合活性。