Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
RNA. 2012 Apr;18(4):844-55. doi: 10.1261/rna.029413.111. Epub 2012 Feb 21.
The transient existence of small RNAs free of binding to the RNA chaperone Hfq is part of the normal dynamic lifecycle of a sRNA. Small RNAs are extremely labile when not associated with Hfq, but the mechanism by which Hfq stabilizes sRNAs has been elusive. In this work we have found that polynucleotide phosphorylase (PNPase) is the major factor involved in the rapid degradation of small RNAs, especially those that are free of binding to Hfq. The levels of MicA, GlmY, RyhB, and SgrS RNAs are drastically increased upon PNPase inactivation in Hfq(-) cells. In the absence of Hfq, all sRNAs are slightly shorter than their full-length species as result of 3'-end trimming. We show that the turnover of Hfq-free small RNAs is growth-phase regulated, and that PNPase activity is particularly important in stationary phase. Indeed, PNPase makes a greater contribution than RNase E, which is commonly believed to be the main enzyme in the decay of small RNAs. Lack of poly(A) polymerase I (PAP I) is also found to affect the rapid degradation of Hfq-free small RNAs, although to a lesser extent. Our data also suggest that when the sRNA is not associated with Hfq, the degradation occurs mainly in a target-independent pathway in which RNase III has a reduced impact. This work demonstrated that small RNAs free of Hfq binding are preferably degraded by PNPase. Overall, our data highlight the impact of 3'-exonucleolytic RNA decay pathways and re-evaluates the degradation mechanisms of Hfq-free small RNAs.
游离于 RNA 分子伴侣 Hfq 的小 RNA 的短暂存在是 sRNA 正常动态生命周期的一部分。当小 RNA 不与 Hfq 结合时，其极不稳定，但 Hfq 稳定 sRNA 的机制一直难以捉摸。在这项工作中，我们发现多核苷酸磷酸化酶（PNPase）是参与小 RNA 快速降解的主要因素，特别是那些游离于 Hfq 的 sRNA。在 Hfq(-)细胞中失活 PNPase 后，MicA、GlmY、RyhB 和 SgrS RNA 的水平显著增加。在没有 Hfq 的情况下，所有 sRNA 的 3'-末端修剪都会导致其比全长物种略短。我们表明，游离于 Hfq 的 sRNA 的周转是生长阶段调节的，并且 PNPase 活性在静止期尤为重要。事实上，PNPase 的作用比普遍认为是小 RNA 降解主要酶的 RNase E 更大。缺乏多聚（A）聚合酶 I（PAP I）也会影响游离于 Hfq 的小 RNA 的快速降解，尽管影响较小。我们的数据还表明，当 sRNA 不与 Hfq 结合时，降解主要发生在一种非靶标依赖的途径中，其中 RNase III 的影响降低。这项工作表明，游离于 Hfq 结合的 sRNA 优先被 PNPase 降解。总的来说，我们的数据强调了 3'-外切核酸酶 RNA 降解途径的影响，并重新评估了游离于 Hfq 的 sRNA 的降解机制。
