Taggart James C, Dierksheide Kathryn Julia, LeBlanc Hannah J, Lalanne Jean-Benoît, Durand Sylvain, Braun Frédérique, Condon Ciarán, Li Gene-Wei
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Nucleic Acids Res. 2025 Jan 24;53(3). doi: 10.1093/nar/gkaf030.
RNA endonucleases are the rate-limiting initiator of decay for many bacterial mRNAs. However, the positions of cleavage and their sequence determinants remain elusive even for the well-studied Bacillus subtilis. Here we present two complementary approaches-transcriptome-wide mapping of endoribonucleolytic activity and deep mutational scanning of RNA cleavage sites-that reveal distinct rules governing the specificity among B. subtilis endoribonucleases. Detection of RNA terminal nucleotides in both 5'- and 3'-exonuclease-deficient cells revealed >103 putative endonucleolytic cleavage sites with single-nucleotide resolution. We found a surprisingly weak consensus for RNase Y targets, a contrastingly strong primary sequence motif for EndoA targets, and long-range intramolecular secondary structures for RNase III targets. Deep mutational analysis of RNase Y cleavage sites showed that the specificity is governed by many disjointed sequence features. Our results highlight the delocalized nature of mRNA stability determinants and provide a strategy for elucidating endoribonuclease specificity in vivo.
RNA 内切核酸酶是许多细菌 mRNA 降解的限速起始因子。然而，即使对于深入研究的枯草芽孢杆菌，切割位点及其序列决定因素仍不清楚。在这里，我们提出了两种互补方法——核糖核酸内切酶活性的全转录组图谱绘制和 RNA 切割位点的深度突变扫描——揭示了枯草芽孢杆菌内切核酸酶之间不同的特异性调控规则。在 5' 和 3' 核酸外切酶缺陷型细胞中检测 RNA 末端核苷酸，以单核苷酸分辨率揭示了 >103 个推定的内切核酸酶切割位点。我们发现 RNase Y 靶标的共有序列出人意料地弱，EndoA 靶标的一级序列基序则相反地强，以及 RNase III 靶标的长程分子内二级结构。对 RNase Y 切割位点的深度突变分析表明，特异性由许多不连续的序列特征控制。我们的结果突出了 mRNA 稳定性决定因素的离域性质，并提供了一种在体内阐明核糖核酸内切酶特异性的策略。
