Grad-seq in a Gram-positive bacterium reveals exonucleolytic sRNA activation in competence control

RNA-protein interactions crucially underlie many steps of bacterial gene expression including post-transcriptional control by small regulatory RNAs (sRNAs). In stark contrast with recent progress in Gram-negative bacteria, knowledge about RNA and protein complexes in Gram-positive species remains scarce. Here, we used Grad-seq to draft a landscape of such complexes in Streptococcus pneumoniae, determining the sedimentation profiles of ~88% of the transcripts and ~62% of the proteins of this important human pathogen. Analysis of in-gradient distributions and subsequent tag-based protein capture identified interactions of the exoribonuclease Cbf1 (a.k.a. YhaM) with sRNAs that control bacterial competence. Contrary to expectation, the nucleolytic activity of Cbf1 stabilized these sRNAs, thereby promoting their function as repressors of competence. These results illustrate how this first RNA/protein complexome resource for a Gram-positive species can be utilized to identify new molecular factors in RNA-based regulation of pathways with relevance to bacterial virulence. Overall design: 6 CLIP-seq samples from S. pneumoniae TIGR4 cbf1-3xFLAG including 3 crosslinked samples and 3 non-crosslinked control samples.

RNA-蛋白质相互作用（RNA-protein interactions）是细菌基因表达诸多关键步骤的核心基础，涵盖由小型调控RNA（small regulatory RNAs, sRNAs）介导的转录后调控过程。与革兰氏阴性细菌领域的近期研究进展形成鲜明对比的是，学界对革兰氏阳性物种中RNA与蛋白质复合物的认知仍极为有限。本研究采用Grad-seq技术，绘制了肺炎链球菌（Streptococcus pneumoniae）中此类复合物的初步图谱，明确了这一重要人类致病菌约88%的转录本与约62%的蛋白质的沉降分布特征。通过对梯度分离分布的分析及后续基于标签的蛋白质捕获实验，本研究鉴定出外切核糖核酸酶Cbf1（又名YhaM）与调控细菌感受态的小型调控RNA之间的相互作用。与预期相悖的是，Cbf1的核酸酶活性反而稳定了这些小型调控RNA，进而增强其作为感受态抑制因子的功能。本研究结果证实，这款首个针对革兰氏阳性物种的RNA/蛋白质复合物组资源，可用于发掘与细菌毒力相关通路的RNA调控新分子因子。总体实验设计：取自肺炎链球菌TIGR4 cbf1-3xFLAG菌株的6个CLIP-seq样本，其中包含3个交联样本与3个非交联对照样本。