Global identification of RsmA/N binding sites in <i>Pseudomonas aeruginosa</i> by <i>in vivo</i> UV CLIP-seq

<i>Pseudomonas aeruginosa</i> harbours two redundant RNA-binding proteins RsmA/RsmN (RsmA/N), which play a critical role in balancing acute and chronic infections. However, <i>in vivo</i> binding sites on target transcripts and the overall impact on the physiology remains unclear. In this study, we applied <i>in vivo</i> UV crosslinking immunoprecipitation followed by RNA-sequencing (UV CLIP-seq) to detect RsmA/N-binding sites at single-nucleotide resolution and mapped more than 500 binding sites to approximately 400 genes directly bound by RsmA/N in <i>P. aeruginosa</i>. This also verified the ANGGA sequence in apical loops skewed towards 5ʹUTRs as a consensus motif for RsmA/N binding. Genetic analysis combined with CLIP-seq results suggested previously unrecognized RsmA/N targets involved in LPS modification. Moreover, the RsmA/N-titrating RNAs RsmY/RsmZ may be positively regulated by the RsmA/N-mediated translational repression of their upstream regulators, thus providing a possible mechanistic explanation for homoeostasis of the Rsm system. Thus, our study provides a detailed view of RsmA/N-RNA interactions and a resource for further investigation of the pleiotropic effects of RsmA/N on gene expression in <i>P. aeruginosa</i>.铜绿假单胞菌（Pseudomonas aeruginosa）携带有两种功能冗余的RNA结合蛋白RsmA/RsmN（RsmA/N），其在平衡急性与慢性感染进程中发挥关键调控作用。然而，目前针对其在靶标转录本上的体内（in vivo）结合位点，以及该蛋白对菌体生理的整体影响仍不明确。本研究采用紫外交联免疫沉淀结合RNA测序（UV CLIP-seq）技术，以单核苷酸分辨率检测RsmA/N的结合位点，在铜绿假单胞菌中定位到超过500个结合位点，对应约400个直接受RsmA/N结合的靶基因。本研究同时验证了位于顶端环区的ANGGA序列偏好富集于5'非翻译区（5'UTR），可作为RsmA/N结合的保守基序。结合CLIP-seq结果开展的遗传分析，揭示了此前未被报道的、参与脂多糖（LPS）修饰的RsmA/N新靶标。此外，作为RsmA/N诱饵RNA的RsmY/RsmZ，可能通过RsmA/N介导的上游调控因子翻译抑制过程实现正向调控，这为解释Rsm系统的稳态（homoeostasis）维持机制提供了潜在的分子途径。综上，本研究清晰阐明了RsmA/N与RNA的互作特征，为后续探究RsmA/N对铜绿假单胞菌基因表达的多效性影响提供了重要研究资源。