Global identification of RsmA/N binding sites inPseudomonas aeruginosabyin vivoUV CLIP-seq

Abstract

Posttranscriptional regulation of gene expression in bacteria is performed by a complex and hierarchical signaling cascade. Pseudomonas aeruginosa harbors two redundant RNA-binding proteins RsmA/RsmN (RsmA/N), which play a critical role in balancing acute and chronic infections. However, in vivo binding sites on target transcripts and the overall impact on the physiology remains unclear. In this study, we applied in vivo 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 peaks to approximately 400 genes directly bound by RsmA/N in P. aeruginosa. This also demonstrated the ANGGA sequence in apical loops skewed towards 5'UTRs as a consensus motif for RsmA/N binding. Genetic analysis combined with CLIP-seq results identified previously unrecognized RsmA/N targets involved in LPS modification. Moreover, the small non-coding RNAs RsmY/RsmZ, which sequester RsmA/N away from target mRNAs, are positively regulated by the RsmA/N-mediated translational repression of hptB, encoding a histidine phosphotransfer protein, and cafA, encoding a cytoplasmic axial filament protein, thus providing a possible mechanistic explanation for homeostasis of the Rsm system. Our findings present the global RsmA/N-RNA interaction network that exerts pleiotropic effects on gene expression in P. aeruginosa.

A Grad-seq View of RNA and Protein Complexes in Pseudomonas aeruginosa under Standard and Bacteriophage Predation Conditions

“… P. aeruginosa is known to possess two large posttranscriptional networks that depend on globally acting RBPs: one governed by Hfq and the other by two CsrA-like Rsm proteins. The mRNA and ncRNA ligands of these RBPs have been mapped in a transcriptome-wide fashion ( 8 , 9 , 17 , 19 ), albeit not in the growth phase studied here.…”

“…RsmA blocks translation by binding to GGA motifs in mRNAs; its own activity is also regulated at the RNA level, by the ncRNAs RsmY/Z/V/W, which sponge RsmA by presenting multiple GGA motifs ( 60 ). The homologous RBP RsmN acts and is acted upon in the same fashion ( 15 , 17 , 18 ). Here, we observe well-correlated sedimentation of the two Rsm proteins and its four-known decoy RNAs, suggesting that in exponentially growing Pseudomonas cells ( Fig.…”

“…The other major regulatory RBPs include the CsrA-like proteins RsmA and RsmN, which act as global translational repressors through recognizing GGA motifs in the 59 region of mRNAs (14)(15)(16)(17)(18), partly so in a combinatorial fashion with Hfq (19). In contrast, much less is known about a putative homolog of FinO/ProQ-like proteins, which is an emerging new family of small regulatory RNA (sRNA)-related RBPs in Gram-negative bacteria (20)(21)(22)(23)(24).…”