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. IMPORTANCEThe ubiquitous bacterium Pseudomonas aeruginosa is notorious as an opportunistic pathogen causing life-threatening acute and chronic infections in immunocompromised patients. P. aeruginosa infection processes are governed by two major gene regulatory systems, namely, the GacA/GacS (GacAS) twocomponent system and the RNA-binding proteins RsmA/RsmN (RsmA/N). RsmA/N basically function as a translational repressor or activator directly by competing with the ribosome. In this study, we .

Specific and Global RNA Regulators in Pseudomonas aeruginosa

“…The CsrA/RsmA family of RNA-binding proteins governs virulence gene expression in pathogenic bacteria [173][174][175][176][177][178], including Pae [139,179]. Members of this family usually negatively regulate translation of target mRNAs through binding to stem-loop structures present in their 5 UTR that contain exposed GGA motifs [27,139,173,[180][181][182][183][184]. Rsm proteins can also act as indirect activators and repressors of a large subset of genes by altering the expression of key regulators (Figure 4a) [139,179,185].…”

“…Rsm proteins can also act as indirect activators and repressors of a large subset of genes by altering the expression of key regulators (Figure 4a) [139,179,185]. Moreover, Rsm proteins can stabilize transcripts, and thereby promote their translation [184,186,187]. Analogously to the Hfq/Crc/CrcZ entity, the activity of these translational regulators can be countered by RsmA/N-binding RNAs, which act as a decoy to abrogate RsmA/N-mediated translational repression and thus, lead to de-repression of RsmA/N controlled genes (Figures 2 and 4b).…”

“…RsmN/F protein synthesis is negatively controlled by RsmA at the translational level. The scheme was drawn based on previous reports [139,143,184]. (b) Synthesis and turnover of RsmY/Z RNAs.…”

“…Although the absence of RsmN/F has little or no impact on the expression of RsmA-regulated genes, the deletion of both rsmA and rsmN/F causes additional phenotypes when compared with a single rsmA deletion [182,218]. In addition, RsmN/F and RsmA exhibit distinct binding affinities for some of their RNA targets [142,[182][183][184]218,219]. RsmN/F binds only to targets that contain at least two Rsm consensus sequences, while RsmA requires only a single GGA motif for efficient interactions [183,184].…”

“…In addition, RsmN/F and RsmA exhibit distinct binding affinities for some of their RNA targets [142,[182][183][184]218,219]. RsmN/F binds only to targets that contain at least two Rsm consensus sequences, while RsmA requires only a single GGA motif for efficient interactions [183,184]. Moreover, RsmA functions as a translational repressor of RsmN/F (Figure 4a) [182].…”