Functional Analyses of the RsmY and RsmZ Small Noncoding Regulatory RNAs in Pseudomonas aeruginosa

Abstract

is a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA. CsrA homologs are present in many bacteria. The opportunistic pathogen uses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activity Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activity and are more stringent than those for RsmA.

RsmV a small non-coding regulatory RNA in Pseudomonas aeruginosa that sequesters RsmA and RsmF from target mRNAs

“…5A-B), the binding affinity of RsmA and RsmF was relatively unaffected by the single GGA substitutions (Table 1). A similar trend was observed in a previous mutagenesis study of RsmY and RsmZ wherein the in vivo activity did not strictly correlate with in vitro binding (37). It was speculated that other RNA binding proteins, such as Hfq, may prevent binding to suboptimal sites in vivo.…”

Bactericidal Type IV Secretion System Homeostasis in Xanthomonas citri

“…Microarray data of an X. citri strain harbouring a mutation in the global regulator CsrA (also 82 called RsmA) indicated its involvement in the regulation of over a hundred genes, including 83 the virB operon that encodes the T4SS proteins VirB1-11 (Andrade et al 2014 CsrA (Weilbacher et al 2003;Janssen et al 2018). Production of these small RNAs in E. coli is 96 controlled by several regulatory pathways, including the BarA/UvrY two-component system 97 that responds to molecules such as formate and acetate, the catabolite repression pathway 98 mediated by cAMP-CRP and the stringent response governed by RelA and SpoT-mediated 99 production of (p)ppGpp (Romeo & Babitzke 2018).…”

RNase E Promotes Expression of Type III Secretion System Genes in Pseudomonas aeruginosa

“…RNase E-mediated regulation of the T3SS is not dependent on the small regulatory RNA RsmY, RsmZ, RsmV, or RsmW. In P. aeruginosa, the small regulatory RNAs RsmY and RsmZ have been shown to inhibit T3SS gene expression (15,29,30). We reasoned that depletion of RNase E might inhibit the expression of T3SS genes by increasing the stability of either RsmY, RsmZ, or both of these small RNAs (sRNAs).…”