Kayley H. Schulmeyer, Manisha R. Diaz, Thomas B. Bair, Wes Sanders, Cindy J. Gode, Alain Laederach, Matthew C. Wolfgang, Timothy L. Yahr

CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the post-transcriptional level. Pseudomonas aeruginosa has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62 nt RNA transcripts with a central core randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CAnGGAyG) that was positioned in a hexaloop region of stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with tssA1, an in vivo regulatory target of both RsmA and RsmF. Our finding suggest RsmF binding requires two GGA-containing sites while RsmA binding requirements are less stringent. The CsrA family of RNA-binding proteins is widely conserved in bacteria and play important roles in post-transcriptional regulation of protein synthesis. P. aeruginosa has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets in common, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to P. aeruginosa physiology and virulence.