Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

Abstract

R NA-binding proteins play an integral role in the posttranscriptional regulation of protein synthesis by altering translation initiation, mRNA stability, and/or RNA processing. The CsrA family of RNA-binding proteins regulates carbon metabolism, virulence factor production, and motility in a number of Gramnegative bacteria (1-5). CsrA proteins usually bind sites on target mRNAs that overlap the Shine-Dalgarno sequence to prevent translation initiation (6-8). Although considerable sequence variability exists between natural CsrA-binding sites, a common feature is a core GGA sequence that is usually presented in the loop portion of a stem-loop structure (9, 10). High-affinity interactions between CsrA and RNA targets have been analyzed by two powerful techniques. First, nuclear magnetic resonance (NMR) spectroscopy was used to show that RsmE, a CsrA homolog in Pseudomonas fluorescens, makes optimal contact with the sequence 5=-(A/U)CANGGANG(U/A), where N is any nucleotide (10). RsmE functions as a molecular clamp and gathers the ANGGAN core into a hexaloop, with the flanking nucleotides forming a 3-bp stem (10). The second approach, a systematic evolution of ligands by exponential enrichment (SELEX), was used to identify high-affinity RNA ligands of Escherichia coli CsrA (9). SELEX is a method used to select for RNA ligands that bind proteins of interest (11). Evolution of the ligands is based on repeated cycles of in vitro selection (12). The selection is driven toward optimized RNA targets that bind to the protein of interest with Citation Schulmeyer KH, Diaz MR, Bair TB, Sanders W, Gode CJ, Laederach A, Wolfgang MC, Yahr TL. 2016. Primary and secondary sequence structure requirements for recognition and discrimination of target RNAs by Pseudomonas aeruginosa RsmA and RsmF.

Global Regulation by CsrA and Its RNA Antagonists

“…This study established that the primary RNA sequence is most important for CsrA binding, and that the presentation of the GGA motif in a loop increases the affinity of CsrA-RNA interaction. More recently, a SELEX method that exploited deep sequencing of RNAs identified a consensus for Pseudomonas aeruginosa RsmA and RsmF as CANGGAYG, with the GGA motif typically found in a hexaloop (43). This sequence and structural arrangement is remarkably similar to that determined for E. coli CsrA.…”

“…Crosslinking studies with purified CsrA established that E. coli CsrA functions as a homodimer (5, 43). Subsequent structural studies demonstrated that each subunit of the dimer contains five β-strands (β 1 -β 5 ), an α-helix, and a flexible C terminus (44).…”

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

“…We used the previously described P tssA1’-‘lacZ translational reporter as surrogate for regulatory control of T6SS (13) and P exsD-lacZ transcriptional reporter as marker for the T3SS (30). RsmA/RsmF directly bind the tssA1 leader region to inhibit translation (12, 13) and positively regulate T3SS gene expression through a mechanism that remains to be defined (13). In a mutant lacking rsmV , rsmY , and rsmZ , RsmA/RsmF availability is high resulting in repression of P tssA1’-‘lacZ reporter activity and high levels of P exsD-lacZ reporter activity (Fig.…”

“…RsmA and RsmF both bind to RsmV with high affinity in vitro (Fig. 3A), and the affinity of RsmF for RsmV is at least 10-fold higher than for RsmY and RsmZ (12, 13). Although the affinity of RsmF for RsmV is higher in vitro, RsmV does not seem to show preferential activity towards RsmF over RsmA in vivo (Fig.…”

“…RsmA and RsmF directly interact with mRNA targets to positively or negatively alter translation efficiency and/or mRNA stability (8, 10, 11). The RsmA and RsmF bindings site on target mRNAs commonly overlap the ribosome binding site and consist of a conserved 5’- CAN GGA YG sequence motif (where N is any nucleotide, the underlined GGA is 100% conserved, and Y is either a cytosine or uracil) that presents the GGA sequence in the loop portion of a stem-loop structure (12-15). While RsmA is able to bind mRNA targets with a single CAN GGA YG sequence (12), RsmF differs in that high affinity binding is only observed with mRNAs targets possessing at least two CAN GGA YG consensus binding sites (12).…”