Location: Emerging Pests and Pathogens ResearchTitle: Interactions among rsmX ncRNAs and Rsm RNA-binding proteins in the plant pathogen Pseudomonas syringae DC3000 Author
|Rouse, Clarissa - St Mary’s College|
|Fishman, Maxwell - Cornell University - New York|
Submitted to: American Society for Biochemistry and Molecular Biology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 1/19/2016
Publication Date: 4/2/2016
Citation: Rouse, C., Fishman, M., Filiatrault, M.J. 2016. Interactions among rsmX ncRNAs and Rsm RNA-binding proteins in the plant pathogen Pseudomonas syringae DC3000. American Society for Biochemistry and Molecular Biology Annual Meeting. Online: C31 1054.1.
Technical Abstract: In response to changing environmental stimuli, many bacterial species utilize the Csr/Rsm system of posttranscriptional gene expression regulation to control metabolism, motility, biofilm formation, and quorum sensing. Most Csr/Rsm RNA binding proteins are thought to bind near the 5’ end of mRNA transcripts to prevent translation. Small noncoding RNA molecules (ncRNAs), containing conserved binding sites, can sequester the Csr/Rsm proteins to allow translation of mRNA targets. This system consists of one to two proteins and two to three ncRNAs in most bacteria. However, the genome of Pseudomonas syringae pv. tomato strain DC3000 is predicted to encode five Rsm proteins and five ncRNAs and it is currently unknown which ncRNAs and mRNAs targets bind to each protein. This project sought to determine the conditions of protein expression and to identify which ncRNAs and mRNAs targets bind to each protein. DC3000 strains were designed to express each of the RNA-binding proteins with a peptide tag, to facilitate immunoprecipitation and isolation of bound mRNAs and ncRNAs. We found that RsmA1, 4, and 5 were not expressed under the conditions tested. RsmA2 and RsmA3 were both expressed in stationary phase in Mannitol-glutamate media. RNA immunoprecipitation (RIP) followed by qRT-PCR showed that RsmA2 binds to (in order of decreasing abundance) ncRNAs rsmX-1, rsmX-2, rsmX-3, and rsmY; RsmA3 binds to rsmX-1, rsmX-2, and rsmY. Transcripts for rsmZ and rsmX-4 were not detected. The ncRNA rsmX-5 was expressed but not significantly enriched in RIP, indicating that it does not bind to RsmA2 or 3 under these conditions. Additionally, mRNA targets were identified based on homology to computationally predicted targets in Pseudomonas aeruginosa. None of the predicted mRNA targets (PSPTO 1951, 0823, 3382, 5128, 0816, 1994, 4224) showed enrichment. It may be that these are not targets, or that the proteins were sequestered by the ncRNAs and not binding to mRNA under the experimental conditions. These results have shed light on the highly complex Rsm system in P. syringae and provide a foundation for future experimentation on Rsm protein associations with mRNAs and ncRNAs under various conditions in order to delineate the mechanisms of this global system of gene regulation.