RNA Interactome of cold shock proteins, CspA and CspE, in Salmonella Typhimurium
McGibbon, Louise Claire
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RNA-dependent control of gene expression is crucial for bacterial adaptation to environmental stresses, such as fluctuations in ambient temperature. In the enteric pathogen Salmonella Typhimurium, a drastic downshift in temperature immediately triggers the “cold shock response” in which selective expression of cold shock proteins (CSPs) aids acclimatisation. The major cold shock protein, CspA, and some of its homologues function as RNA chaperones and play critical roles in destabilising aberrant RNA secondary structures that form at reduced temperatures. However, the precise roles and targets of this protein family remain unclear. With the aim of generating a genome-wide map of protein-RNA interactions, in vivo UV cross-linking and analysis of cDNA (CRAC) was performed. This novel, high-throughput technique allows identification of all RNA targets for a particular protein, which in this case was the cold-induced protein CspA, and constitutively expressed CspE. CRAC results reveal a remarkable number and diversity in the RNA targets of these CSPs. For example, CspA targets approximately 25% of the RNA encoded by the Salmonella genome. CspA and CspE were shown to target mRNAs encoding proteins involved in metabolism, stress, cell division and RNA turnover, as well as a number of mRNAs that are cold shock-inducible. Bioinformatic analyses have shown that mainly protein coding regions are targeted and, interestingly, 5’ untranslated regions (UTR) and small RNAs, which often play roles as regulators of translational control. There also appears to be a reproducible pattern of repeated binding along mRNA transcripts, suggesting a role for these Csps in maintaining mRNAs in a linear conformation, which is required for efficient translation. To validate targets, phenotypic analyses were performed, including growth studies during amino acid starvation, and the response to heat shock and UV DNA damage. These experiments confirmed involvement of the paralogues, and further bioinformatic analysis revealed that these proteins were targeting key regulatory regions on some specific targets. A more in-depth analysis was carried out on one target – the general stress response sigma factor RpoS (σS) and a model of CspA paralogue involvement in regulating the mRNA of this target is presented. Overall, the in vivo data from this study suggests that these cold shock proteins are crucial for modulating key cellular processes beyond that which their name implies.