Large-scale identification of trans-translation substrates targeted by tmRNA in Aeromonas veronii

Transfer-messenger RNA (tmRNA) is ubiquitous in bacteria, acting as the core component for the trans-translation system that contributes to label the aberrantly synthesized peptides for degradation and to release the stalled ribosomes. Deletion of tmRNA causes a variety of phenotypes related to important physiological processes in bacteria. To illustrate the molecular mechanism of the versatility of tmRNA in aquatic pathogen Aeromonas veronii, we mutated the C-terminal nucleotides of tmRNA (MutmRNA) for encoding a tag containing six histidine residues (His₆tag), so as to capture and enrich the trans-translation substrates from the cell lysates through a Ni²⁺-NTA affinity chromatograph. The results showed that the concentrated substrates were detected as distinct and specific bands in western blotting using anti-His antibody, demonstrating that specific defective mRNAs were frequently and intensively rescued by trans-translation during the translation process in A. veronii. The substrates were analyzed by LC-MS/MS and further identified by searching a theoretically constructed database specific for A. veronii. Total of 24 potential substrates were identified, with various functions involved in metabolism, as well as structure and signal-based cellular events. Among the identified substrates, PspA and AsmA were labeled by Flag, and expressed in the presence of the modified trans-translation system in E. coli. Their labelings with MutmRNA were validated by purification through Ni²⁺-NTA column followed by western blotting using anti-Flag antibody. This study provided the most abundant set of endogenous targets for tmRNA in A. veronii, and facilitated further investigations about the molecular mechanism and signal pathway of tmRNA-mediated trans-translation.