A method for in vivo identification of bacterial small RNA-binding proteins.
Bottom Line: Subsequently, PrrF- and PrrH-protein complexes were enriched using cDNA "bait", and enriched RNA-protein complexes were analyzed by tandem mass spectrometry to identify PrrF and PrrH associated proteins.Interestingly, Hfq was identified more often in samples probed with the PrrF cDNA "bait" as compared to the PrrH cDNA "bait", suggesting Hfq has a stronger binding affinity for the PrrF sRNAs in vivo.As such, this study demonstrates that in vivo cross-linking coupled with sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS) is an effective methodology for unbiased identification of bacterial sRNA-binding proteins.
Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland.Show MeSH
Mentions: We next used UV irradiation to cross-link RNA-protein complexes in PAO1 grown in low-iron conditions. Maximal PrrF and PrrH expression was observed by qPCR at 18 h growth in M9 minimal media (Fig. 1C), so we used this time point for UV cross-linking and SSAC-MS/MS analysis, as outlined in Figure 2. Iron-depleted cultures of PAO1 and the ΔprrF1,2 mutant were UV-irradiated to irreversibly cross-link RNA-protein complexes, and irradiated cells were harvested for RNA isolation. Purified RNA was hybridized to either biotinylated PrrF1 or PrrH cDNA probes (“bait”), which are specific for the PrrF1 and PrrH sRNAs, respectively (Fig. 1). PrrF1- and PrrH-protein complexes were then enriched using streptavidin-coated magnetic beads. Enriched protein-RNA complexes were heated to disrupt the biotin-streptavidin linkage, trypsinized, and analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The majority of samples analyzed by this methodology resulted in greater than 15 peptides corresponding to P. aeruginosa proteins, with some runs producing more than 100 P. aeruginosa peptides. To prioritize the corresponding protein hits that were most likely to be PrrF or PrrH-binding proteins, we used the following criteria: (1) peptides corresponding to the protein had to be detected in at least three independent PAO1 samples analyzed with the same bait, and (2) peptides corresponding to the protein must not have been detected in more than one ΔprrF1,2 mutant sample analyzed with the same bait. Samples that produced fewer than 10 peptides in total were considered failed runs and not included in further analyses. A summary of the proteins meeting our criteria for either the PrrF1 or PrrH bait are shown in Table 2, and a complete compilation of our mass spectrometry results are provided in the (Tables S2, S3).
Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland.