RNA-RNA Interactomes of ProQ and Hfq Reveal Overlapping and Competing Roles

Abstract

Highlights d RNA-RNA interactomes for RNA chaperones ProQ and Hfq are identified by RIL-seq d A significant fraction of the RNA-RNA pairs on ProQ also are found on Hfq d sRNA-mediated regulation is impacted by growth conditions d ProQ blocks RNase III-and Hfq-mediated downregulation by a novel sRNA sponge

Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

“…We wondered whether the ProQ-cspE B3H interaction might be limited by competition with endogenous ProQ or cellular Hfq, given that co-immunoprecipitation studies have suggested that ProQ and Hfq may compete for a subset of their RNA substrates. (22,37) While no additional stimulation of transcription over basal levels was observed when the ProQ-cspE B3H experiment was repeated in ΔproQ reporter cells ( Fig 1C; 1.2), we found that the fold-stimulation of β-gal transcription indeed increased in Δhfq reporter cells ( Fig 1D; 2.3x). This Δhfq reporter strain was previously used for detecting Hfq-RNA interactions, (30) and was used throughout the remainder of this study.…”

“…Given that ProQ has been found to interact with dozens of sRNAs and hundreds of mRNAs inside of Salmonella and E. coli cells, (17) we sought to determine whether our B3H system could detect ProQ interactions with additional RNAs beyond cspE. We tested three additional RNAs that had been found to interact in vivo with ProQ: sRNAs SibB, RyjB and the 3'UTR of fbaA (hereafter, fbaA); (17,22,37) as well as four sRNAs for which we had previously detected B3H interactions with Hfq: ChiX, OxyS, ArcZ, and MgrR ( Fig S3). (30) While a cspE hybrid RNA consistently produced the highest stimulation of transcription above basal levels when present with α-ProQ Δ CTD , we observed a ≥1.5-fold increase in fold-stimulation of β-gal activity when each of these hybrid RNAs was present in reporter cells (Fig 2A; full b-gal data in Fig S4A).…”

“…There has been interest in the overlap of the subset of cellular RNAs bound by ProQ and Hfq, (22,37) another global RNA-binding protein that stabilizes dozens of sRNAs and catalyzes annealing with mRNAs in E. coli. In this study, ProQ was found to bind to a wider range of RNAs than Hfq, producing B3H interactions with RNAs found to interact both with ProQ as well as with Hfq in vivo (Fig 2).…”

“…(41) Collectively, our data are consistent with ProQ and Hfq sharing a subset of RNA targets, as has been suggested by previous studies. (22,37) It is well established that Hfq has multiple RNA-binding surfaces that possess distinct RNA-binding specificity and contribute to RNA annealing. (6,42) While E. coli FinO and N. meningitidis NMB1681 have been shown to catalyze RNA annealing and strand exchange, (18,19,21,40) these activities have not yet been demonstrated for ProQ itself.…”

Determinants of RNA recognition by the FinO domain of theEscherichia coliProQ protein

“…Global identification of new RNA/protein interactions has been enabled by deep-sequencing approaches (7), such as Grad-seq, which uses glycerol gradients to identify novel RNA/protein complexes (8), CLIP-seq, which uses crosslinking to define protein binding sites in the transcriptome (9), and RIL-seq which identifies protein-dependent RNA-RNA interactions (10). Recent studies using these approaches showed that another protein, named ProQ, is a global RNA binding protein in Escherichia coli and Salmonella enterica (11-13), and is involved in sRNA interactions with other RNA molecules (12,14,15). The pool of RNA ligands of ProQ is mostly distinct from that of Hfq and contains more mRNAs than sRNAs (11,12).While ProQ was originally discovered in a search for genes that affect proline transport (16), this protein contributes to several physiological processes in E.coli and S. enterica (17)including DNA metabolism (13,14), bacterial virulence (15), and adaptation to osmotic stress (12) and resource limitation (18).…”