Dynamic interactions between the RNA chaperone Hfq, small regulatory RNAs, and mRNAs in live bacterial cells

Abstract

RNA-binding proteins play myriad roles in regulating RNAs and RNA-mediated functions. In bacteria, the RNA chaperone Hfq is an important post-transcriptional gene regulator. Using live-cell super-resolution imaging, we can distinguish Hfq binding to different sizes of cellular RNAs. We demonstrate that under normal growth conditions, Hfq exhibits widespread mRNA-binding activity, with the distal face of Hfq contributing mostly to the mRNA binding in vivo. In addition, sRNAs can either co-occupy Hfq with the mRNA as a ternary complex, or displace the mRNA from Hfq in a binding face-dependent manner, suggesting mechanisms through which sRNAs rapidly access Hfq to induce sRNA-mediated gene regulation. Finally, our data suggest that binding of Hfq to certain mRNAs through its distal face can recruit RNase E to promote turnover of these mRNAs in an sRNA-independent manner, and such regulatory function of Hfq can be decoyed by sRNA competitors that bind strongly at the distal face.

Diversity of bacterial small RNAs drives competitive strategies for a mutual chaperone

“…The least replacement occurred when DsrA searched for a place on Hfq pre-bound to ChiX (H-CX vs DA; 87% resident remaining at 5 nM competitor). These results agreed with previous reports that ChiX sRNA is a powerful competitor for Hfq 6 , 12 , 28 , and with the notion that sRNAs can compete more robustly for Hfq when, like ChiX, they interact with both its proximal and distal faces 29 .…”

“…As a result, the stabilities of RNA-protein complexes depend on the number of surfaces an RNA contacts, as well as the strength of each contact. Conversely, each RBP can usually recognize many RNAs, resulting in competition amongst similar RNAs for limited copies of shared proteins 3 – 12 . Our results show that binding to empty Hfq after passive dissociation of a previously bound RNA is rare, as previously proposed 26 .…”

“…The functions of cellular RNAs are influenced by their interactions with RNA-binding proteins (RBPs), which may exchange or remodel the RNAs over their life cycle 1 , 2 . When RBPs are limiting in number, their RNA ligands compete for binding 3 – 12 . This competition is often thought to depend on the order of recruitment, or the binding energetics.…”

Kinetic modeling reveals additional regulation at co-transcriptional level by post-transcriptional sRNA regulators

“…Because the stability of SgrS and RyhB, as well as their target binding, are Hfq dependent, the close match between the kinetic parameters measured here and the numbers reported for endogenous RNAs suggests that the plasmid expression of the target mRNAs did not cause Hfq to become a limiting factor. In addition, a recent study reveals that sRNAs can effectively gain access to Hfq even if it is already occupied by mRNAs in vivo ( Park et al, 2021 ), further arguing against Hfq becoming the limiting factor in our measurements.…”

“…Here, using single-molecule localization microscopy (SMLM), we confirmed the unbiased localization for endogenously expressed SgrS and RyhB ( Figures 7B – 7E ). In addition, the chaperone protein, Hfq, was observed to diffuse freely into the nucleoid region ( Park et al, 2021 ; Persson et al, 2013 ) and to bind to nascent transcripts ( Kambara et al, 2018 ). It is likely that at least part of the Hfq binding to the nascent transcripts is mediated by sRNAs.…”