Project description:The RNA chaperone Hfq is regarded as a critical effector promoting interaction between small regulatory RNAs (sRNAs) and cognate target mRNAs. While much of this interpretation is based on in vitro assays, no in vivo evidence actually exists to support this model. Here we report that Hfq is typically unnecessary for binding of various sRNA-mRNA complexes in vivo. Data obtained from pull-downs of MS2-tagged RyhB, RybB and DsrA sRNAs followed by RNAseq identification of target mRNAs suggest that absence of Hfq is not essential for sRNA-mRNA complex formation in vivo.
Project description:The RNA chaperone Hfq is regarded as a critical effector promoting interaction between small regulatory RNAs (sRNAs) and cognate target mRNAs. While much of this interpretation is based on in vitro assays, no in vivo evidence actually exists to support this model. Here we report that Hfq is typically unnecessary for binding of various sRNA-mRNA complexes in vivo. Data obtained from pull-downs of MS2-tagged RyhB, RybB and DsrA sRNAs followed by RNAseq identification of target mRNAs suggest that absence of Hfq is not essential for sRNA-mRNA complex formation in vivo.
Project description:The RNA chaperone Hfq is regarded as a critical effector promoting interaction between small regulatory RNAs (sRNAs) and cognate target mRNAs. While much of this interpretation is based on in vitro assays, no in vivo evidence actually exists to support this model. Here we report that Hfq is typically unnecessary for binding of various sRNA-mRNA complexes in vivo. Data obtained from pull-downs of MS2-tagged RyhB, RybB and DsrA sRNAs followed by RNAseq identification of target mRNAs suggest that absence of Hfq is not essential for sRNA-mRNA complex formation in vivo.
Project description:The Sm protein Hfq chaperones small non-coding RNAs (sRNAs) in bacteria, facilitating sRNA regulation of target mRNAs. Hfq acts in part by remodeling the sRNA and mRNA structures, yet the basis for this remodeling activity is not understood. To understand how Hfq remodels RNA, we used single-molecule Förster resonance energy transfer (smFRET) to monitor conformational changes in OxyS sRNA upon Hfq binding. The results show that E. coli Hfq first compacts OxyS, bringing its 5' and 3 ends together. Next, Hfq destabilizes an internal stem-loop in OxyS, allowing the RNA to adopt a more open conformation that is stabilized by a conserved arginine on the rim of Hfq. The frequency of transitions between compact and open conformations depend on interactions with Hfqs flexible C-terminal domain (CTD), being more rapid when the CTD is deleted, and slower when OxyS is bound to Caulobacter crescentus Hfq, which has a shorter and more stable CTD than E. coli Hfq. We propose that the CTDs gate transitions between OxyS conformations that are stabilized by interaction with one or more arginines. These results suggest a general model for how basic residues and intrinsically disordered regions of RNA chaperones act together to refold RNA.
Project description:Many bacteria use small RNAs (sRNAs) and the RNA chaperone Hfq to regulate mRNA stability and translation. Hfq, a ring-shaped homohexamer, has multiple faces that can bind both sRNAs and their mRNA targets. We find that Hfq has at least two distinct ways in which it interacts with sRNAs; these different binding properties have strong effects on the stability of the sRNA in vivo and the sequence requirements of regulated mRNAs. Class I sRNAs depend on proximal and rim Hfq sites for stability and turn over rapidly. Class II sRNAs are more stable and depend on the proximal and distal Hfq sites for stabilization. Using deletions and chimeras, we find that while Class I sRNAs regulate mRNA targets with previously defined ARN repeats, Class II sRNAs regulate mRNAs carrying UA-rich rim-binding sites. We discuss how these different binding modes may correlate with different roles in the cell, with Class I sRNAs acting as emergency responders and Class II sRNAs acting as silencers.
