Abstract
In all bacterial species examined thus far, small regulatory RNAs (sRNAs) contribute to intricate patterns of dynamic genetic regulation. Many of the actions of these nucleic acids are mediated by well-characterized chaperones such as the Hfq protein, but genetic screens have also recently identified the 3′-to-5′ exoribonuclease polynucleotide phosphorylase (PNPase) as an unexpected stabilizer and facilitator of sRNAs in vivo. To understand how a ribonuclease might mediate these effects, we tested the interactions of PNPase with sRNAs and found that the enzyme can readily degrade these nucleic acids in vitro but, nonetheless, copurifies from cell extracts with the same sRNAs without discernible degradation or modification to their 3′ ends, suggesting that the associated RNA is protected against the destructive activity of the ribonuclease. In vitro, PNPase, Hfq, and sRNA can form a ternary complex in which the ribonuclease plays a nondestructive, structural role. Such ternary complexes might be formed transiently in vivo, but could help to stabilize particular sRNAs and remodel their population on Hfq. Taken together, our results indicate that PNPase can be programmed to act on RNA in either destructive or stabilizing modes in vivo and may form complex, protective ribonucleoprotein assemblies that shape the landscape of sRNAs available for action.
Highlights
Small noncoding regulatory RNAs are versatile regulators of gene expression throughout all branches of life (Wagner and Romby 2015)
We demonstrate that E. coli polynucleotide phosphorylase (PNPase) can sequester some small regulatory RNAs (sRNAs) without degrading them, and that PNPase impacts the distribution of sRNAs associated with Hfq in vivo
We explored if sRNAs dependent on PNPase for stability are physically associated with this protein in vivo, using an affinity-tagged enzyme
Summary
Small noncoding regulatory RNAs (sRNAs) are versatile regulators of gene expression throughout all branches of life (Wagner and Romby 2015). These molecules can change gene expression by altering mRNA stability and/or translational efficiency, and they achieve specificity for defined transcripts through complementary base-pairing (Thomason and Storz 2010; Pasquinelli 2012; De Lay et al 2013). Two nucleic acid binding domains at the C terminus, belonging to the conserved KH and S1 families, decorate the entrance to a central pore Once engaged by those domains, the RNA threads into the central pore to one of the active sites; there, its 3′ terminal phosphodiester bond can be cleaved by a phosphate molecule orientated for attack. Previous in vitro studies of the E. coli PNPase have shown that the S1 and KH domains and the core play important roles in substrate capture (Stickney et al 2005; MatusOrtega et al 2007; Wong et al 2013), and recent evidence supports a model for S1 contributing to PNPase auto-regulation by binding the 5′ UTR and repressing translation (Robert-Le Meur and Portier 1994; Carzaniga et al 2015)
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