The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and Escherichia coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5′ and 3′ untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ’s interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ’s NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an RNA duplex.

The sequence-specific RNA-binding proteins polypyrimidine tract–binding protein 1 (PTBP1) and heterogeneous nuclear ribonucleoprotein L (HNRNPL) protect mRNAs from nonsense-mediated decay (NMD) by preventing the UPF1 RNA helicase from associating with potential decay targets. Here, by analyzing in vitro helicase activity, dissociation of UPF1 from purified mRNPs, and transcriptome-wide UPF1 RNA binding, we present the mechanistic basis for inhibition of NMD by PTBP1. Unlike mechanisms of RNA stabilization that depend on direct competition for binding sites among protective RNA-binding proteins and decay factors, PTBP1 promotes displacement of UPF1 already bound to potential substrates. Our results show that PTBP1 directly exploits the tendency of UPF1 to release RNA upon ATP binding and hydrolysis. We further find that UPF1 sensitivity to PTBP1 is coordinated by a regulatory loop in domain 1B of UPF1. We propose that the UPF1 regulatory loop and protective proteins control kinetic proofreading of potential NMD substrates, presenting a new model for RNA helicase regulation and target selection in the NMD pathway.

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and E. coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein interacting with many sRNAs and mRNAs, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs.Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an A-form RNA duplex. INTRODUCTION Regulatory, small RNAs (sRNAs) are found in nearly all bacterial species and implicated in important processes such as virulence, biofilm formation, host interactions and antibiotic resistance.(1-3) These sRNAs typically regulate messenger RNA (mRNA) translation through imperfect base pairing near an mRNA's ribosomal binding site.(2, 4-6) In many bacterial species, the stability and function of sRNAs are supported by global RNA-binding proteins, such as the protein Hfq.(1, 4, 7-9) Given that Hfq is not present in all bacterial species and that not all sRNAs depend on Hfq for their function, there is increasing interest in other RNA-binding proteins that may play a role in global gene-regulation in bacteria,(2, 10-13) including a class of proteins that contain FinO domains.(14-17) The Escherichia coli protein FinO is the founding member of the FinO structural class of RNA-binding proteins. In E. coli, FinO binds the FinP sRNA and regulates the 5´ untranslated region (UTR) of traJ.(18, 19) Similarly, Legionella pneumophila RocC contains a FinO-domain and binds the sRNA RocR along with at least four 5' UTRs of mRNAs involved in competence.(20) In E. coli, another FinO-domain-containing protein called ProQ was initially characterized as an RNA-binding protein contributing to osmoregulation through expression of proP.(21) ProQ was recently identified through Grad-Seq experiments to bind to dozens of cellular RNAs,(17) including a large number of sRNAs and mRNA 3'UTRs in Samonella and E. coli.(22) ProQ binding has been shown to regulate mRNA-expression levels through interactions with both 5' and 3' UTRs. It has been shown to form a ternary complex with an sRNA (RaiZ) and an mRNA (hupA), to support RaiZ's repression of hupA,(23) and to protect mRNAs from exonucleolytic degradation by binding to 3' ends.(22) Further, ProQ supports the sRNA SraL in preventin...

The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machinery and the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (<200 nt) in physiological contexts and undergoes ATPase-stimulated dissociation from RNA. We combine an in silico screen with these assays to identify and characterize known and novel UPF1 mutants with altered helicase, ATPase, and RNA binding properties. We find that UPF1 mutants with substantially impaired processivity (E797R, G619K/A546H), faster (G619K) or slower (K547P, E797R, G619K/A546H) unwinding rates, and/or reduced mechanochemical coupling (i.e. the ability to harness ATP hydrolysis for work; K547P, R549S, G619K, G619K/A546H) can still support efficient NMD of well-characterized targets in human cells. These data are consistent with a central role for UPF1 ATPase activity in driving cycles of RNA binding and dissociation to ensure accurate NMD target selection.