PhD student, Monash University
We would like to determine the role of ProQ in sRNA regulation and RNA binding.
Pasteurella multocida is the causative agent of many diseases, including; fowl cholera in chickens. Riboregulation is an important mechanism by which bacteria regulate transcript abundance and protein production. This mechanism involves small RNA molecules binding to mRNA transcripts to alter transcript stability and/or translational efficiency. Hfq is a well-characterized RNA chaperone that is required for many of these sRNA/mRNA interactions. Recently, ProQ has been identified as a second RNA chaperone that plays a critical role in stabilizing some sRNA/mRNA interactions. To assess the role of ProQ in P. multocida, we constructed a proQ mutant strain by TargeTron® insertional mutagenesis. The proteome of the proQ mutant was compared with the wild-type VP161 strain using high-throughput liquid proteomics, where 21 proteins showed differential production in the proQ mutant. Of these; seven proteins showed increased production, 14 showed decreased production, and 11 of the 21 were involved in transport or metabolism of amino acids, lipids or carbohydrates. The transcriptome of the proQ mutant was also analysed using RNA-seq; 35 transcripts showed increased expression and 96 showed decreased expression. Of these, 18 were predicted sRNAs. Direct interaction between ProQ and two sRNAs of this group (Prrc10 and Prrc37) and a further three sRNAs (Prrc14, Prrc13, and Prrc02), was confirmed using UV-CLASH (UV-crosslinking, ligation, and sequencing of hybrids). The hfq gene showed decreased expression in the proQ mutant, while 17 tRNA genes showed increased expression. Thus, P. multocida ProQ may be a master regulator of genes/proteins involved in the regulation of protein production.
Abstract: The functional annotation of transcriptomes and identification of noncoding RNA (ncRNA) classes has been greatly facilitated by the advent of next-generation RNA sequencing which, by reading the nucleotide order of transcripts, theoretically allows the rapid profiling of all transcripts in a cell. However, primary sequence per se is a poor predictor of function, as ncRNAs dramatically vary in length and structure and often lack identifiable motifs. Therefore, to visualize an informative RNA landscape of organisms with potentially new RNA biology that are emerging from microbiome and environmental studies requires the use of more functionally relevant criteria. One such criterion is the association of RNAs with functionally important cognate RNA-binding proteins. Here we analyze the full ensemble of cellular RNAs using gradient profiling by sequencing (Grad-seq) in the bacterial pathogen Salmonella enterica, partitioning its coding and noncoding transcripts based on their network of RNA–protein interactions. In addition to capturing established RNA classes based on their biochemical profiles, the Grad-seq approach enabled the discovery of an overlooked large collective of structured small RNAs that form stable complexes with the conserved protein ProQ. We show that ProQ is an abundant RNA-binding protein with a wide range of ligands and a global influence on Salmonella gene expression. Given its generic ability to chart a functional RNA landscape irrespective of transcript length and sequence diversity, Grad-seq promises to define functional RNA classes and major RNA-binding proteins in both model species and genetically intractable organisms.
Pub.: 26 Sep '16, Pinned: 25 Aug '17
Abstract: The stability and function of regulatory small RNAs (sRNAs) often require a specialized RNA-binding protein called an RNA chaperone. Recent findings show that proteins containing a ProQ/FinO domain constitute a new class of RNA chaperones that could play key roles in post-transcriptional gene regulation throughout bacterial species.
Pub.: 13 Feb '17, Pinned: 25 Aug '17
Abstract: The protein ProQ has recently been identified as a global RNA chaperone in Salmonella, and a similar role is anticipated for its numerous homologues in divergent bacterial species. We report the solution structure of Escherichia coli ProQ, revealing an N-terminal FinO-like domain, a C-terminal domain that unexpectedly has a Tudor-domain fold commonly found in eukaryotes, and an elongated bridging intra-domain linker that is flexible but nonetheless incompressible. Structure based sequence analysis suggests that the Tudor domain was acquired through horizontal gene transfer and gene fusion to the ancestral FinO-like domain. Through a combination of biochemical and biophysical approaches, we have mapped putative RNA binding surfaces on all three domains of ProQ and modelled the protein's conformation in the apo and RNA-bound forms. Taken together, these data suggest how the FinO, Tudor and linker domains of ProQ cooperate to recognise complex RNA structures and serve to promote RNA-mediated regulation.
Pub.: 15 Feb '17, Pinned: 25 Aug '17
Abstract: Research into post-transcriptional control of mRNAs by small noncoding RNAs (sRNAs) in the model bacteria Escherichia coli and Salmonella enterica has mainly focused on sRNAs that associate with the RNA chaperone Hfq. However, the recent discovery of the protein ProQ as a common binding partner that stabilizes a distinct large class of structured sRNAs suggests that additional RNA regulons exist in these organisms. The cellular functions and molecular mechanisms of these new ProQ-dependent sRNAs are largely unknown. Here, we report in Salmonella Typhimurium the mode-of-action of RaiZ, a ProQ-dependent sRNA that is made from the 3' end of the mRNA encoding ribosome-inactivating protein RaiA. We show that RaiZ is a base-pairing sRNA that represses in trans the mRNA of histone-like protein HU-α. RaiZ forms an RNA duplex with the ribosome-binding site of hupA mRNA, facilitated by ProQ, to prevent 30S ribosome loading and protein synthesis of HU-α. Similarities and differences between ProQ- and Hfq-mediated regulation will be discussed.
Pub.: 25 Mar '17, Pinned: 25 Aug '17
Abstract: Small RNAs (sRNAs), particularly those that act by limited base pairing with mRNAs, are part of most regulatory networks in bacteria. In many cases, the base-pairing interaction is facilitated by the RNA chaperone Hfq. However, not all bacteria encode Hfq and some base-pairing sRNAs do not require Hfq raising the possibility of other RNA chaperones. Candidates are proteins with homology to FinO, a factor that promotes base pairing between the FinP antisense sRNA and the traJ mRNA to control F plasmid transfer. Recent papers have shown that the Salmonella enterica FinO-domain protein ProQ binds a large suite of sRNAs, including the RaiZ sRNA, which represses translation of the hupA mRNA, and the Legionella pneumophila protein RocC binds the RocR sRNA, which blocks expression of competence genes. Here we discuss what is known about FinO-domain structures, including the recently solved Escherichia coli ProQ structure, as well as the RNA binding properties of this family of proteins and evidence they act as chaperones. We compare these properties with those of Hfq. We further summarize what is known about the physiological roles of FinO-domain proteins and enumerate outstanding questions whose answers will establish whether they constitute a second major class of RNA chaperones. This article is protected by copyright. All rights reserved.
Pub.: 04 Apr '17, Pinned: 25 Aug '17
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