A pinboard by
Emily Gulliver

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.


Grad-seq guides the discovery of ProQ as a major small RNA-binding protein

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