Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

Mouali

Kuper

et al. 2020

RNA

RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq, and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinOdomain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalog of microbial RBPs.

Section: Discussionmentioning

confidence: 99%

Section: A Candidate Fino-domain Rbpmentioning

confidence: 99%

Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein

Mouali

Kuper

et al. 2020

RNA

“…Their role as new RNA matchmakers, mechanistically distinct from Hfq, is being intensively studied, but the molecular details of the interplay between ProQ and its natural RNA ligands only begin to emerge ( Melamed et al, 2020 ). Structural and functional studies highlighted the importance of the conserved N-terminal FinO-like domain, which harbors most of the RNA-binding activity and places ProQ into a larger family of FinO-like RNA chaperones ( Chaulk et al, 2010 , 2011 ; Glover et al, 2015 ; Gonzalez et al, 2017b ; Eidelpes et al, 2020 ; Gerovac et al, 2020 , 2021 ; Immer et al, 2020 ; Pandey et al, 2020 ; Stein et al, 2020 ). However, the role of the C-terminal “Tudor-like” domain ( Figure 6 ), that contributes to the RNA chaperone activities of ProQ ( Chaulk et al, 2011 ; Gonzalez et al, 2017b ), does not cease to intrigue researchers.…”

Section: Profiling Stable Rnps With Grad-seqmentioning

confidence: 99%

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

Vogel

Smirnov

2021

Front. Mol. Biosci.

Macromolecular complexes of proteins and RNAs are essential building blocks of cells. These stable supramolecular particles can be viewed as minimal biochemical units whose structural organization, i.e., the way the RNA and the protein interact with each other, is directly linked to their biological function. Whether those are dynamic regulatory ribonucleoproteins (RNPs) or integrated molecular machines involved in gene expression, the comprehensive knowledge of these units is critical to our understanding of key molecular mechanisms and cell physiology phenomena. Such is the goal of diverse complexomic approaches and in particular of the recently developed gradient profiling by sequencing (Grad-seq). By separating cellular protein and RNA complexes on a density gradient and quantifying their distributions genome-wide by mass spectrometry and deep sequencing, Grad-seq charts global landscapes of native macromolecular assemblies. In this review, we propose a function-based ontology of stable RNPs and discuss how Grad-seq and related approaches transformed our perspective of bacterial and eukaryotic ribonucleoproteins by guiding the discovery of new RNA-binding proteins and unusual classes of noncoding RNAs. We highlight some methodological aspects and developments that permit to further boost the power of this technique and to look for exciting new biology in understudied and challenging biological models.

“…Acting as an RNA chaperone, RocC uses its FinO domain to recognize an inhibitory sRNA (RocR) and several trans -encoded target mRNAs in the competence regulon ( 10 , 11 ). From these and other recent functional studies of ProQ ( 12 , 13 ) or ProQ-associated sRNAs ( 14 , 15 ), a working model has emerged whereby the FinO domain enables these RBPs to act in a transcriptome-wide manner to impact on various aspects of bacterial physiology. Whether the FinO domain recognizes a specific nucleotide sequence or structural element in these many cellular targets is currently unclear; no informative consensus binding motif could be extracted from the large target suite of Salmonella ProQ ( 7 ).…”

Section: Introductionmentioning

confidence: 99%

“…As efforts to understand the molecular basis of RNA target recognition and selectivity by FinO domain proteins gain new momentum ( 12 , 13 , 29 , 30 ), we must also determine the actual target suite of FinO itself. Based on early genetics ( 17 , 31 ), FinP and traJ have been regarded as the primary cellular ligands of FinO, yet this assumption has not been tested with global methods as used for FopA, ProQ or RocC ( 4 , 6 , 10 ).…”

Section: Introductionmentioning

confidence: 99%

In vivotargets ofSalmonellaFinO include a FinP-like small RNA controlling copy number of a cohabitating plasmid

Mouali

Mineikaitė

et al. 2021

Nucleic Acids Research

FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.