| 1. |
- Andresen, Liis, et al.
(författare)
-
CLIP-Seq in Bacteria : Global Recognition Patterns of Bacterial RNA-Binding Proteins
- 2018
-
Ingår i: High-Density Sequencing Applications in Microbial Molecular Genetics. - : ELSEVIER ACADEMIC PRESS INC. - 9780128159934 ; , s. 127-145
-
Bokkapitel (refereegranskat)abstract
- RNA-protein interactions are at the heart of many central cellular processes, and RNA-binding proteins (RBPs) associate with virtually all RNA molecules in a cell. In bacteria, global RBPs, often in conjunction with small regulatory RNAs, affect physiology and virulence by controlling transcription, translation, and RNA decay. To understand how these regulatory proteins orchestrate global gene expression, detailed maps of their cellular RNA binding sites are required. To this end, cross-linking and immunoprecipitation followed by deep sequencing (CLIP-seq) has revolutionized RBP studies by providing knowledge about global recognition patterns of RBPs in both eukaryotic and bacterial cells. In this chapter, we provide a step-by-step protocol for global mapping of bona fide RBP binding sites using CLIP-seq in bacteria. This protocol has been successfully applied for charting the binding sites of Hfq, CsrA, and ProQ, three global regulatory RBPs in Salmonella enterica and Escherichia coli, and should be readily applicable to other RBPs and bacterial species.
|
|
| 2. |
- Gonzalez, Grecia M., et al.
(författare)
-
Structure of the Escherichia coli ProQ RNA-binding protein
- 2017
-
Ingår i: RNA. - : COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. - 1355-8382 .- 1469-9001. ; 23:5, s. 696-711
-
Tidskriftsartikel (refereegranskat)abstract
- The protein ProQ has recently been identified as a global small noncoding RNA-binding protein in Salmonella, and a similar role is anticipated for its numerous homologs in divergent bacterial species. We report the solution structure of Escherichia call 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 intradomain 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 modeled 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 recognize complex RNA structures and serve to promote RNA-mediated regulation.
|
|
| 3. |
- Hoekzema, Mirthe, et al.
(författare)
-
Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation
- 2019
-
Ingår i: EMBO Journal. - : EMBO Press. - 0261-4189 .- 1460-2075. ; 38:7
-
Tidskriftsartikel (refereegranskat)abstract
- Small RNAs post-transcriptionally regulate many processes inbacteria. Base-pairing of sRNAs near ribosome-binding sites inmRNAs inhibits translation, often requiring the RNA chaperoneHfq. In the canonical model, Hfq simultaneously binds sRNAs andmRNA targets to accelerate pairing. Here, we show that theEscher-ichia colisRNAs OmrA and OmrB inhibit translation of the diguany-late cyclase DgcM (previously: YdaM), a player in biofilmregulation. In OmrA/B repression ofdgcM, Hfq is not required as anRNA interaction platform, but rather unfolds an inhibitory RNAstructure that impedes OmrA/B binding. This restructuring involvesdistal face binding of Hfq and is supported by RNA structuremapping. A corresponding mutant protein cannot support inhibi-tionin vitroandin vivo; proximal and rim mutations have negligi-ble effects. Strikingly, OmrA/B-dependent translational inhibitionin vitrois restored, in complete absence of Hfq, by a deoxyoligori-bonucleotide that base-pairs to the biochemically mapped Hfq siteindgcMmRNA. We suggest that Hfq-dependent RNA structureremodeling can promote sRNA access, which represents a mecha-nism distinct from an interaction platform model.
|
|
| 4. |
- Holmqvist, Erik, 1977-, et al.
(författare)
-
Global Maps of ProQ Binding In Vivo Reveal Target Recognition via RNA Structure and Stability Control at mRNA 3 ' Ends
- 2018
-
Ingår i: Molecular Cell. - : CELL PRESS. - 1097-2765 .- 1097-4164. ; 70:5, s. 971-982
-
Tidskriftsartikel (refereegranskat)abstract
- The conserved RNA-binding protein ProQ has emerged as the centerpiece of a previously unknown third large network of post-transcriptional control in enterobacteria. Here, we have used in vivo UV cross-linking and RNA sequencing (CLIP-seq) to map hundreds of ProQ binding sites in Salmonella enterica and Escherichia coli. Our analysis of these binding sites, many of which are conserved, suggests that ProQ recognizes its cellular targets through RNA structural motifs found in small RNAs (sRNAs) and at the 3' end of mRNAs. Using the cspE mRNA as a model for 3' end targeting, we reveal a function for ProQ in protecting mRNA against exoribonucleolytic activity. Taken together, our results underpin the notion that ProQ governs a post-transcriptional network distinct from those of the well-characterized sRNA-binding proteins, CsrA and Hfq, and suggest a previously unrecognized, sRNA-independent role of ProQ in stabilizing mRNAs.
|
|
| 5. |
- Holmqvist, Erik, 1977-, et al.
(författare)
-
Impact of bacterial sRNAs in stress responses
- 2017
-
Ingår i: Biochemical Society Transactions. - 0300-5127 .- 1470-8752. ; 45, s. 1203-1212
-
Forskningsöversikt (refereegranskat)abstract
- Bacterial life is harsh and involves numerous environmental and internal challenges that are perceived as stresses. Consequently, adequate responses to survive, cope with, and counteract stress conditions have evolved. In the last few decades, a class of small, non-coding RNAs (sRNAs) has been shown to be involved as key players in stress responses. This review will discuss - primarily from an enterobacterial perspective - selected stress response pathways that involve antisense-type sRNAs. These include themes of how bacteria deal with severe envelope stress, threats of DNA damage, problems with poisoning due to toxic sugar intermediates, issues of iron homeostasis, and nutrient limitation/starvation. The examples discussed highlight how stress relief can be achieved, and how sRNAs act mechanistically in regulatory circuits. For some cases, we will propose scenarios that may suggest why contributions from post-transcriptional control by sRNAs, rather than transcriptional control alone, appear to be a beneficial and universally selected feature.
|
|
| 6. |
- Holmqvist, Erik, 1977-, et al.
(författare)
-
RNA-binding proteins in bacteria
- 2018
-
Ingår i: Nature Reviews Microbiology. - : Nature Publishing Group. - 1740-1526 .- 1740-1534. ; 16:10, s. 601-615
-
Forskningsöversikt (refereegranskat)abstract
- RNA-binding proteins (RBPs) are central to most if not all cellular processes, dictating the fate of virtually all RNA molecules in the cell. Starting with pioneering work on ribosomal proteins, studies of bacterial RBPs have paved the way for molecular studies of RNA-protein interactions. Work over the years has identified major RBPs that act on cellular transcripts at the various stages of bacterial gene expression and that enable their integration into post-transcriptional networks that also comprise small non-coding RNAs. Bacterial RBP research has now entered a new era in which RNA sequencing-based methods permit mapping of RBP activity in a truly global manner in vivo. Moreover, the soaring interest in understudied members of host-associated microbiota and environmental communities is likely to unveil new RBPs and to greatly expand our knowledge of RNA-protein interactions in bacteria.
|
|
