ORCID Profile
0000-0002-7924-2744
Current Organisation
The University of Edinburgh
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Publisher: EMBO
Date: 11-11-2016
Abstract: RNA sequencing studies have identified hundreds of non‐coding RNA s in bacteria, including regulatory small RNA ( sRNA ). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high‐throughput analysis of RNA – RNA interactions in bacteria. Here we demonstrate that in vivo sRNA – mRNA duplexes can be recovered using UV ‐crosslinking, ligation and sequencing of hybrids ( CLASH ). Many sRNA s recruit the endoribonuclease, RN ase E, to facilitate processing of mRNA s. We were able to recover base‐paired sRNA – mRNA duplexes in association with RN ase E, allowing proximity‐dependent ligation and sequencing of cognate sRNA – mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA – mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co‐regulated target mRNA s. We identified multiple mRNA targets for the pathotype‐specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli . Numerous sRNA interactions were also identified with non‐coding RNA s, including sRNA s and tRNA s, demonstrating the high complexity of the sRNA interactome.
Publisher: Springer Science and Business Media LLC
Date: 10-09-2018
Publisher: Springer Science and Business Media LLC
Date: 22-06-2022
DOI: 10.1038/S41467-022-31173-Y
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen responsible for significant human morbidity and mortality. Post-transcriptional regulation by small RNAs (sRNAs) has emerged as an important mechanism for controlling virulence. However, the functionality of the majority of sRNAs during infection is unknown. To address this, we performed UV cross-linking, ligation, and sequencing of hybrids (CLASH) in MRSA to identify sRNA-RNA interactions under conditions that mimic the host environment. Using a double-stranded endoribonuclease III as bait, we uncovered hundreds of novel sRNA-RNA pairs. Strikingly, our results suggest that the production of small membrane-permeabilizing toxins is under extensive sRNA-mediated regulation and that their expression is intimately connected to metabolism. Additionally, we also uncover an sRNA sponging interaction between RsaE and RsaI. Taken together, we present a comprehensive analysis of sRNA-target interactions in MRSA and provide details on how these contribute to the control of virulence in response to changes in metabolism.
Publisher: Proceedings of the National Academy of Sciences
Date: 19-12-2011
Abstract: In idual microRNAs (miRNAs) are rapidly down-regulated during conditions of cellular activation and infection, but factors mediating miRNA turnover are poorly understood. Infection of mouse cells with murine cytomegalovirus (MCMV) induces the rapid down-regulation of an antiviral cellular miRNA, miR-27. Here, we identify a transcript produced by MCMV that binds to miR-27 and mediates its degradation. UV-crosslinking and high-throughput sequencing [CRAC (UV-crosslinking and analysis of cDNA)] identified MCMV RNA segments associated with the miRNA-binding protein Argonaute 2 (Ago2). A cluster of hits mapped to a predicted miR-27-binding site in the 3′UTR of the previously uncharacterized ORF, m169. The expression kinetics of the m169 transcript correlated with degradation of miR-27 during infection, and m169 expression inhibited miR-27 functional activity in a reporter assay. siRNA knockdown of m169 demonstrated its requirement for miR-27 degradation following infection and did not affect other host miRNAs. Substitution of the miR-27-binding site in m169 to create complementarity to a different cellular miRNA, miR-24, resulted in down-regulation of only miR-24 following infection. The m169 transcript is cytoplasmic, capped, polyadenylated, and interacts with miRNA-27 through seed pairing: characteristic features of the normal messenger RNA (mRNA) targets of miRNAs. This virus–host interaction reveals a mode of miRNA regulation in which a mRNA directs the degradation of a miRNA. We speculate that RNA-mediated miRNA degradation could be a more general viral strategy for manipulating host cells.
Publisher: Cold Spring Harbor Laboratory
Date: 17-05-2021
DOI: 10.1101/2021.05.13.444021
Abstract: RNA homodimerization is important for various physiological processes, including the assembly of membraneless organelles, RNA subcellular localization, and packaging of viral genomes. However, understanding of RNA homodimerization has been h ered by the lack of systematic in vivo detection methods. Here we show that PARIS, COMRADES, and other RNA proximity ligation methods can detect RNA homodimers transcriptome-wide as “overlapping” chimeric reads that contain more than one copy of the same sequence. Analysing published proximity ligation datasets, we show that RNA:RNA homodimers mediated by direct base-pairing interactions are rare across the transcriptome, but highly enriched in specific transcripts, including U8 snoRNA, U2 snRNA and a subset of tRNAs. Analysis of data from virus-infected cells reveals homodimerization of SARS-CoV-2 and Zika genomes, mediated by specific palindromic sequences located within protein-coding regions of N protein in SARS-CoV-2 and NS2A gene in Zika. We speculate that regions of viral genomes involved in homodimerization may constitute effective targets for antiviral therapies.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Grzegorz Kudla.