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Interplay between 3′-to-5′ exoRNases and RNase Y in Streptococcus pyogenes
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Unit for the Science of Pathogens, D-10117 Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, D-10117 Berlin, Germany..
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2018 (English)Manuscript (preprint) (Other academic)
Place, publisher, year, edition, pages
2018.
National Category
Biochemistry and Molecular Biology Microbiology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-153182OAI: oai:DiVA.org:umu-153182DiVA, id: diva2:1261702
Available from: 2018-11-08 Created: 2018-11-08 Last updated: 2018-11-08
In thesis
1. Post-transcriptional regulation by RNases in Streptococcus pyogenes
Open this publication in new window or tab >>Post-transcriptional regulation by RNases in Streptococcus pyogenes
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ribonucleases (RNases) are proteins that adjust cellular RNA levels by processing RNA transcripts, leading to their stabilization or degradation. RNases are grouped based on their ability to cleave the transcript internally (endoRNases) or degrade the transcript starting from the ends (exoRNases). Specificities of RNA degradation vary among bacterial species, attributable to different sets of endo- and exoRNases. Most of the current knowledge gathered about the roles of RNases and their targets relies on the study of a few model bacteria, such as Escherichia coli and Bacillus subtilis. The aim of this thesis was to understand how Streptococcus pyogenes, a strict human pathogen, controls and adjusts gene expression by characterizing in vivo RNase activities.

The transcriptome of S. pyogenes was inspected to identify cleavages in vivo performed by RNases of interest using RNA sequencing. For this purpose, we developed a method to compare transcript 5′ and 3′ ends in RNase deletion mutants with those in the parental strain. We first applied our method for the study of endoRNase III, which cleaves ds RNA, and endoRNase Y, which is specific for ss RNA. We accurately retrieved RNase III cleavage positions in structured regions, characterized by 2 nucleotide (nt) 3′ overhangs, and we showed RNase III nicking activity in vivo. We observed that RNase Y processed transcripts after a guanosine. The upstream and downstream fragments generated by a single cleavage event were never both identified, indicating that RNase Y processing always led to the degradation of one of the two fragments. To investigate further the degradation of the upstream fragment subsequent to RNase Y processing, we characterized the 3′-to-5′ exoRNases R, YhaM, and PNPase. RNase R did not have any detectable activity in standard laboratory conditions. YhaM is an intriguing enzyme that removed on average 3 nt of the majority of cellular transcripts. PNPase fully degraded fragments originating from endoRNase processing and is the main 3′-to-5′ exoRNase involved in RNA decay in S. pyogenes.

To conclude, in this work, we developed a novel method to analyze RNA sequencing data. This method was successfully applied to the study of both endo- and exoRNases. Most importantly, we identified the targetomes of RNases III, Y, R, YhaM, and PNPase and we highlighted the distinctive features of these enzymes.

Place, publisher, year, edition, pages
Umeå: Umeå University, Department of Molecular Biology, 2018. p. 58
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1993
Keywords
Post-transcriptional regulation, mRNA decay, RNA sequencing, RNase Y, RNase III, YhaM, PNPase, RNase R, RNase J1, Streptococcus pyogenes.
National Category
Biochemistry and Molecular Biology Microbiology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-153176 (URN)978-91-7601-950-4 (ISBN)
Public defence
2018-12-06, Norrlands universitetssjukhus, Unod R1, Hörsal E04,, Umeå, 13:00 (English)
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Available from: 2018-11-13 Created: 2018-11-08 Last updated: 2018-11-16Bibliographically approved

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Lécrivain, Anne-LaureLe Rhun, AnaïsCharpentier, Emmanuelle

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Lécrivain, Anne-LaureLe Rhun, AnaïsCharpentier, Emmanuelle
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Molecular Infection Medicine Sweden (MIMS)Umeå Centre for Microbial Research (UCMR)Department of Molecular Biology (Faculty of Medicine)
Biochemistry and Molecular BiologyMicrobiology

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