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Post-transcriptional regulation by RNases 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).ORCID iD: 0000-0001-8957-3857
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 [en]
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: urn:nbn:se:umu:diva-153176ISBN: 978-91-7601-950-4 (print)OAI: oai:DiVA.org:umu-153176DiVA, id: diva2:1261772
Public defence
2018-12-06, Norrlands universitetssjukhus, Unod R1, Hörsal E04,, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2018-11-13 Created: 2018-11-08 Last updated: 2018-11-16Bibliographically approved
List of papers
1. Identification of endoribonuclease specific cleavage positions reveals novel targets of RNase III in Streptococcus pyogenes
Open this publication in new window or tab >>Identification of endoribonuclease specific cleavage positions reveals novel targets of RNase III in Streptococcus pyogenes
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2017 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 5, p. 2329-2340Article in journal (Refereed) Published
Abstract [en]

A better understanding of transcriptional and post-transcriptional regulation of gene expression in bacteria relies on studying their transcriptome. RNA sequencing methods are used not only to assess RNA abundance but also the exact boundaries of primary and processed transcripts. Here, we developed a method, called identification of specific cleavage position (ISCP), which enables the identification of direct endoribonuclease targets in vivo by comparing the 5' and 3' ends of processed transcripts between wild type and RNase deficient strains. To demonstrate the ISCP method, we used as a model the double-stranded specific RNase III in the human pathogen Streptococcus pyogenes. We mapped 92 specific cleavage positions (SCPs) among which, 48 were previously described and 44 are new, with the characteristic 2 nucleotides 3' overhang of RNase III. Most SCPs were located in untranslated regions of RNAs. We screened for RNase III targets using transcriptomic differential expression analysis (DEA) and compared those with the RNase III targets identified using the ISCP method. Our study shows that in S. pyogenes, under standard growth conditions, RNase III has a limited impact both on antisense transcripts and on global gene expression with the expression of most of the affected genes being downregulated in an RNase III deletion mutant.

National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-133755 (URN)10.1093/nar/gkw1316 (DOI)000397286600018 ()
Available from: 2017-05-05 Created: 2017-05-05 Last updated: 2018-11-08Bibliographically approved
2. In vivo 3′-to-5′ exoribonuclease targetomes of Streptococcus pyogenes
Open this publication in new window or tab >>In vivo 3′-to-5′ exoribonuclease targetomes of Streptococcus pyogenes
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2018 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 46, p. 11814-11819Article in journal (Refereed) Published
Abstract [en]

mRNA decay plays an essential role in the control of gene expression in bacteria. Exoribonucleases (exoRNases), which trim transcripts starting from the 5′ or 3′ end, are particularly important to fully degrade unwanted transcripts and renew the pool of nucleotides available in the cell. While recent techniques have allowed genome-wide identification of ribonuclease (RNase) targets in bacteria in vivo, none of the 3′-to-5′ exoRNase targetomes (i.e., global processing sites) have been studied so far. Here, we report the targetomes of YhaM, polynucleotide phosphorylase (PNPase), and RNase R of the human pathogen Streptococcus pyogenes. We determined that YhaM is an unspecific enzyme that trims a few nucleotides and targets the majority of transcript ends, generated either by transcription termination or by endonucleolytic activity. The molecular determinants for YhaM-limited processivity are yet to be deciphered. We showed that PNPase clears the cell from mRNA decay fragments produced by endoribonucleases (endoRNases) and is the major 3′-to-5′ exoRNase for RNA turnover in S. pyogenes. In particular, PNPase is responsible for the degradation of regulatory elements from 5′ untranslated regions. However, we observed little RNase R activity in standard culture conditions. Overall, our study sheds light on the very distinct features of S. pyogenes 3′-to-5′ exoRNases.

Place, publisher, year, edition, pages
National Academy of Sciences, 2018
Keywords
3′-to-5′ exoRNase, 5′-end sequencing, 3′-end sequencing, Streptococcus pyogenes, RNA degradation
National Category
Microbiology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-153170 (URN)10.1073/pnas.1809663115 (DOI)000449934400052 ()30381461 (PubMedID)
Available from: 2018-11-08 Created: 2018-11-08 Last updated: 2018-12-17Bibliographically approved
3. Interplay between 3′-to-5′ exoRNases and RNase Y in Streptococcus pyogenes
Open this publication in new window or tab >>Interplay between 3′-to-5′ exoRNases and RNase Y in Streptococcus pyogenes
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2018 (English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Microbiology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-153182 (URN)
Available from: 2018-11-08 Created: 2018-11-08 Last updated: 2018-11-08

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Lécrivain, Anne-Laure

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Molecular Infection Medicine Sweden (MIMS)Umeå Centre for Microbial Research (UCMR)Department of Molecular Biology (Faculty of Medicine)
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