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In vivo 3′-to-5′ exoribonuclease targetomes of 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, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; nstitute for Biology, Humboldt University, Berlin, Germany .
Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
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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. Vol. 115, no 46, p. 11814-11819
Keywords [en]
3′-to-5′ exoRNase, 5′-end sequencing, 3′-end sequencing, Streptococcus pyogenes, RNA degradation
National Category
Microbiology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-153170DOI: 10.1073/pnas.1809663115ISI: 000449934400052PubMedID: 30381461OAI: oai:DiVA.org:umu-153170DiVA, id: diva2:1261620
Available from: 2018-11-08 Created: 2018-11-08 Last updated: 2018-12-17Bibliographically approved
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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Supervisors
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)
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