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RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Helmholtz Centre for Infection Research (HZI), Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany.ORCID iD: 0000-0003-2211-2153
Helmholtz Centre for Infection Research (HZI), Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany.
Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, S-75003 Uppsala, Sweden.
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Max F. Perutz Laboratories (MFPL), University of Vienna, A-1030 Vienna, Austria.
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2016 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 13, no 2, 177-195 p.Article in journal (Refereed) Published
Abstract [en]

Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

Place, publisher, year, edition, pages
2016. Vol. 13, no 2, 177-195 p.
Keyword [en]
RNA sequencing, Streptococcus pyogenes, small RNAs, antisense RNAs, riboswitches, T-boxes, leader RNAs, gene expression regulation
National Category
URN: urn:nbn:se:umu:diva-111088DOI: 10.1080/15476286.2015.1110674ISI: 000371745100010PubMedID: 26580233OAI: diva2:868027

Originally published in manuscript form.

Available from: 2015-11-09 Created: 2015-11-04 Last updated: 2016-05-16Bibliographically approved
In thesis
1. Multifaceted RNA-mediated regulatory mechanisms in Streptococcus pyogenes
Open this publication in new window or tab >>Multifaceted RNA-mediated regulatory mechanisms in Streptococcus pyogenes
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bacterial pathogens rely on precise regulation of gene expression to coordinate host infection processes and resist invasion by mobile genetic elements. An interconnected network of protein and RNA regulators dynamically controls the expression of virulence factors using a variety of mechanisms. In this thesis, the role of selected regulators, belonging to the class of small RNAs (sRNAs), is investigated.

Streptococcus pyogenes is a pathogen responsible for a wide range of human diseases. Genome-wide screenings have indicated that S. pyogenes encodes numerous sRNAs, yet only a limited number have been characterized. A major goal of this study was to identify and characterize novel sRNAs and antisense RNAs (asRNAs) using RNA sequencing analysis. We validated 30 novel sRNAs and asRNAs, and identified 9 sRNAs directly cleaved by the ribonucleases RNase III and/or RNase Y.

Previous work from the laboratory has highlighted the role of sRNAs from the type II Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) systems in S. pyogenes. CRISPR-Cas systems provide adaptive immunity to prokaryotes against infection by mobile genetic elements. Two sRNAs, forming a complementary duplex (dual-RNA), are effectors of this system: the mature CRISPR RNAs (crRNAs) and the trans-activating crRNA (tracrRNA). The dual-RNA guides the Cas9 endonuclease to cleave both strands of the invading DNA in a sequence-specific manner. This RNA-programmable CRISPR-Cas9 system is now utilized for genome editing and engineering in a wide range of cells and organisms. To expand the potentialities of this tool, we both, searched for Cas9 orthologs and predicted numerous tracrRNA orthologs. We defined tracrRNA as a new family of sRNAs sharing the ability to base-pair to cognate crRNAs, without conservation of structure, sequence or location. We show that Cas9 and the dual tracrRNA:crRNAs are only interchangeable between closely related type II CRISPR-Cas systems.

In summary, this thesis presents new insights into RNA-mediated regulatory mechanisms in S. pyogenes. We identified and described the expression of novel sRNAs, highlighting potential antisense RNAs. Focusing on the dual-RNA programmable type II CRISPR-Cas system, we provided evidence for co-evolution of the Cas9 enzyme with tracrRNA:crRNA, a basis for Cas9 multiplexing in genome editing.

Place, publisher, year, edition, pages
Umeå: Umeå university, 2015. 75 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1732
Streptococcus pyogenes, small RNAs, CRISPR, Cas9, tracrRNA, RNases, gene expression, RNA sequencing
National Category
Microbiology Biochemistry and Molecular Biology
Research subject
Infectious Diseases; Microbiology
urn:nbn:se:umu:diva-111090 (URN)978-91-7601-304-5 (ISBN)
Public defence
2015-12-14, Unod R1, Hörsal E04, Byggnad 6E, NUS - Norrlands universitetssjukhus, Umeå, 09:00 (English)
Available from: 2015-11-10 Created: 2015-11-04 Last updated: 2015-11-09Bibliographically approved

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