Change search
ReferencesLink to record
Permanent link

Direct link
Multifaceted RNA-mediated regulatory mechanisms in Streptococcus pyogenes
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research (HZI), Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany.ORCID iD: 0000-0003-2211-2153
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
Keyword [en]
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: urn:nbn:se:umu:diva-111090ISBN: 978-91-7601-304-5OAI: diva2:868035
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
List of papers
1. RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes
Open this publication in new window or tab >>RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes
Show others...
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.

RNA sequencing, Streptococcus pyogenes, small RNAs, antisense RNAs, riboswitches, T-boxes, leader RNAs, gene expression regulation
National Category
urn:nbn:se:umu:diva-111088 (URN)10.1080/15476286.2015.1110674 (DOI)000371745100010 ()26580233 (PubMedID)

Originally published in manuscript form.

Available from: 2015-11-09 Created: 2015-11-04 Last updated: 2016-05-16Bibliographically approved
2. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems
Open this publication in new window or tab >>The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems
2013 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 10, no 5, 726-737 p.Article in journal (Refereed) Published
Abstract [en]

CRISPR-Cas is a rapidly evolving RNA-mediated adaptive immune system that protects bacteria and archaea against mobile genetic elements. The system relies on the activity of short mature CRISPR RNAs (crRNAs) that guide Cas protein(s) to silence invading nucleic acids. A set of CRISPR-Cas, type II, requires a trans-activating small RNA, tracrRNA, for maturation of precursor crRNA (pre-crRNA) and interference with invading sequences. Following co-processing of tracrRNA and pre-crRNA by RNase III, dual-tracrRNA:crRNA guides the CRISPR-associated endonuclease Cas9 (Csn1) to cleave site-specifically cognate target DNA. Here, we screened available genomes for type II CRISPR-Cas loci by searching for Cas9 orthologs. We analyzed 75 representative loci, and for 56 of them we predicted novel tracrRNA orthologs. Our analysis demonstrates a high diversity in cas operon architecture and position of the tracrRNA gene within CRISPR-Cas loci. We observed a correlation between locus heterogeneity and Cas9 sequence diversity, resulting in the identification of various type II CRISPR-Cas subgroups. We validated the expression and co-processing of predicted tracrRNAs and pre-crRNAs by RNA sequencing in five bacterial species. This study reveals tracrRNA family as an atypical, small RNA family with no obvious conservation of structure, sequence or localization within type II CRISPR-Cas loci. The tracrRNA family is however characterized by the conserved feature to base-pair to cognate pre-crRNA repeats, an essential function for crRNA maturation and DNA silencing by dual-RNA:Cas9. The large panel of tracrRNA and Cas9 ortholog sequences should constitute a useful database to improve the design of RNA-programmable Cas9 as genome editing tool.

Place, publisher, year, edition, pages
Landes Bioscience, 2013
tracrRNA, CRISPR-Cas, type II system, Cas9 (Csn1), RNA processing, RNA maturation, small non-coding RNA, bacteria, adaptive immunity, mobile genetic elements
National Category
Cell and Molecular Biology
urn:nbn:se:umu:diva-80075 (URN)10.4161/rna.24321 (DOI)000323176900011 ()
Available from: 2013-09-09 Created: 2013-09-09 Last updated: 2015-11-09Bibliographically approved
3. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems
Open this publication in new window or tab >>Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems
Show others...
2014 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 4, 2577-2590 p.Article in journal (Refereed) Published
Abstract [en]

The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA: crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA: Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

National Category
Biochemistry and Molecular Biology
urn:nbn:se:umu:diva-87653 (URN)10.1093/nar/gkt1074 (DOI)000332381000048 ()
Available from: 2014-04-08 Created: 2014-04-07 Last updated: 2015-11-09Bibliographically approved

Open Access in DiVA

fulltext(2706 kB)256 downloads
File information
File name FULLTEXT01.pdfFile size 2706 kBChecksum SHA-512
Type fulltextMimetype application/pdf
cover(2117 kB)31 downloads
File information
File name COVER01.pdfFile size 2117 kBChecksum SHA-512
Type coverMimetype application/pdf
spikblad(21 kB)2 downloads
File information
File name SPIKBLAD01.pdfFile size 21 kBChecksum SHA-512
Type spikbladMimetype application/pdf

Search in DiVA

By author/editor
Le Rhun, Anaïs
By organisation
Molecular Infection Medicine Sweden (MIMS)Department of Molecular Biology (Faculty of Medicine)Umeå Centre for Microbial Research (UCMR)
MicrobiologyBiochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 256 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 1800 hits
ReferencesLink to record
Permanent link

Direct link