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Lécrivain, A.-L., Le Rhun, A., Renault, T. T., Ahmed-Begrich, R., Hahnke, K. & Charpentier, E. (2018). In vivo 3′-to-5′ exoribonuclease targetomes of Streptococcus pyogenes. Proceedings of the National Academy of Sciences of the United States of America, 115(46), 11814-11819
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
Lécrivain, A.-L., Broglia, L., Renault, T., Hahnke, K., Ahmed-Begrich, R., Le Rhun, A. & Charpentier, E. (2018). 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
Broglia, L., Materne, S., Lecrivain, A.-L., Hahnke, K., Le Rhun, A. & Charpentier, E. (2018). RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B. RNA Biology, 15(10), 1336-1347
Open this publication in new window or tab >>RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B
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2018 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 15, no 10, p. 1336-1347Article in journal (Refereed) Published
Abstract [en]

Endoribonuclease Y (RNase Y) is a crucial regulator of virulence in Gram-positive bacteria. In the human pathogen Streptococcus pyogenes, RNase Y is required for the expression of the major secreted virulence factor streptococcal pyrogenic exotoxin B (SpeB), but the mechanism involved in this regulation remains elusive. Here, we demonstrate that the 5′ untranslated region of speB mRNA is processed by several RNases including RNase Y. In particular, we identify two RNase Y cleavage sites located downstream of a guanosine (G) residue. To assess whether this nucleotide is required for RNase Y activity in vivo, we mutated it and demonstrate that the presence of this G residue is essential for the processing of the speB mRNA 5′ UTR by RNase Y. Although RNase Y directly targets and processes speB, we show that RNase Y-mediated regulation of speB expression occurs primarily at the transcriptional level and independently of the processing in the speB mRNA 5′ UTR. To conclude, we demonstrate for the first time that RNase Y processing of an mRNA target requires the presence of a G. We also provide new insights on the speB 5′ UTR and on the role of RNase Y in speB regulation.

Place, publisher, year, edition, pages
Taylor & Francis, 2018
Keywords
Streptococcus pyogenes, speB, RNase Y, virulence, 5' untranslated region, transcriptional regulation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-154094 (URN)10.1080/15476286.2018.1532253 (DOI)000450608900008 ()30290721 (PubMedID)
Funder
Göran Gustafsson Foundation for Research in Natural Sciences and MedicineThe Kempe FoundationsSwedish Research Council, K2013-57X-21436-04-3
Available from: 2018-12-11 Created: 2018-12-11 Last updated: 2018-12-11Bibliographically approved
Resch, U., Tsatsaronis, J. A., Le Rhun, A., Stuebiger, G., Rohde, M., Kasvandik, S., . . . Charpentier, E. (2016). A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus. mBio, 7(6), Article ID e00207-16.
Open this publication in new window or tab >>A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus
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2016 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 6, article id e00207-16Article in journal (Refereed) Published
Abstract [en]

Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to "anchorless surface proteins." Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response. IMPORTANCE Group A streptococcus (GAS) is a Gram-positive bacterial pathogen responsible for more than 500,000 deaths annually. Establishment of GAS infection is dependent on a suite of proteins exported via the general secretory pathway. Here, we show that GAS naturally produces extracellular vesicles with a unique lipid composition that are laden with proteins and RNAs. Interestingly, both virulence-associated proteins and RNA species were found to be differentially abundant in vesicles relative to the bacteria. Furthermore, we show that genetic disruption of the virulence-associated two-component regulator CovRS leads to an increase in vesicle production. This study comprehensively describes the protein, RNA, and lipid composition of GAS-secreted MVs and alludes to a regulatory system impacting this process.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-131666 (URN)10.1128/mBio.00207-16 (DOI)000392079500013 ()
Available from: 2017-02-24 Created: 2017-02-24 Last updated: 2018-06-09Bibliographically approved
Le Rhun, A., Beer, Y. Y., Reimegård, J., Chylinski, K. & Charpentier, E. (2016). RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes. RNA Biology, 13(2), 177-195
Open this publication in new window or tab >>RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes
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2016 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 13, no 2, p. 177-195Article 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.

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

Originally published in manuscript form.

Available from: 2015-11-09 Created: 2015-11-04 Last updated: 2018-06-07Bibliographically approved
Fonfara, I., Richter, H., Bratovic, M., Le Rhun, A. & Charpentier, E. (2016). The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature, 532(7600), 517-520
Open this publication in new window or tab >>The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA
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2016 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 532, no 7600, p. 517-520Article in journal (Refereed) Published
Abstract [en]

CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA(1). The well-studied types I, II and III utilize a set of distinct CRISPR-associated ( Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA)(2-5) and the mature crRNAs then guide a complex of Cas proteins ( Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA(6-12). In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA-crRNA duplex then guides Cas9 to cleave target DNA15. Here, we demonstrate a novel mechanism in CRISPR-Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5'-YTN- 3' protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5' overhang(16). The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V- A constitutes the most minimalistic of the CRISPR- Cas systems so far described.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-120803 (URN)10.1038/nature17945 (DOI)000374815900051 ()
Available from: 2016-05-25 Created: 2016-05-23 Last updated: 2018-06-07Bibliographically approved
Le Rhun, A. (2015). Multifaceted RNA-mediated regulatory mechanisms in Streptococcus pyogenes. (Doctoral dissertation). Umeå: Umeå university
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. p. 75
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1732
Keywords
Streptococcus pyogenes, small RNAs, CRISPR, Cas9, tracrRNA, RNases, gene expression, RNA sequencing
National Category
Microbiology Biochemistry and Molecular Biology
Research subject
Infectious Diseases; Microbiology
Identifiers
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)
Opponent
Supervisors
Available from: 2015-11-10 Created: 2015-11-04 Last updated: 2018-06-07Bibliographically approved
Fonfara, I., Le Rhun, A., Chylinski, K., Makarova, K. S., Lécrivain, A.-L., Bzdrenga, J., . . . Charpentier, E. (2014). Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Research, 42(4), 2577-2590
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
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2014 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 4, p. 2577-2590Article 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
Identifiers
urn:nbn:se:umu:diva-87653 (URN)10.1093/nar/gkt1074 (DOI)000332381000048 ()
Available from: 2014-04-08 Created: 2014-04-07 Last updated: 2018-06-08Bibliographically approved
Chylinski, K., Le Rhun, A. & Charpentier, E. (2013). The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biology, 10(5), 726-737
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, p. 726-737Article 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
Keywords
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
Identifiers
urn:nbn:se:umu:diva-80075 (URN)10.4161/rna.24321 (DOI)000323176900011 ()
Available from: 2013-09-09 Created: 2013-09-09 Last updated: 2018-06-08Bibliographically approved
Le Rhun, A. & Charpentier, E. (2012). Small RNAs in streptococci. RNA Biology, 9(4), 414-426
Open this publication in new window or tab >>Small RNAs in streptococci
2012 (English)In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 9, no 4, p. 414-426Article in journal (Refereed) Published
Abstract [en]

The group of streptococci includes species responsible for severe diseases in humans. To adapt to their environment and infect their hosts, streptococci depend on precise regulation of gene expression. The last decade has witnessed increasing findings of small RNAs (sRNAs) having regulatory functions in bacteria. More recently, genome-wide screens revealed that streptococcal genomes also encode multiple sRNAs. Some sRNAs including the class of CRISPR RNAs (crRNAs) play critical roles in streptococcal adaptation and virulence. Analysis of sRNA mechanisms uncovered three sRNAs that target in trans mRNA (FasX), sRNA (tracrRNA) and DNA (crRNA). Overall, the current understanding of sRNA-mediated regulation in streptococci remains very limited. Given the complexity of regulatory networks and the number of recently predicted sRNAs, future research should reveal new functions and mechanisms for the streptococcal sRNAs. Here, we provide a comprehensive summary of the information available on the topic.

Keywords
streptococci, small RNAs, CRISPR/Cas, trans-acting RNAs, gene regulation, adaptation, pathogenicity, virulence, bacterial immunity
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-55740 (URN)10.4161/rna.20104 (DOI)000304098100007 ()22546939 (PubMedID)
Available from: 2012-06-01 Created: 2012-05-29 Last updated: 2018-06-08Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-2211-2153

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