scholarly journals Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity

Cell ◽  
2015 ◽  
Vol 161 (5) ◽  
pp. 1164-1174 ◽  
Author(s):  
Poulami Samai ◽  
Nora Pyenson ◽  
Wenyan Jiang ◽  
Gregory W. Goldberg ◽  
Asma Hatoum-Aslan ◽  
...  
Keyword(s):  
Rna Cleavage ◽  
Type Iii ◽  
Dna And Rna

scholarly journals A Type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity

2019 ◽  
Author(s):  
Lucy Chou-Zheng ◽  
Asma Hatoum-Aslan
Keyword(s):  
Immune Response ◽  
Staphylococcus Epidermidis ◽  
Mobile Genetic Elements ◽  
Genetic Elements ◽  
Type Iii ◽  
Robust Immune Response ◽  
Functional Assays ◽  
Dna And Rna ◽  
Biochemical Analyses ◽  
Phage Population

AbstractCRISPR-Cas systems provide sequence-specific immunity against phages and mobile genetic elements using CRISPR-associated nucleases guided by short CRISPR RNAs (crRNAs). Type III systems exhibit a robust immune response that can lead to the extinction of a phage population, a feat coordinated by a multi-subunit effector complex that destroys invading DNA and RNA. Here, we demonstrate that a model Type III system in Staphylococcus epidermidis relies upon the activities of two degradosome-associated nucleases, PNPase and RNase J2, to mount a successful defense. Genetic, molecular, and biochemical analyses reveal that PNPase promotes crRNA maturation, and both nucleases are required for efficient clearance of phage-derived nucleic acids. Furthermore, functional assays show that RNase J2 is essential for immunity against diverse mobile genetic elements originating from plasmid and phage. Altogether, our observations reveal the evolution of a critical collaboration between two nucleic acid degrading machines which ensures cell survival when faced with phage attack.

scholarly journals Type III-A CRISPR-Cas Csm Complexes: Assembly, Periodic RNA Cleavage, DNase Activity Regulation, and Autoimmunity

2019 ◽  
Vol 73 (2) ◽  
pp. 264-277.e5 ◽  
Author(s):  
Ning Jia ◽  
Charlie Y. Mo ◽  
Chongyuan Wang ◽  
Edward T. Eng ◽  
Luciano A. Marraffini ◽  
...  
Keyword(s):  
Dnase Activity ◽  
Rna Cleavage ◽  
Activity Regulation ◽  
Type Iii

scholarly journals Topoisomerase 3B (TOP3B) DNA and RNA Cleavage Complexes and Pathway to Repair TOP3B-Linked RNA and DNA Breaks

2020 ◽  
Author(s):  
Sourav Saha ◽  
Yilun Sun ◽  
Shar-Yin Huang ◽  
Ukhyun Jo ◽  
Hongliang Zhang ◽  
...  
Keyword(s):  
Rna Cleavage ◽  
Dna Breaks ◽  
Dna And Rna ◽  
Rna And Dna

scholarly journals A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Lucy Chou-Zheng ◽  
Asma Hatoum-Aslan
Keyword(s):  
Immune Response ◽  
Staphylococcus Epidermidis ◽  
Mobile Genetic Elements ◽  
Genetic Elements ◽  
Type Iii ◽  
Robust Immune Response ◽  
Functional Assays ◽  
Dna And Rna ◽  
Biochemical Analyses ◽  
Phage Population

CRISPR-Cas systems provide sequence-specific immunity against phages and mobile genetic elements using CRISPR-associated nucleases guided by short CRISPR RNAs (crRNAs). Type III systems exhibit a robust immune response that can lead to the extinction of a phage population, a feat coordinated by a multi-subunit effector complex that destroys invading DNA and RNA. Here, we demonstrate that a model type III system in Staphylococcus epidermidis relies upon the activities of two degradosome-associated nucleases, PNPase and RNase J2, to mount a successful defense. Genetic, molecular, and biochemical analyses reveal that PNPase promotes crRNA maturation, and both nucleases are required for efficient clearance of phage-derived nucleic acids. Furthermore, functional assays show that RNase J2 is essential for immunity against diverse mobile genetic elements originating from plasmid and phage. Altogether, our observations reveal the evolution of a critical collaboration between two nucleic acid degrading machines which ensures cell survival when faced with phage attack.

scholarly journals Control of cyclic oligoadenylate synthesis in a type III CRISPR system

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Christophe Rouillon ◽  
Januka S Athukoralage ◽  
Shirley Graham ◽  
Sabine Grüschow ◽  
Malcolm F White
Keyword(s):  
Transcription Factors ◽  
Rna Binding ◽  
Rna Cleavage ◽  
Type Iii ◽  
Rna Transcripts ◽  
Crispr System ◽  
Cyclase Domain ◽  
Activated State ◽  
Target Rna ◽  
Oligoadenylate Synthesis

The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3’ end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The ‘RNA shredding’ activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence.

scholarly journals The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Januka S Athukoralage ◽  
Shirley Graham ◽  
Christophe Rouillon ◽  
Sabine Grüschow ◽  
Clarissa M Czekster ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Second Messengers ◽  
Sulfolobus Solfataricus ◽  
Comprehensive Analysis ◽  
Cyclase Activity ◽  
Rna Cleavage ◽  
Abortive Infection ◽  
Type Iii ◽  
Dynamic Interplay

Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an ‘off-switch’ for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.

scholarly journals DNA and RNA Cleavage Complexes and Repair Pathway for TOP3B RNA- and DNA-Protein Crosslinks

Cell Reports ◽  
2020 ◽  
Vol 33 (13) ◽  
pp. 108569
Author(s):  
Sourav Saha ◽  
Yilun Sun ◽  
Shar-yin Naomi Huang ◽  
Simone Andrea Baechler ◽  
Lorinc Sandor Pongor ◽  
...  
Keyword(s):  
Rna Cleavage ◽  
Repair Pathway ◽  
Dna And Rna ◽  
Protein Crosslinks ◽  
Rna And Dna

scholarly journals Type III-A CRISPR systems as a versatile gene knockdown technology

2020 ◽  
Author(s):  
Walter T. Woodside ◽  
Nikita Vantsev ◽  
Michael P. Terns
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Bacterial Species ◽  
Distinct Type ◽  
Gene Knockdown ◽  
Control Of Gene Expression ◽  
Type Iii ◽  
Dna And Rna ◽  
Pathway Analyses

AbstractCRISPR-Cas systems are functionally diverse prokaryotic anti-viral defense systems, which encompass six distinct types (I-VI) that each encode different effector Cas nucleases with distinct nucleic acid cleavage specificities. By harnessing the unique attributes of the various CRISPR-Cas systems, a range of innovative CRISPR-based DNA and RNA targeting tools and technologies have been developed. Here, we exploit the ability of type III-A CRISPR-Cas systems to carry out RNA-guided and sequence-specific target RNA cleavage for establishment of research tools for post-transcriptional control of gene expression. Type III-A systems from three bacterial species (L. lactis, S. epidermidis and S. thermophilus) were each expressed on a single plasmid in E. coli and the efficiency and specificity of gene knockdown was assessed by Northern blot analysis. We show that engineered type III-A modules can be programmed using tailored CRISPR RNAs to efficiently knock down gene expression of both coding and non-coding RNAs in vivo. Moreover, simultaneous degradation of multiple cellular mRNA transcripts can be directed by utilizing a CRISPR array expressing corresponding gene-targeting crRNAs. Our results demonstrate the utility of distinct type III-A modules to serve as effective gene knockdown platforms in heterologous cells. This transcriptome engineering technology has the potential to be further refined and exploited for key applications including gene discovery and gene pathway analyses in additional prokaryotic and perhaps eukaryotic cells and organisms.

scholarly journals The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling

2020 ◽  
Author(s):  
Januka S. Athukoralage ◽  
Shirley Graham ◽  
Christophe Rouillon ◽  
Sabine Grüschow ◽  
Clarissa M. Czekster ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Second Messengers ◽  
Sulfolobus Solfataricus ◽  
Comprehensive Analysis ◽  
Cyclase Activity ◽  
Rna Cleavage ◽  
Abortive Infection ◽  
Type Iii ◽  
Dynamic Interplay

AbstractCyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an “off-switch” for the cyclase activity (Rouillon et al., 2018). Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system, and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.

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