scholarly journals Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

2021 ◽  
Author(s):  
Sabine Gruschow ◽  
Catherine S Adamson ◽  
Malcolm F White
Keyword(s):  
Rna Binding ◽  
Limit Of Detection ◽  
Flanking Sequence ◽  
Guide Rna ◽  
Type Iii ◽  
Specificity And Sensitivity ◽  
Virus Rna ◽  
Messenger Molecules ◽  
Target Rna

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

scholarly journals Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR–Cas immunity

2020 ◽  
Vol 48 (8) ◽  
pp. 4418-4434 ◽  
Author(s):  
Kawanda Foster ◽  
Sabine Grüschow ◽  
Scott Bailey ◽  
Malcolm F White ◽  
Michael P Terns
Keyword(s):  
Rna Binding ◽  
Pyrococcus Furiosus ◽  
Type Iii ◽  
Second Messenger Signaling ◽  
Biochemical Analyses ◽  
Rna Interaction ◽  
Dual Mechanism ◽  
Target Rna ◽  
Rna And Dna

Abstract Type III CRISPR–Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3′ protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.

scholarly journals Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases

2017 ◽  
Author(s):  
Ole Niewoehner ◽  
Carmela Garcia-Doval ◽  
Jakob T. Rostøl ◽  
Christian Berk ◽  
Frank Schwede ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Rna Binding ◽  
Second Messengers ◽  
Allosteric Activation ◽  
Conceptual Similarity ◽  
Type Iii ◽  
Rna Transcripts ◽  
Rna Targeting ◽  
Target Rna

ABSTRACTIn many prokaryotes, type III CRISPR–Cas systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex that possesses dual RNase and DNase activities. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone ribonuclease that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the type III interference complex, the Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CARF domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to a hitherto unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.

scholarly journals SCOPE: Flexible targeting and stringent CARF activation enables type III CRISPR-Cas diagnostics

2021 ◽  
Author(s):  
Jurre A. Steens ◽  
Yifan Zhu ◽  
David W. Taylor ◽  
Jack P.K. Bravo ◽  
Stijn H.P Prinsen ◽  
...  
Keyword(s):  
Natural Variation ◽  
Nasal Swab ◽  
Rna Binding ◽  
Seed Region ◽  
Specific Detection ◽  
Unique Region ◽  
Type Iii ◽  
Single Host ◽  
Messenger Molecules ◽  
Target Rna

SummaryCharacteristic properties of type III CRISPR-Cas systems include recognition of target RNA (rather than DNA) and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of a target RNA and production of cyclic oligoadenylate (cOA) second messenger molecules that may trigger dormancy or cell death. In this study, we discovered that a largely exposed seed region at the 3’ end of the crRNA is essential for target RNA binding and cleavage, whereas base pairing at a unique region at the 5’ end of the guide is required to trigger cOA production. Moreover, we uncovered that the natural variation in the composition of type III complexes within a single host results in different guide lengths, and hence variable seed regions. This shifting seed may prevent escape by invading genetic elements, while controlling cOA production very tightly to prevent unnecessary damage to the host. Lastly, we used these findings to develop a new diagnostic tool, named SCOPE, which was used for the specific detection of SARS-CoV-2 from human nasal swab samples, showing sensitivities in the atto-molar range.

scholarly journals Structure and Function of an in vivo Assembled Type III-A CRISPR-Cas Complex Reveal Critical Roles of Dynamics in Activity Control

2021 ◽  
Author(s):  
Sagar Sridhara ◽  
Jay Rai ◽  
Charlisa Whyms ◽  
Walter Woodside ◽  
Michael P Terns ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Structure And Function ◽  
Flanking Sequence ◽  
Electron Cryomicroscopy ◽  
Graphic Art ◽  
Type Iii ◽  
And Function ◽  
Target Rna

AbstractThe small RNA-mediated immunity in bacteria depends on foreign RNA-activated and self RNA-inhibited enzymatic activities. The multi-subunit Type III-A CRISPR-Cas effector complex (Csm) exemplifies this principle, but its molecular basis for regulation remains unexplained. Recognition of the foreign RNA, or cognate target RNA (CTR), triggers its single-stranded deoxyribonuclease (DNase) and cyclic oligoadenylate (cOA) synthesis activities. The same activities remain dormant in the presence of the self-RNA, or noncognate target RNA (NTR) that differs from CTR only in its 3’-protospacer flanking sequence. Here we captured four structures of in vivo assembled Lactococcus lactis Csm (LlCsm) by electron cryomicroscopy representing both the active and the inactive states. Surprisingly, in absence of bound RNA, LlCsm largely forms a minimal assembly lacking the Csm2 subunit with a stably bound catalytic subunit Csm1. Comparison of the minimal LlCsm structure and activities, both in vitro and in vivo, with those of fully assembled LlCsm reveals a molecular mechanism responsible for the viral RNA-activated and self RNA-inhibited activity of Csm1 through protein dynamics.Graphic Art Summary

scholarly journals Structural Organization of a Type III-A CRISPR Effector Subcomplex Determined by X-ray Crystallography and Cryo-EM

2018 ◽  
Author(s):  
Bryan W. Dorsey ◽  
Lei Huang ◽  
Alfonso Mondragón
Keyword(s):  
Staphylococcus Epidermidis ◽  
Rna Binding ◽  
Guide Rna ◽  
Type Iii ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Class 1 ◽  
Target Rna ◽  
Resolution Single ◽  
Cas Proteins

AbstractClustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types. Class 1 CRISPR-Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, as well as to provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.

scholarly journals SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jurre A. Steens ◽  
Yifan Zhu ◽  
David W. Taylor ◽  
Jack P. K. Bravo ◽  
Stijn H. P. Prinsen ◽  
...  
Keyword(s):  
Immune Response ◽  
Diagnostic Tool ◽  
Nasal Swab ◽  
Rna Binding ◽  
Seed Region ◽  
Base Pairing ◽  
Specific Detection ◽  
Type Iii ◽  
Single Host ◽  
Target Rna

AbstractCharacteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3′ end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5′ end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.

scholarly journals Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels

2017 ◽  
Vol 114 (31) ◽  
pp. 8283-8288 ◽  
Author(s):  
Jun Liu ◽  
Yong Juan Zhao ◽  
Wan Hua Li ◽  
Yun Nan Hou ◽  
Ting Li ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Bimolecular Fluorescence Complementation ◽  
Knock Out ◽  
Guide Rna ◽  
Type Iii ◽  
Cyclic Adp Ribose ◽  
Human Multiple Myeloma ◽  
Hybrid Screening

CD38 catalyzes the synthesis of the Ca2+ messenger, cyclic ADP-ribose (cADPR). It is generally considered to be a type II protein with the catalytic domain facing outside. How it can catalyze the synthesis of intracellular cADPR that targets the endoplasmic Ca2+ stores has not been resolved. We have proposed that CD38 can also exist in an opposite type III orientation with its catalytic domain facing the cytosol. Here, we developed a method using specific nanobodies to immunotarget two different epitopes simultaneously on the catalytic domain of the type III CD38 and firmly established that it is naturally occurring in human multiple myeloma cells. Because type III CD38 is topologically amenable to cytosolic regulation, we used yeast-two-hybrid screening to identify cytosolic Ca2+ and integrin-binding protein 1 (CIB1), as its interacting partner. The results from immunoprecipitation, ELISA, and bimolecular fluorescence complementation confirmed that CIB1 binds specifically to the catalytic domain of CD38, in vivo and in vitro. Mutational studies established that the N terminus of CIB1 is the interacting domain. Using shRNA to knock down and Cas9/guide RNA to knock out CIB1, a direct correlation between the cellular cADPR and CIB1 levels was demonstrated. The results indicate that the type III CD38 is functionally active in producing cellular cADPR and that the activity is specifically modulated through interaction with cytosolic CIB1.

scholarly journals Combinatorial recognition of clustered RNA elements by a multidomain RNA-binding protein, IMP3

2018 ◽  
Author(s):  
Tim Schneider ◽  
Lee-Hsueh Hung ◽  
Masood Aziz ◽  
Anna Wilmen ◽  
Stephanie Thaum ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Binding Domains ◽  
Systematic Strategy ◽  
Rna Elements ◽  
Target Rna

AbstractHow multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We have established an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, providing a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

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 An essential RNA-binding lysine residue in the Nab3 RRM domain undergoes mono and trimethylation

2019 ◽  
Author(s):  
Kwan Yin Lee ◽  
Anand Chopra ◽  
Kyle Biggar ◽  
Marc D. Meneghini
Keyword(s):  
Rna Binding ◽  
Growth Defect ◽  
Rna Sequences ◽  
Rna Recognition Motifs ◽  
Rrm Domain ◽  
Lysine Residues ◽  
Recognition Motifs ◽  
Target Rna

AbstractThe Nrd1-Nab3-Sen1 (NNS) complex integrates molecular inputs to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by methylation of histone H3 lysine-4 as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3, and Sen1 that are mono-, di-, or trimethylated, suggesting novel molecular inputs for NNS regulation. One of these methylated residues, Nab3 lysine-363 (K363), resides within its RRM, and is known to physically contact target RNA. Although mutation of Nab3-K363 to arginine (Nab3-K363R) causes a severe growth defect, it nevertheless produces a stable protein that is incorporated into the NNS complex, suggesting that RNA binding through Nab3-K363 is crucial for NNS function. Consistent with this hypothesis, K363R mutation decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects in vivo. These findings reveal crucial roles for Nab3-K363 and suggest that methylation of this residue may modulate NNS activity through its impact on Nab3 RNA binding.

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