scholarly journals tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

2021 ◽  
Author(s):  
Ekaterina Semenova ◽  
Ishita Jain ◽  
Matvey Kolesnik ◽  
Leonid Minakhin ◽  
Natalia Morozova ◽  
...  
Keyword(s):  
Defense Mechanism ◽  
Specific Binding ◽  
Rna Degradation ◽  
Host Population ◽  
Bacterial Toxin ◽  
Rna Recognition ◽  
Trna Species ◽  
Necessary And Sufficient ◽  
Cell Dormancy ◽  
Target Rna

Abstract Type VI CRISPR-Cas systems are the only CRISPR variety that cleaves exclusively RNA1,2. In addition to the CRISPR RNA (crRNA)-guided, sequence-specific binding and cleavage of target RNAs, such as phage transcripts, the type VI effector, Cas13, causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from phage spread3,4. We show here that the principal form of collateral RNA degradation elicited by Cas13a protein from Leptotrichia shahii upon target RNA recognition is the cleavage of anticodons of multiple tRNA species, primarily those with anticodons containing uridines. This tRNA cleavage is necessary and sufficient for bacterial dormancy induction by Cas13a. In addition, Cas13a activates the RNases of bacterial toxin-antitoxin modules, thus indirectly causing mRNA and rRNA cleavage, which could provide a back-up defense mechanism. The identified mode of action of Cas13a resembles that of bacterial anticodon nucleases involved in antiphage defense5, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module6,7 encompassing an anticodon nuclease.

scholarly journals tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

2021 ◽  
Author(s):  
Ishita Jain ◽  
Matvey Kolesnik ◽  
Leonid Minakhin ◽  
Natalia Morozova ◽  
Anna Shiriaeva ◽  
...  
Keyword(s):  
Defense Mechanism ◽  
Specific Binding ◽  
Rna Degradation ◽  
Host Population ◽  
Bacterial Toxin ◽  
Rna Recognition ◽  
Trna Species ◽  
Necessary And Sufficient ◽  
Cell Dormancy ◽  
Target Rna

Type VI CRISPR-Cas systems are the only CRISPR variety that cleaves exclusively RNA. In addition to the CRISPR RNA (crRNA)-guided, sequence-specific binding and cleavage of target RNAs, such as phage transcripts, the type VI effector, Cas13, causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from phage spread. We show here that the principal form of collateral RNA degradation elicited by Cas13a protein from Leptotrichia shahii upon target RNA recognition is the cleavage of anticodons of multiple tRNA species, primarily those with anticodons containing uridines. This tRNA cleavage is necessary and sufficient for bacterial dormancy induction by Cas13a. In addition, Cas13a activates the RNases of bacterial toxin-antitoxin modules, thus indirectly causing mRNA and rRNA cleavage, which could provide a back-up defense mechanism. The identified mode of action of Cas13a resembles that of bacterial anticodon nucleases involved in antiphage defense, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease.

scholarly journals Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Katsura Minegishi ◽  
Benjamin Rothé ◽  
Kaoru R. Komatsu ◽  
Hiroki Ono ◽  
Yayoi Ikawa ◽  
...  
Keyword(s):  
Fluid Flow ◽  
Mouse Embryo ◽  
Untranslated Region ◽  
Mrna Decay ◽  
Rna Binding ◽  
Specific Binding ◽  
Flow Direction ◽  
Rna Degradation ◽  
Immotile Cilia ◽  
Necessary And Sufficient

AbstractMolecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3′ untranslated region (3′-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3′-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3′-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node.

scholarly journals Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

2020 ◽  
Author(s):  
Carmela Garcia-Doval ◽  
Frank Schwede ◽  
Christian Berk ◽  
Jakob T. Rostøl ◽  
Ole Niewoehner ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Second Messengers ◽  
Rna Degradation ◽  
Rna Recognition ◽  
Crispr Interference ◽  
Type Iii ◽  
Functional Assays ◽  
Downstream Effectors ◽  
Target Rna

AbstractUpon target RNA recognition, type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers to activate downstream effectors including Csm6-family ribonucleases via their CARF domains. Here we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator and report the crystal structure of EiCsm6 bound to a cA6 mimic. The structure, combined with biochemical and in vivo functional assays, reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.

Only two of the five zinc fingers of the eukaryotic transcriptional repressor PRDI-BF1 are required for sequence-specific DNA binding

1992 ◽  
Vol 12 (5) ◽  
pp. 1940-1949
Author(s):  
A D Keller ◽  
T Maniatis
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Specific Binding ◽  
Regulatory Element ◽  
Zinc Fingers ◽  
Transcriptional Repressor ◽  
Gene Promoter ◽  
Binding Activity ◽  
Necessary And Sufficient ◽  
Sequence Requirements

The eukaryotic transcriptional repressor PRDI-BF1 contains five zinc fingers of the C2H2 type, and the protein binds specifically to PRDI, a 14-bp regulatory element of the beta interferon gene promoter. We have investigated the amino acid sequence requirements for specific binding to PRDI and found that the five zinc fingers and a short stretch of amino acids N terminal to the first finger are necessary and sufficient for PRDI-specific binding. The contribution of individual zinc fingers to DNA binding was investigated by inserting them in various combinations into another zinc finger-containing DNA-binding protein whose own fingers had been removed. We found that insertion of PRDI-BF1 zinc fingers 1 and 2 confer PRDI-binding activity on the recipient protein. In contrast, the insertion of PRDI-BF1 zinc fingers 2 through 5, the insertion of zinc finger 1 or 2 alone, and the insertion of zinc fingers 1 and 2 in reverse order did not confer PRDI-binding activity. We conclude that the first two PRDI-BF1 zinc fingers together are sufficient for the sequence-specific recognition of PRDI.

scholarly journals Molecular Basis for Target RNA Recognition and Cleavage by Human RISC

Cell ◽  
2007 ◽  
Vol 130 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Stefan Ludwig Ameres ◽  
Javier Martinez ◽  
Renée Schroeder
Keyword(s):  
Molecular Basis ◽  
Rna Recognition ◽  
Target Rna

A Sense Oligonucleotide Specifically Reverses the Prothrombin Depletion and Anticoagulation Caused by ISIS 401025, an Antisense Oligonucleotide Targeting Prothrombin RNA in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 926-926
Author(s):  
Chenguang Zhao ◽  
Brett P. Monia ◽  
Andy Siwkowski ◽  
Sue Freier ◽  
Donna Witchell ◽  
...  
Keyword(s):  
Anticoagulant Activity ◽  
Rna Degradation ◽  
Subsequent Treatment ◽  
Weekly Dose ◽  
Mrna Levels ◽  
Transcript Levels ◽  
Sense Strand ◽  
Long Acting ◽  
First Time ◽  
Target Rna

Abstract The ability to safely and effectively neutralize anticoagulant activity is of particular importance in the case of long acting drugs. While antisense oligonucleotides (ASOs) benefit from their slow clearance rate from liver (t1/2 approx 10 days in mice) which permits infrequent dosing, the potential necessity to reverse ASO drug activity presents a challenge. ISIS 401025 is a second generation ASO targeting prothrombin RNA that has previously been shown to reduce thrombin generation and prolong PT and aPTT as a result of depleting prothrombin mRNA levels in mice. To explore the possibility of developing an “antidote” strategy for ASO-mediated anticoagulation, we designed a sense oligonucleotide, ISIS 405277, complementary to the ISIS 401025 sequence and examined its ability to reverse the effects of prior treatment with ISIS 401025 in mice. A PT-INR of 3.0 was established after 3 weeks of treatment with ISIS 401025 at a weekly dose of 60 mg/kg, corresponding to a reduction of prothrombin mRNA transcript levels of 96%. A single injection of the sense oligonucleotide, ISIS 405277, resulted in a dose-dependent reversal of INR to 1.6, 1.1, and 0.9 three days after injection of 30, 60, and 90 mg/kg, respectively. Corresponding prothrombin transcript levels in liver tissue were returned to 18.4%, 27.8%, and 38.9% of normal levels, respectively. Results from a study designed to determine the kinetics of reversal of anticoagulation indicated that a 50% reduction of PT-INR 4.3, established following 3 weeks of treatment with ISIS 401025, required 11 days in the absence of subsequent treatment, while the reduction was achieved in 2.2 days following a single administration of 90 mg/kg of sense oligonucleotide. When mice were pretreated with an ASO targeting prothrombin that was not complementary to the sense oligonucleotide (ISIS 40527), sense oligonucleotide treatment was unable to reverse either target depletion or anticoagulation, indicating a sequence-specific antidote effect. These results demonstrate for the first time that subsequent administration of a sense strand oligonucleotide can neutralize ASO mediated target RNA degradation in animals, and demonstrates its potential utility in reversing ASO-based activity for anticoagulation.

scholarly journals A crucial RNA-binding lysine residue in the Nab3 RRM domain undergoes SET1 and SET3-responsive methylation

2020 ◽  
Vol 48 (6) ◽  
pp. 2897-2911 ◽  
Author(s):  
Kwan Yin Lee ◽  
Anand Chopra ◽  
Giovanni L Burke ◽  
Ziyan Chen ◽  
Jack F Greenblatt ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Recognition ◽  
Rna Sequences ◽  
Rna Recognition Motifs ◽  
Rrm Domain ◽  
Lysine Residues ◽  
Recognition Motifs ◽  
Nascent Transcripts ◽  
Target Rna

Abstract The Nrd1–Nab3–Sen1 (NNS) complex integrates molecular cues to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by histone methylation 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 methylated, suggesting novel molecular inputs for NNS regulation. We identify mono-methylation of one these residues (Nab3-K363me1) as being partly dependent on the H3K4 methyltransferase, Set1, a known regulator of NNS function. Moreover, the accumulation of Nab3-K363me1 is essentially abolished in strains lacking SET3, a SET domain containing protein that is positively regulated by H3K4 methylation. Nab3-K363 resides within its RRM and physically contacts target RNA. Mutation of Nab3-K363 to arginine (Nab3-K363R) decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects and slow growth. These findings identify SET3 as a potential contextual regulator of Nab3 function through its role in methylation of Nab3-K363. Consistent with this hypothesis, we report that SET3 exhibits genetic activation of NAB3 that is observed in a sensitized context.

scholarly journals High Levels of Interleukin-10 Impair Resistance to Pulmonary Coccidioidomycosis in Mice in Part through Control of Nitric Oxide Synthase 2 Expression

2006 ◽  
Vol 74 (6) ◽  
pp. 3387-3395 ◽  
Author(s):  
Maria del Pilar Jimenez ◽  
Lorraine Walls ◽  
Joshua Fierer
Keyword(s):  
Nitric Oxide ◽  
Transgenic Mice ◽  
Defense Mechanism ◽  
Inbred Mice ◽  
Interleukin 10 ◽  
Coccidioides Immitis ◽  
Recombinant Inbred Mice ◽  
Necessary And Sufficient ◽  
Nitric Oxide Synthase 2 ◽  
Oxide Synthase

ABSTRACT We have shown previously that there is a direct correlation between IL-10 levels and susceptibility to Coccidioides immitis peritonitis in C57BL/6 (B6), DBA/2, and BXD recombinant inbred mice. We now show that B6 mice are also more susceptible to C. immitis pneumonia and that interleukin-10 (IL-10)-deficient (IL-10−/−) B6 mice are more resistant to C. immitis pneumonia. In addition, we established that high levels of IL-10 are sufficient to make genetically resistant mice susceptible to both C. immitis peritonitis and pneumonia by infecting h.IL-10 transgenic mice. Infected h.IL-10 transgenic mice express lower levels of gamma interferon, IL-12 p40, and inducible nitric oxide synthetase 2 (NOS2) mRNA in their lungs, implicating inducible NOS as a defense mechanism in this disease. We treated DBA/2 mice with aminoguanidine, and they became more susceptible to C. immitis peritonitis and pneumonia. We conclude that high levels of IL-10 are both necessary and sufficient to make mice susceptible to C. immitis, regardless of the genetic background of the mice, and that IL-10 impairs resistance to C. immitis in part by suppressing NO synthesis.

scholarly journals A B Cell Superantigen–Induced Persistent “Hole” in the B-1 Repertoire

2000 ◽  
Vol 192 (1) ◽  
pp. 87-98 ◽  
Author(s):  
Gregg J. Silverman ◽  
Stephen P. Cary ◽  
Denise C. Dwyer ◽  
Linda Luo ◽  
Raymond Wagenknecht ◽  
...  
Keyword(s):  
Immune System ◽  
B Cell ◽  
Specific Binding ◽  
Protein A ◽  
Adaptive Immune System ◽  
Variable Region ◽  
Natural Antibodies ◽  
Bacterial Toxin ◽  
Antigen Receptors ◽  
Host Immune Responses

The bacterial toxin protein A from Staphylococcus aureus (SpA) interacts with B cell antigen receptors encoded by variable region heavy chain (VH) clan III genes via a V region framework surface that has been highly conserved during the evolution of the adaptive immune system. We have investigated the consequences of exposure to this prototypic B cell superantigen, and found that treatment of neonates or adults induces a T cell–independent deletion of a large supraclonal set of susceptible B cells that includes clan III/VH S107 family–expressing lymphocytes. In studies of different SpA forms, the magnitude of the induced deletion directly correlated with the VH-specific binding affinity/avidity. Upon cessation of SpA exposure, the representation of conventional splenic (B-2 subset) lymphocytes normalized; however, we found that the VH family–restricted deficit of peritoneal B-1 cells persisted. SpA treatment also induced a persistent loss of splenic S107-μ transcripts, with a loss of certain natural antibodies and specific tolerance to phosphorylcholine immunogens that normally recruit protective antimicrobial responses dominated by the S107-expressing B-1 clone, T15. These studies illustrate how a B cell superantigen can exploit a primordial Achilles heel in the immune system, for which B-1 cells, an important source of natural antibodies and host immune responses, have special susceptibility.

scholarly journals Expanding the binding specificity for RNA recognition by a PUF domain

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wei Zhou ◽  
Daniel Melamed ◽  
Gabor Banyai ◽  
Cindy Meyer ◽  
Thomas Tuschl ◽  
...  
Keyword(s):  
Rna Binding ◽  
Amino Acid Substitutions ◽  
Rna Recognition ◽  
Specific Interactions ◽  
Wild Type ◽  
Modular Architecture ◽  
Recognition Sequence ◽  
Puf Domain ◽  
Target Rna ◽  
Type Sequence

AbstractThe ability to design a protein to bind specifically to a target RNA enables numerous applications, with the modular architecture of the PUF domain lending itself to new RNA-binding specificities. For each repeat of the Pumilio-1 PUF domain, we generate a library that contains the 8,000 possible combinations of amino acid substitutions at residues critical for RNA contact. We carry out yeast three-hybrid selections with each library against the RNA recognition sequence for Pumilio-1, with any possible base present at the position recognized by the randomized repeat. We use sequencing to score the binding of each variant, identifying many variants with highly repeat-specific interactions. From these data, we generate an RNA binding code specific to each repeat and base. We use this code to design PUF domains against 16 RNAs, and find that some of these domains recognize RNAs with two, three or four changes from the wild type sequence.

Sign in / Sign up

Export Citation Format

Share Document