scholarly journals CRISPR interference: a structural perspective

2013 ◽  
Vol 453 (2) ◽  
pp. 155-166 ◽  
Author(s):  
Judith Reeks ◽  
James H. Naismith ◽  
Malcolm F. White
Keyword(s):  
Structural Biology ◽  
Recent Progress ◽  
Rna Binding ◽  
Protein Domain ◽  
Type I ◽  
Dependent Manner ◽  
Structural Studies ◽  
Future Studies ◽  
Associated Proteins ◽  
Cas Proteins

CRISPR (cluster of regularly interspaced palindromic repeats) is a prokaryotic adaptive defence system, providing immunity against mobile genetic elements such as viruses. Genomically encoded crRNA (CRISPR RNA) is used by Cas (CRISPR-associated) proteins to target and subsequently degrade nucleic acids of invading entities in a sequence-dependent manner. The process is known as ‘interference’. In the present review we cover recent progress on the structural biology of the CRISPR/Cas system, focusing on the Cas proteins and complexes that catalyse crRNA biogenesis and interference. Structural studies have helped in the elucidation of key mechanisms, including the recognition and cleavage of crRNA by the Cas6 and Cas5 proteins, where remarkable diversity at the level of both substrate recognition and catalysis has become apparent. The RNA-binding RAMP (repeat-associated mysterious protein) domain is present in the Cas5, Cas6, Cas7 and Cmr3 protein families and RAMP-like domains are found in Cas2 and Cas10. Structural analysis has also revealed an evolutionary link between the small subunits of the type I and type III-B interference complexes. Future studies of the interference complexes and their constituent components will transform our understanding of the system.

scholarly journals UBA1 and UBA2, Two Proteins That Interact with UBP1, a Multifunctional Effector of Pre-mRNA Maturation in Plants

2002 ◽  
Vol 22 (12) ◽  
pp. 4346-4357 ◽  
Author(s):  
Mark H. L. Lambermon ◽  
Yu Fu ◽  
Dominika A. Wieczorek Kirk ◽  
Marcel Dupasquier ◽  
Witold Filipowicz ◽  
...  
Keyword(s):  
Steady State ◽  
Rna Binding ◽  
Rna Recognition Motif ◽  
Dependent Manner ◽  
Binding Domains ◽  
Mrna Maturation ◽  
Associated Proteins ◽  
Steady State Levels ◽  
Transient Overexpression

ABSTRACT Nicotiana plumbaginifolia UBP1 is an hnRNP-like protein associated with the poly(A)+ RNA in the cell nucleus. Consistent with a role in pre-mRNA processing, overexpression of UBP1 in N. plumabaginifolia protoplasts enhances the splicing of suboptimal introns and increases the steady-state levels of reporter mRNAs, even intronless ones. The latter effect of UBP1 is promoter specific and appears to be due to UBP1 binding to the 3′ untranslated region (3′-UTR) and protecting the mRNA from exonucleolytic degradation (M. H. L. Lambermon, G. G. Simpson, D. A. Kirk, M. Hemmings-Mieszczak, U. Klahre, and W. Filipowicz, EMBO J. 19:1638-1649, 2000). To gain more insight into UBP1 function in pre-mRNA maturation, we characterized proteins interacting with N. plumbaginifolia UBP1 and one of its Arabidopsis thaliana counterparts, AtUBP1b, by using yeast two-hybrid screens and in vitro pull-down assays. Two proteins, UBP1-associated proteins 1a and 2a (UBA1a and UBA2a, respectively), were identified in A. thaliana. They are members of two novel families of plant-specific proteins containing RNA recognition motif-type RNA-binding domains. UBA1a and UBA2a are nuclear proteins, and their recombinant forms bind RNA with a specificity for oligouridylates in vitro. As with UBP1, transient overexpression of UBA1a in protoplasts increases the steady-state levels of reporter mRNAs in a promoter-dependent manner. Similarly, overexpression of UBA2a increases the levels of reporter mRNAs, but this effect is promoter independent. Unlike UBP1, neither UBA1a nor UBA2a stimulates pre-mRNA splicing. These and other data suggest that UBP1, UBA1a, and UBA2a may act as components of a complex recognizing U-rich sequences in plant 3′-UTRs and contributing to the stabilization of mRNAs in the nucleus.

scholarly journals Further investigation of the characteristics and biological function of Eimeria tenella apical membrane antigen 1

Parasite ◽  
2020 ◽  
Vol 27 ◽  
pp. 70
Author(s):  
Qingjie Wang ◽  
Qiping Zhao ◽  
Shunhai Zhu ◽  
Bing Huang ◽  
Shuilan Yu ◽  
...  
Keyword(s):  
Rna Binding ◽  
Apical Membrane ◽  
Mapk Signaling ◽  
Eimeria Tenella ◽  
Host Cells ◽  
Differentially Expressed ◽  
Type I ◽  
Dependent Manner ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen

Apical membrane antigen 1 (AMA1) is a type I integral membrane protein that is highly conserved in apicomplexan parasites. Previous studies have shown that Eimeria tenella AMA1 (EtAMA1) is critical for sporozoite invasion of host cells. Here, we show that EtAMA1 is a microneme protein secreted by sporozoites, confirming previous results. Individual and combined treatment with antibodies of EtAMA1 and its interacting proteins, E. tenella rhoptry neck protein 2 (EtRON2) and Eimeria-specific protein (EtESP), elicited significant anti-invasion effects on the parasite in a concentration-dependent manner. The overexpression of EtAMA1 in DF-1 cells showed a significant increase of sporozoite invasion. Isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS were used to screen differentially expressed proteins (DEPs) in DF-1 cells transiently transfected with EtAMA1. In total, 3953 distinct nonredundant proteins were identified and 163 of these were found to be differentially expressed, including 91 upregulated proteins and 72 downregulated proteins. The DEPs were mainly localized within the cytoplasm and were involved in protein binding and poly(A)-RNA binding. KEEG analyses suggested that the key pathways that the DEPs belonged to included melanogenesis, spliceosomes, tight junctions, and the FoxO and MAPK signaling pathways. The data in this study not only provide a comprehensive dataset for the overall protein changes caused by EtAMA1 expression, but also shed light on EtAMA1’s potential molecular mechanisms during Eimeria infections.

scholarly journals Recent advances in structural studies of the CRISPR-Cas-mediated genome editing tools

2018 ◽  
Vol 6 (3) ◽  
pp. 438-451 ◽  
Author(s):  
Yuwei Zhu ◽  
Zhiwei Huang
Keyword(s):  
Nucleic Acids ◽  
Genome Editing ◽  
Adaptive Immunity ◽  
Structural Biology ◽  
Structural Studies ◽  
Guide Rnas ◽  
Recent Advances ◽  
Cas Proteins

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR) and accompanying CRISPR-associated (Cas) proteins provide RNA-guided adaptive immunity for prokaryotes to defend themselves against viruses. The CRISPR-Cas systems have attracted much attention in recent years for their power in aiding the development of genome editing tools. Based on the composition of the CRISPR RNA-effector complex, the CRISPR-Cas systems can be divided into two classes and six types. In this review, we summarize recent advances in the structural biology of the CRISPR-Cas-mediated genome editing tools, which helps us to understand the mechanism of how the guide RNAs assemble with diverse Cas proteins to cleave target nucleic acids.

scholarly journals Challenges and perspectives for structural biology of lncRNAs—the example of the Xist lncRNA A-repeats

2019 ◽  
Vol 11 (10) ◽  
pp. 845-859 ◽  
Author(s):  
Alisha N Jones ◽  
Michael Sattler
Keyword(s):  
High Resolution ◽  
Structural Biology ◽  
Recent Progress ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Features ◽  
Molecular Features

Abstract Following the discovery of numerous long non-coding RNA (lncRNA) transcripts in the human genome, their important roles in biology and human disease are emerging. Recent progress in experimental methods has enabled the identification of structural features of lncRNAs. However, determining high-resolution structures is challenging as lncRNAs are expected to be dynamic and adopt multiple conformations, which may be modulated by interaction with protein binding partners. The X-inactive specific transcript (Xist) is necessary for X inactivation during dosage compensation in female placental mammals and one of the best-studied lncRNAs. Recent progress has provided new insights into the domain organization, molecular features, and RNA binding proteins that interact with distinct regions of Xist. The A-repeats located at the 5′ end of the transcript are of particular interest as they are essential for mediating silencing of the inactive X chromosome. Here, we discuss recent progress with elucidating structural features of the Xist lncRNA, focusing on the A-repeats. We discuss the experimental and computational approaches employed that have led to distinct structural models, likely reflecting the intrinsic dynamics of this RNA. The presence of multiple dynamic conformations may also play an important role in the formation of the associated RNPs, thus influencing the molecular mechanism underlying the biological function of the Xist A-repeats. We propose that integrative approaches that combine biochemical experiments and high-resolution structural biology in vitro with chemical probing and functional studies in vivo are required to unravel the molecular mechanisms of lncRNAs.

scholarly journals Programmable mammalian translational modulators by CRISPR-associated proteins

2021 ◽  
Author(s):  
Shunsuke Kawasaki ◽  
Hiroki Ono ◽  
Moe Hirosawa ◽  
Takeru Kuwabara ◽  
Hirohide Saito
Keyword(s):  
Mammalian Cells ◽  
Translational Regulation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genetic Circuits ◽  
Genomic Engineering ◽  
Associated Proteins ◽  
Translational Regulators ◽  
Rna Motif ◽  
Cas Proteins

The complexity of synthetic genetic circuits relies on repertories of biological circuitry with high orthogonality. Although post-transcriptional circuitry relying on RNA-binding proteins (RBPs) qualifies as a repertory, the limited pool of regulatory devices hinders network modularity and scalability. Here we propose CaRTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Genomic Engineering) to repurpose CRISPR-associated (Cas) proteins as translational modulators. We demonstrate that a set of Cas proteins are able to repress (OFF) or activate (ON) the translation of mRNAs that contain a Cas-binding RNA motif in the 5'-UTR. We designed 81 different types of translation OFF and ON switches and verified their functional characteristics. Many of them functioned as efficient translational regulators and showed orthogonality in mammalian cells. By interconnecting these switches, we designed and built artificial circuits, including 60 translational AND gates. Moreover, we show that various CRISPR-related technologies, including anti-CRISPR and split-Cas9 platforms, can be repurposed to control translation. Our Cas-mediated translational regulation is compatible with transcriptional regulation by Cas proteins and increases the complexity of synthetic circuits with fewer elements. CaRTRIDGE builds protein-responsive mRNA switches more than ever and leads to the development of both Cas-mediated genome editing and translational regulation technologies.

scholarly journals Human DICER helicase domain recruits PKR and modulates its antiviral activity

2021 ◽  
Vol 17 (5) ◽  
pp. e1009549
Author(s):  
Thomas C. Montavon ◽  
Morgane Baldaccini ◽  
Mathieu Lefèvre ◽  
Erika Girardi ◽  
Béatrice Chane-Woon-Ming ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Mammalian Cells ◽  
Sindbis Virus ◽  
Rna Binding ◽  
Semliki Forest Virus ◽  
Rna Binding Proteins ◽  
Helicase Domain ◽  
Type I ◽  
Dependent Manner ◽  
Rna Helicases

The antiviral innate immune response mainly involves type I interferon (IFN) in mammalian cells. The contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the ribonuclease DICER can generate viral siRNAs in specific conditions. It has also been proposed that type I IFN and RNA silencing could be mutually exclusive antiviral responses. In order to decipher the implication of DICER during infection of human cells with alphaviruses such as the Sindbis virus and Semliki forest virus, we determined its interactome by proteomics analysis. We show that DICER specifically interacts with several double-stranded RNA binding proteins and RNA helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase RNA-activated (PKR) are enriched with DICER in virus-infected cells. We demonstrate that the helicase domain of DICER is essential for this interaction and that its deletion confers antiviral properties to this protein in an RNAi-independent, PKR-dependent, manner.

scholarly journals Arginine Methylation Regulates SARS-CoV-2 Nucleocapsid Protein Function and Viral Replication

2021 ◽  
Author(s):  
Ting Cai ◽  
Zhenbao Yu ◽  
Zhen Wang ◽  
Chen Liang ◽  
Stephane Richard
Keyword(s):  
Protein Function ◽  
Rna Binding ◽  
Binding Capacity ◽  
Viral Proteins ◽  
Arginine Methylation ◽  
Host Protein ◽  
Hek293 Cells ◽  
Type I ◽  
Dependent Manner ◽  
N Protein

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) playing critical roles during viral infections. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG sequences. Arginine methylation of N protein was confirmed by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated by cell culture. We demonstrate that arginine methylation of N protein is required for its RNA binding capacity, since treatment with a type I PRMT inhibitor (MS023) or substitution of R95K or R177K inhibited interaction with the 5'-UTR of the SARS-CoV-2 genomic RNA. We defined the N interactome in HEK293 cells with or without MS023 treatment and identified PRMT1 and many of its RGG/RG substrates including the known interactor, G3BP1, and other components of stress granules (SG). Methylation of N protein at R95 regulates another function namely its property to suppress the formation of SGs. MS023 treatment or R95K substitution blocked N-mediated suppression of SGs. Also, the co-expression of methylarginine reader TDRD3 quenched N-mediated suppression of SGs in a dose-dependent manner. Finally, pre-treatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. With type I PRMT inhibitors being in clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may be an additional therapeutic application of these drugs.

scholarly journals Bud13 Promotes a Type I Interferon Response by Countering Intron Retention in Irf7

2018 ◽  
Author(s):  
Luke S. Frankiw ◽  
Devdoot Majumdar ◽  
Christian Burns ◽  
Annie Moradian ◽  
Michael J. Sweredoski ◽  
...  
Keyword(s):  
Rna Binding ◽  
Type I Interferon ◽  
Intron Retention ◽  
Global Analysis ◽  
Interferon Response ◽  
Type I ◽  
Dependent Manner ◽  
Protein Levels ◽  
Expression Of Genes ◽  
Gene Expression Control

SUMMARYIntron retention (IR) has emerged as an important mechanism of gene expression control. Despite this, the factors that control IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified Bud13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in Bud13 led to increased IR, decreased mature Irf7 transcript and protein levels, and consequently to a dampened type I interferon response. This impairment of Irf7 production in Bud13-deficient cells compromised their ability to withstand VSV infection. Global analysis of Bud13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a Bud13-dependent manner. Deficiency of Bud13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, Bud13 facilitates the expression of genes at which IR occurs.

scholarly journals The Oncogene Metadherin Interacts with the Known Splicing Proteins YTHDC1, Sam68 and T-STAR and Plays a Novel Role in Alternative mRNA Splicing

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1233 ◽  
Author(s):  
Hayley J. Luxton ◽  
Benjamin S. Simpson ◽  
Ian G. Mills ◽  
Nicola R. Brindle ◽  
Zeba Ahmed ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Cancer Tissue ◽  
Dependent Manner ◽  
Nuclear Speckles ◽  
Alternative Mrna Splicing ◽  
Associated Proteins ◽  
Reporter Assays ◽  
Poor Outcomes

Oncogenic metadherin is a key contributor to tumourigenesis with metadherin expression and cytoplasmic localisation previously linked to poor survival. A number of reports have shown metadherin localises specifically to nuclear speckles known to be rich in RNA-binding proteins including the splicing proteins YTHDC1, Sam68 and T-STAR, that have been shown to select alternative splice sites in mRNA of tumour-associated proteins including BRCA, MDM2 and VEGF. Here we investigate the interaction and relationship between metadherin and the splice factors YTHDC1, T-STAR and Sam68. Using a yeast two-hybrid assay and immunoprecipitation we show that metadherin interacts with YTHDC1, Sam68 and T-STAR and demonstrate that T-STAR is significantly overexpressed in prostate cancer tissue compared to benign prostate tissue. We also demonstrate that metadherin influences splice site selection in a dose-dependent manner in CD44v5-luc minigene reporter assays. Finally, we demonstrate that prostate cancer patients with higher metadherin expression have greater expression of the CD44v5 exon. CD44v5 expression could be used to discriminate patients with poor outcomes following radical prostatectomy. In this work we show for the first time that metadherin interacts with, and modulates, the function of key components of splicing associated with cancer development and progression.

scholarly journals The Arcanobacterium pyogenes Collagen-Binding Protein, CbpA, Promotes Adhesion to Host Cells

2003 ◽  
Vol 71 (8) ◽  
pp. 4368-4374 ◽  
Author(s):  
Paula A. Esmay ◽  
Stephen J. Billington ◽  
Malen A. Link ◽  
J. Glenn Songer ◽  
B. Helen Jost
Keyword(s):  
Epithelial Cells ◽  
Binding Protein ◽  
Collagen Type I ◽  
Protein Domain ◽  
Host Cells ◽  
Type I ◽  
Dependent Manner ◽  
Multicopy Plasmid ◽  
Collagen Binding ◽  
Arcanobacterium Pyogenes

ABSTRACT Arcanobacterium pyogenes is an opportunistic pathogen associated with suppurative diseases in economically important food animals such as cattle, pigs, and turkeys. A. pyogenes adheres to host epithelial cells, and adhesion is promoted by the action of neuraminidase, which is expressed by this organism. However, a neuraminidase-deficient mutant of A. pyogenes only had a reduced ability to adhere to host epithelial cells, indicating that other factors are involved in adhesion. Far Western blotting revealed the presence of an approximately 120-kDa A. pyogenes cell wall protein that binds collagen type I. The 3.5-kb gene that encodes the 124.7-kDa CbpA protein was cloned, and sequence analysis indicated that CbpA contains a typical MSCRAMM protein domain structure. Recombinant, six-His-tagged CbpA (HIS-CbpA) was capable of binding collagen types I, II, and IV but not fibronectin. In addition, CbpA was involved in the ability of A. pyogenes to adhere to HeLa and 3T6 cells, as a cbpA knockout strain had 38.2 and 57.0% of wild-type adhesion, respectively. This defect could be complemented by providing cbpA on a multicopy plasmid. Furthermore, HIS-CbpA blocked A. pyogenes adhesion to HeLa or 3T6 cells in a dose-dependent manner. cbpA was only present in 48% of the A. pyogenes strains tested (n = 75), and introduction of plasmid-encoded cbpA into a naturally cbpA-deficient strain increased the ability of this strain to bind to HeLa and 3T6 cells 2.9- and 5.7-fold, respectively. These data indicate that CbpA, a collagen-binding protein of A. pyogenes, plays a role in the adhesion of this organism to host cells.

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