Structures of Human ADAR2 Bound to DsRNA Reveal Base-flipping Mechanism and Basis for Site Selectivity PDF Download

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Publisher:
ISBN:
Category :
Languages : en
Pages : 8

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Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. In this paper, we describe four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis of the ADAR deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus rationalizing differences in selectivity observed between different ADARs. In conclusion, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.

Author:
Publisher: Academic Press
ISBN: 0128117788
Category : Science
Languages : en
Pages : 370

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Book Description
RNA Modification, Volume 41 examines the powerful ability to regulate the function of RNA molecules or modify the message transmitted by RNA molecules. Chapters in this newly released volume include The Importance of Being Modified: Modifications Shape RNA Function through Chemistry, Structure and Dynamics, The evolution of multi-substrate specificity by RNA modification enzymes, TrmD: a methyl transferase for tRNA methylation with m1G37, Structures and activities of the Elongator complex and its co-factors, RNA pseudouridylation: Mechanism and Function, The activity of 5’3' exonucleases on hypo modified tRNA substrates and other structured RNAs, and the Synthesis, heterogeneity and function of post-transcriptional nucleotide modifications in eukaryotic ribosomal RNAs. This field has recently seen a very rapid progress in the understanding of the mechanism and enzymes involved in RNA modification. This volume presents some of the most recent advances in the identification and function of enzymes involved in modifying RNA molecules. Features authoritative expertise from recognized contributors to the field Presents the most recent advances in the rapidly evolving field of RNA modification Covers the identification and function of enzymes involved in modifying RNA molecules

Author: Kui Li
Publisher: Elsevier
ISBN: 0128191007
Category : Technology & Engineering
Languages : en
Pages : 444

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Livestock Genome Editing Tools introduces applications and improvements to a series of new genome editing techniques in livestock, such as pigs, cattle, and sheep. These tools include zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system, as well as the traditional gene targeting tools. The book also summarizes a series of genome editing-related techniques including microinjection and somatic cell nuclear transfer. Written by international experts who have been working on livestock genetic editing field for more than 30 years, this book provides extensively theoretical and practical experience for readers to master the latest developments. This book explores the importance of research and application, as well as operation procedures, of livestock genetic editing tools. The writing of operation details makes this book an accessible read. Livestock Genome Editing Tools is an important resource for researchers interested in genome-edited animals, scientists and technicians in breeding institutions, and is also of interest to students major in animal reproduction and biological engineering. Provides operable experimental procedures of pigs, cattle, and sheep genome editing tools Introduces the evaluation, breeding process, and application of each of the latest and most effective tools Examines the importance of livestock germplasm innovation, breed improvement, and human disease model generation

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Publisher: Academic Press
ISBN: 0323994091
Category : Science
Languages : en
Pages : 328

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Book Description
MRNA-Based Therapeutics, Volume 372 in the International Review of Cell and Molecular Biology series, covers topics surrounding the effect of different metabolic situations, their contribution to metabolic modulation, and their impact on tumor growth. Specific chapters in this release include New era of nucleic acid therapies: Clinical applications and perspectives, Messenger RNA as personalized therapy: time of truth for rare metabolic disease, Applications of Self-Replicating RNA, mRNA therapy in PKU, Advances in gene-editing technologies, mRNA delivery technologies: towards clinical translation, Advances in mRNA vaccines, and more. Provides the authority and expertise of leading contributors from an international board of authors Presents the latest release in International Review of Cell and Molecular Biology serials Updated release includes the latest information on MRNA-Based Therapeutics

Author: Naoki Sugimoto
Publisher: Springer Nature
ISBN: 9811997764
Category : Science
Languages : en
Pages : 2847

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Book Description
This handbook is the first to comprehensively cover nucleic acids from fundamentals to recent advances and applications. It is divided into 10 sections where authors present not only basic knowledge but also recent research. Each section consists of extensive review chapters covering the chemistry, biology, and biophysics of nucleic acids as well as their applications in molecular medicine, biotechnology and nanotechnology. All sections within this book are: Physical Chemistry of Nucleic Acids (Section Editor: Prof. Roland Winter), Structural Chemistry of Nucleic Acids (Section Editor: Prof. Janez Plavec), Organic Chemistry of Nucleic Acids (Section Editor: Prof. Piet Herdewijin), Ligand Chemistry of Nucleic Acids (Section Editor: Prof. Marie-Paule Teulade-Fichou), Nucleic Acids and Gene Expression (Section Editor: Prof. Cynthia Burrows), Analytical Methods and Applications of Nucleic Acids (Section Editor: Prof. Chaoyong Yang), Nanotechnology and Nanomaterial Biology of Nucleic Acids (Section Editor: Prof. Zhen Xi), Nucleic Acids Therapeutics (Section Editor: Prof. Katherine Seley-Radtke), Biotechnology and Synthetic Biology of Nucleic Acids (Section Editor: Prof. Eriks Rozners), Functional Nucleic Acids (Section Editor: Prof. Keith R. Fox). The handbook is edited by outstanding leaders with contributions written by international renowned experts. It is a valuable resource not only for researchers but also graduate students working in areas related to nucleic acids who would like to learn more about their important role and potential applications.

Author: Justin Mclntyre Thomas
Publisher:
ISBN: 9780355462111
Category :
Languages : en
Pages :

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Book Description
Adenosine deaminases acting on RNA (ADARs) are RNA editing enzymes that convert adenosine to inosine in duplex RNA. Because inosine behaves like guanosine in Watson-Crick base pairing, A-to-I editing may have wide ranging consequences in RNA function. The X-ray crystal structure of the deaminase domain of one member of the ADAR family (ADAR2) was solved over a decade ago, however this structure lacked RNA and provided limited information on how ADARs select, bind and edit adenosines within duplex RNA. This dissertation describes solving of X-ray co-crystal structures of ADAR2’s deaminase domain bound to synthetic duplex RNA and subsequent experiments to define the structural basis of the enzymes sequence preferences. Chapter 2 describes the solved crystal structures of ADAR2 deaminase domain-RNA complexes. The deaminase domain’s RNA binding interface is analyzed and new RNA binding residues are identified. In Chapter 3 the importance of newly identified RNA binding residues is examined through in-vitro biochemical assays. The structural basis for ADAR2’s nearest neighbor sequence preferences are also defined by probing contacts made to the RNA using chemically modified RNA substrates. This information may aid the development of systems to direct A-to-I editing of specific adenosines using ADARs. In Chapter 4, RNA binding experiments using ADAR2 constructs bearing double stranded RNA binding domains (dsRBDs) are carried out with the goal of better understanding how the dsRBDs affect the specificity and behavior of the full length ADAR2. Mobility shift assays, RNA cleavage footprinting assays and electron microscopy all provide evidence that ADAR2 undergoes a specific RNA dependent dimerization. Finally, efforts to obtain high resolution structural data for ADAR2 constructs bearing dsRBDs through X-ray crystallography and Cryo-electron microscopy are discussed

Author:
Publisher: Academic Press
ISBN: 0323853021
Category : Science
Languages : en
Pages : 560

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Book Description
Curing Genetic Diseases through Genome Reprogramming, Volume 182 captures an historic moment in the field of gene therapy—the dawn of a new age in which the dream of curing genetic diseases has become realizable. The volume presents the most clinically advanced gene therapy and genome editing approaches for the treatment of genetic diseases in specific organs, including difficult therapeutic targets, futuristic ideas of genetic interventions, and large scale human genome repair. An initial chapter addresses the complex ethical aspects involved in the very idea of modifying the human genome. Provides a comprehensive view of gene therapy and genome editing technologies, including epigenetic editing Describes the state-of-the-art and future directions for the treatment of genetic diseases, also considering economical aspects Presents chapters that each give a thorough review of a specific disease, target organ or visionary approach, including ethical considerations

Author: Tristan Thomas Eifler
Publisher:
ISBN: 9781303791857
Category :
Languages : en
Pages :

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Book Description
ADARs (adenosine deaminases acting on RNA) are a family of RNA editing enzymes that deaminate adenosines (A) in double-stranded RNA (dsRNA), generating inosine, which is read by the cellular machinery as guanosine. Consequently, ADAR editing of messenger RNA can result in amino acid substitutions, thereby increasing the diversity of an organism's proteome. It is still not entirely clear why particular adenosines in dsRNA molecules are selected for ADAR editing. Furthermore, very little is known regarding how ADAR catalytic domains interact with substrate dsRNA during deamination. This dissertation describes experiments that probe the structural and sequential determinants of ADAR2 editing in dsRNA to reach a better understanding of ADAR2 selectivity.We have observed that overexpressing human ADAR2 (hADAR2) in the budding yeast Saccharomyces cerevisiae reduces cell growth to a degree proportional to the deaminase activity of the enzyme. We hypothesized that this is a product of hADAR2 editing endogenous yeast RNA. As described in Chapter 2, we employed next-generation sequencing to sequence the transcriptomes of S. cerevisiae expressing catalytically active and inactive hADAR2 and identified 19 candidate hADAR2 editing sites. Aligning the edited regions revealed both canonical and novel preferred flanking sequences for hADAR2 editing. Specifically, we observed a preference for A two nucleotides downstream of the edited A in addition to previously noted preferences for U and G 5' and 3' of the editing site, respectively. Follow-up analysis of the predicted hADAR2 editing sites in yeast was necessary to confirm editing and test the novel preferred nucleotide neighbor observed in the sequence alignment. We Sanger sequenced reverse-transcription polymerase chain reaction (RT-PCR) products of yeast total RNA and performed in vitro deamination assays on RNA substrates. We confirmed editing at 8 of the 19 candidate sites and determined that mutating the A 2 nucleotides downstream of the edited A in the yeast hADAR2 substrate BDF2 decreases editing efficacy at that site both in vivo and in vitro. This work is described in Chapter 3. Not only did we discover novel hADAR2 substrates but a novel preferred neighbor for hADAR2 editing. This expands understanding of hADAR2 selectivity and provides us with new editing substrates for future study.It has been understood that ADAR binding with dsRNA is mediated by its double-stranded RNA binding domains (dsRBDs). However, as described in Chapter 4, we determined the yeast hADAR2 substrate BDF2 is rapidly edited by the isolated ADAR2 deaminse domain. We also examined an established human hADAR2 substrate with structural and sequential similarities to BDF2, GLI1, and found it too was rapidly edited independently of the hADAR2 dsRBDs. Not only does this have implications in how hADAR2 recognizes, binds, and edits RNA but suggests the hADAR2 deaminase domain could used to form a covalently-linked heterocomplex with substrate RNA suitable for X-ray crystallography. Finally, Chapter 5 describes our attempts to incorporate the unnatural amino acid p-azidophenylalanine into a hADAR2 truncation using an orthogonal tRNA/tRNA synthetase pair for site-specific ligation of probe molecules to the protein and the generation of a hADAR2 quadruple cysteine mutant. We determined that the hADAR2 quadruple cysteine mutant retains deaminase activity and may therefore be useful in linking the protein with probe molecules via disulfide bonds for structural studies.

Author: Charles E. Samuel
Publisher: Springer Science & Business Media
ISBN: 3642228011
Category : Science
Languages : en
Pages : 244

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“The objective of this CTMI volume is to provide readers with a foundation for understanding what ADARs are and how they act to affect gene expression and function. It is becoming increasingly apparent that ADARs may possess roles not only as enzymes that deaminate adenosine to produce inosine in RNA substrates with double-stranded character, but also as proteins independent of their catalytic property. Because A-to-I editing may affect base-pairing and RNA structure, processes including translation, splicing, RNA replication, and miR and siRNA silencing may be affected. Future studies of ADARs no doubt will provide us with additional surprises and new insights into the modulation of biological processes by the ADAR family of proteins.”

Author: Kiyoshi Nagai
Publisher: Oxford University Press, USA
ISBN:
Category : Medical
Languages : en
Pages : 302

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Book Description
The study of RNA-protein interactions is crucial to understanding the mechanisms and control of gene expression and protein synthesis. The realization that RNAs are often far more biologically active than was previously appreciated has stimulated a great deal of new research in this field. Uniquely, in this book, the world's leading researchers have collaborated to produce a comprehensive and current review of RNA-protein interactions for all scientists working in this area. Timely, comprehensive, and authoritative, this new Frontiers title will be invaluable for all researchers in molecular biology, biochemistry and structural biology.