Emerging Paradigms of Regulated Microrna Processing
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Crystal Structure of the Atpase Domain of Translation Initiation
Research Article 671 Crystal structure of the ATPase domain of translation initiation factor 4A from Saccharomyces cerevisiae — the prototype of the DEAD box protein family Jörg Benz1*, Hans Trachsel2 and Ulrich Baumann1* Background: Translation initiation factor 4A (eIF4A) is the prototype of the Addresses: 1Departement für Chemie und DEAD-box family of proteins. DEAD-box proteins are involved in a variety of Biochemie, Universität Bern, Freiestrasse 3, Germany and 2Institut für Biochemie und cellular processes including splicing, ribosome biogenesis and RNA Molekularbiologie, Universität Bern, Bühlstrasse 28, degradation. Energy from ATP hydrolysis is used to perform RNA unwinding CH-3012 Bern, Switzerland. during initiation of mRNA translation. The presence of eIF4A is required for the 43S preinitiation complex to bind to and scan the mRNA. *Corresponding authors. E-mail: [email protected] [email protected] Results: We present here the crystal structure of the nucleotide-binding domain of eIF4A at 2.0 Å and the structures with bound adenosinediphosphate Key words: crystal, DEAD box, NTPase, translation and adenosinetriphosphate at 2.2 Å and 2.4 Å resolution, respectively. The initiation, X-ray structure of the apo form of the enzyme has been determined by multiple Received: 22 February 1999 isomorphous replacement. The ATPase domain contains a central seven- Revisions requested: 18 March 1999 stranded β sheet flanked by nine α helices. Despite low sequence homology to Revisions received: 19 April 1999 the NTPase domains of RNA and DNA helicases, the three-dimensional fold of Accepted: 22 April 1999 eIF4A is nearly identical to the DNA helicase PcrA of Bacillus Published: 1 June 1999 stearothermophilus and to the RNA helicase NS3 of hepatitis C virus. -
MECHANISM of RNA REMODELING by DEAD-BOX HELICASES By
MECHANISM OF RNA REMODELING BY DEAD-BOX HELICASES by QUANSHENG YANG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Dr. Eckhard Jankowsky Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ___________________Quansheng Yang______________________________________ candidate for the__________________Ph.D._____________________degree (signed)__________William Merrick____________________________ (Chair of the Committee) ___________Vernon Anderson___________________________ ___________Eckhard Jankowsky _________________________ ___________Anthony Berdis ____________________________ ____________________________________________________ ____________________________________________________ (date) ___________12/12/2006______________________ 2 Table of Contents Chapter 1: Structures and biochemical activities of DExH/D helicases………………...11 Chapter 2: Initial characterization of Ded1…………….....…………………...................31 Chapter 3: ATP and ADP-dependent modulation of RNA unwinding and strand annealing activities by the DEAD-box helicase Ded1…………………………………...41 Chapter 4: Protein-assisted RNA structure conversion towards and against thermodynamic equilibrium values………………………………………………………68 Chapter 5: Duplex unwinding by a DEAD-box helicase without translocation on the loading strand...………………………………………………………………................101 Chapter 6: Duplex unwinding by DEAD-box helicases -
A Helicase-Independent Activity of Eif4a in Promoting Mrna Recruitment to the Human Ribosome
A helicase-independent activity of eIF4A in promoting mRNA recruitment to the human ribosome Masaaki Sokabea and Christopher S. Frasera,1 aDepartment of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis, CA 95616 Edited by Alan G. Hinnebusch, National Institutes of Health, Bethesda, MD, and approved May 5, 2017 (received for review December 12, 2016) In the scanning model of translation initiation, the decoding site and at the solvent side of the mRNA entry channel (14). Importantly, latch of the 40S subunit must open to allow the recruitment and that study showed that a short mRNA that does not extend into the migration of messenger RNA (mRNA); however, the precise molec- entry channel fails to displace eIF3j. A similar observation was also ular details for how initiation factors regulate mRNA accommodation found for initiation mediated by the hepatitis C virus internal ribo- into the decoding site have not yet been elucidated. Eukaryotic some entry site, where an mRNA truncated after the initiation co- initiation factor (eIF) 3j is a subunit of eIF3 that binds to the mRNA don failed to displace eIF3j (11). Taken together, these studies entry channel and A-site of the 40S subunit. Previous studies have suggest a model in which a full accommodation of mRNA in the shown that a reduced affinity of eIF3j for the 43S preinitiation mRNA entry channel of the 40S subunit corresponds to a reduced complex (PIC) occurs on eIF4F-dependent mRNA recruitment. Because affinity of eIF3j for the 40S subunit. This model has allowed us to eIF3j and mRNA bind anticooperatively to the 43S PIC, reduced eIF3j exploit the change in eIF3j affinity for the 43S PIC to quantitatively affinity likely reflects a state of full accommodation of mRNA into the monitor the process of mRNA recruitment. -
DEAD-Box RNA Helicases in Cell Cycle Control and Clinical Therapy
cells Review DEAD-Box RNA Helicases in Cell Cycle Control and Clinical Therapy Lu Zhang 1,2 and Xiaogang Li 2,3,* 1 Department of Nephrology, Renmin Hospital of Wuhan University, Wuhan 430060, China; [email protected] 2 Department of Internal Medicine, Mayo Clinic, 200 1st Street, SW, Rochester, MN 55905, USA 3 Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 1st Street, SW, Rochester, MN 55905, USA * Correspondence: [email protected]; Tel.: +1-507-266-0110 Abstract: Cell cycle is regulated through numerous signaling pathways that determine whether cells will proliferate, remain quiescent, arrest, or undergo apoptosis. Abnormal cell cycle regula- tion has been linked to many diseases. Thus, there is an urgent need to understand the diverse molecular mechanisms of how the cell cycle is controlled. RNA helicases constitute a large family of proteins with functions in all aspects of RNA metabolism, including unwinding or annealing of RNA molecules to regulate pre-mRNA, rRNA and miRNA processing, clamping protein complexes on RNA, or remodeling ribonucleoprotein complexes, to regulate gene expression. RNA helicases also regulate the activity of specific proteins through direct interaction. Abnormal expression of RNA helicases has been associated with different diseases, including cancer, neurological disorders, aging, and autosomal dominant polycystic kidney disease (ADPKD) via regulation of a diverse range of cellular processes such as cell proliferation, cell cycle arrest, and apoptosis. Recent studies showed that RNA helicases participate in the regulation of the cell cycle progression at each cell cycle phase, including G1-S transition, S phase, G2-M transition, mitosis, and cytokinesis. -
DEAD-Box RNA Helicases Are Among the Onstituents of the Tobacco
iochemis t B try n & la P P h f y o Hafidh et al., J Plant Biochem Physiol 2013, 1:3 s l i Journal of o a l n o DOI: 10.4172/2329-9029.1000114 r g u y o J ISSN: 2329-9029 Plant Biochemistry & Physiology Short Communication OpenOpen Access Access DEAD-Box RNA Helicases are among the Constituents of the Tobacco Pollen mRNA Storing Bodies Said Hafidh1, David Potěšil2,3, Zbyněk Zdráhal2,3 and David Honys1* 1Laboratory of Pollen Biology, Institute of Experimental Botany ASCR, Rozvojova 263, 165 02 Praha 6, Czech Republic 2CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic 3National centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech Republic Keywords: Translation; mRNA storage; RNA helicase; mRNP; Results and Discussion Ribonucleoprotein; Pollen Of the complex EPP proteome, we have identified three isoforms Introduction of the DEAD-box RNA helicase family eukaryotic translation initiation factor 4A (eIF4A)-8,9,13 as designated components of the EPP Male gametogenesis in flowering plants is attributed by RNA granules. eIF4A is required for binding of capped mRNA to substantial changes in cellular reorganization, cell fate specification the 40S ribosomal subunit via eIF3, whilst its ATP-dependent helicase and changes in cellular metabolism including RNA metabolism. activity functions in unwinding the secondary structure of the 5’ UTR Sequestration from immediate translation of protein coding mRNAs to facilitate ribosomal binding and translation. In synapses, eIF4A is has emerged as one aspect of mRNA post transcriptional regulation a target of smRNA (BC1) that blocks the eIF4A helicase activity and that is widespread during gametogenesis. -
Crystal Structure of Conserved Domains 1 and 2 of the Human DEAD-Box Helicase DDX3X in Complex with the Mononucleotide AMP
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ZENODO doi:10.1016/j.jmb.2007.06.050 J. Mol. Biol. (2007) 372, 150–159 Crystal Structure of Conserved Domains 1 and 2 of the Human DEAD-box Helicase DDX3X in Complex with the Mononucleotide AMP Martin Högbom1†, Ruairi Collins1†, Susanne van den Berg1 Rose-Marie Jenvert2, Tobias Karlberg1, Tetyana Kotenyova1 Alex Flores1, Gunilla B. Karlsson Hedestam3 and Lovisa Holmberg Schiavone1⁎ 1Structural Genomics DExD-box helicases are involved in all aspects of cellular RNA metabolism. Consortium, Department of Conserved domains 1 and 2 contain nine signature motifs that are Medical Biochemistry and responsible for nucleotide binding, RNA binding and ATP hydrolysis. Biophysics, Karolinska Institute, The human DEAD-box helicase DDX3X has been associated with several SE-171 77 Stockholm, Sweden different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/ 2School of Life Sciences, partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the Södertörns högskola, crystal structure of conserved domains 1 and 2 of DDX3X, including a SE-141 04 Huddinge, Sweden DDX3-specific insertion that is not generally found in human DExD-box 3Department of Microbiology, helicases, is presented. The N-terminal domain 1 and the C-terminal domain Tumor and Cell Biology, 2 both display RecA-like folds comprising a central β-sheet flanked by Karolinska Institute, α-helices. Interestingly, the DDX3X-specific insertion forms a helical SE-171 77 Stockholm, Sweden element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. -
Post-Transcriptional Control of Mirna Biogenesis
Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Michlewski and Caceres Post-transcriptional control of miRNA biogenesis GRACJAN MICHLEWSKI1,2 and JAVIER F. CÁCERES3 1Division of Infection and Pathway Medicine, University of Edinburgh, The Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK; 2Zhejiang University-University of Edinburgh Institute, Zhejiang University, 718 East Haizhou Rd., Haining, Zhejiang 314400, P.R. China; 3MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK Corresponding authors: [email protected], [email protected] 1 Downloaded from rnajournal.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Michlewski and Caceres ABSTRACT MicroRNAs (miRNAs) are important regulators of gene expression that bind complementary target mRNAs and repress their expression. Precursor miRNA molecules undergo nuclear and cytoplasmic processing events, carried out by the endoribonucleases, DROSHA and DICER, respectively, to produce mature miRNAs that are loaded onto the RISC (RNA-induced silencing complex) to exert their biological function. Regulation of mature miRNA levels is critical in development, differentiation and disease, as demonstrated by multiple levels of control during their biogenesis cascade. Here, we will focus on post- transcriptional mechanisms and will discuss the impact of cis-acting sequences in precursor miRNAs, as well as trans-acting factors that bind to these precursors and influence their processing. In particular, we will highlight the role of general RNA-binding proteins (RBPs) as factors that control the processing of specific miRNAs, revealing a complex layer of regulation in miRNA production and function. -
Microprocessor Dynamics and Interactions at Endogenous Imprinted C19MC Microrna Genes
Research Article 2709 Microprocessor dynamics and interactions at endogenous imprinted C19MC microRNA genes Cle´ment Bellemer1,2, Marie-Line Bortolin-Cavaille´ 1,2, Ute Schmidt3, Stig Mølgaard Rask Jensen4,*, Jørgen Kjems4, Edouard Bertrand3 and Je´roˆ me Cavaille´ 1,2,` 1Laboratoire de Biologie Mole´culaire Eucaryote (LBME), Universite´ Paul Sabatier (UPS), Universite´ de Toulouse, 31000 Toulouse, France 2CNRS, LBME, 31000 Toulouse, France 3Institute of Molecular Genetics of Montpellier, CNRS UMR 5535, 34293 Montpellier, France 4Interdisciplinary Nanoscience Center iNANO, Department of Molecular Biology, Aarhus University, C.F. Møllers Alle´, Building 1130, Room 404, 8000 Aarhus, Denmark *Present address: National Cancer Institute, Frederick Building 567, Room 261/275, Frederick, MD 21702, USA `Author for correspondence ([email protected]) Accepted 2 February 2012 Journal of Cell Science 125, 2709–2720 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.100354 Summary Nuclear primary microRNA (pri-miRNA) processing catalyzed by the DGCR8–Drosha (Microprocessor) complex is highly regulated. Little is known, however, about how microRNA biogenesis is spatially organized within the mammalian nucleus. Here, we image for the first time, in living cells and at the level of a single microRNA cluster, the intranuclear distribution of untagged, endogenously-expressed pri-miRNAs generated at the human imprinted chromosome 19 microRNA cluster (C19MC), from the environment of transcription sites to single molecules of fully released DGCR8-bound pri-miRNAs dispersed throughout the nucleoplasm. We report that a large fraction of Microprocessor concentrates onto unspliced C19MC pri-miRNA deposited in close proximity to their genes. Our live-cell imaging studies provide direct visual evidence that DGCR8 and Drosha are targeted post-transcriptionally to C19MC pri-miRNAs as a preformed complex but dissociate separately. -
Mirna Targeting and Alternative Splicing in the Stress Response – Events Hosted by Membrane-Less Compartments Mariya M
© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs202002. doi:10.1242/jcs.202002 REVIEW miRNA targeting and alternative splicing in the stress response – events hosted by membrane-less compartments Mariya M. Kucherenko and Halyna R. Shcherbata* ABSTRACT 2012; Suh et al., 2002); in the long term, they globally alter Stress can be temporary or chronic, and mild or acute. Depending on multiple intracellular signaling pathways that control almost its extent and severity, cells either alter their metabolism, and adopt a every aspect of cellular physiology. Such persistent stresses cause new state, or die. Fluctuations in environmental conditions occur cells to adjust their metabolism and even cellular architecture to frequently, and such stress disturbs cellular homeostasis, but in survive. general, stresses are reversible and last only a short time. There is The speed and scale of gene expression readjustment are crucial increasing evidence that regulation of gene expression in response to parameters for optimal cell survival upon stress, and this adjustment temporal stress happens post-transcriptionally in specialized can happen at either the transcriptional or the post-transcriptional subcellular membrane-less compartments called ribonucleoprotein level (Fig. 1). During persistent stress, coordinated transcription (RNP) granules. RNP granules assemble through a concentration- factor networks are prominent regulators of the adaptive responses dependent liquid–liquid phase separation of RNA-binding proteins that result in global changes in gene expression, which is important that contain low-complexity sequence domains (LCDs). Interestingly, for slow but long-lasting adaptation and recovery (de Nadal et al., many factors that regulate microRNA (miRNA) biogenesis and 2011; Gray et al., 2014; Novoa et al., 2003). -
Mutual Regulation of RNA Silencing and the IFN Response As an Antiviral Defense System in Mammalian Cells
International Journal of Molecular Sciences Review Mutual Regulation of RNA Silencing and the IFN Response as an Antiviral Defense System in Mammalian Cells Tomoko Takahashi 1,2,* and Kumiko Ui-Tei 1,3,* 1 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan 2 Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan 3 Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8561, Japan * Correspondence: [email protected] (T.T.); [email protected] (K.U.-T.); Tel.: +81-48-858-3404 (T.T.); +81-3-5841-3044 (K.U.-T.) Received: 23 December 2019; Accepted: 15 February 2020; Published: 17 February 2020 Abstract: RNA silencing is a posttranscriptional gene silencing mechanism directed by endogenous small non-coding RNAs called microRNAs (miRNAs). By contrast, the type-I interferon (IFN) response is an innate immune response induced by exogenous RNAs, such as viral RNAs. Endogenous and exogenous RNAs have typical structural features and are recognized accurately by specific RNA-binding proteins in each pathway. In mammalian cells, both RNA silencing and the IFN response are induced by double-stranded RNAs (dsRNAs) in the cytoplasm, but have long been considered two independent pathways. However, recent reports have shed light on crosstalk between the two pathways, which are mutually regulated by protein–protein interactions triggered by viral infection. This review provides brief overviews of RNA silencing and the IFN response and an outline of the molecular mechanism of their crosstalk and its biological implications. -
Virus-Encoded Micrornas: an Overview and a Look to the Future
Review Virus-Encoded microRNAs: An Overview and a Look to the Future Rodney P. Kincaid, Christopher S. Sullivan* The University of Texas at Austin, Molecular Genetics & Microbiology, Austin, Texas, United States of America harmful nucleic acids such as endogenous transposons or Abstract: MicroRNAs (miRNAs) are small RNAs that play exogenous viruses (reviewed in [4–6]). While the antiviral role of important roles in the regulation of gene expression. First RNAi is well-established in plants, insects, and nematodes, this described as posttranscriptional gene regulators in does not seem to be the case in most (if not all) mammalian cell eukaryotic hosts, virus-encoded miRNAs were later contexts. When compared to some plants and invertebrates, strong uncovered. It is now apparent that diverse virus families, experimental evidence supporting an antiviral role for mammalian most with DNA genomes, but at least some with RNA RNAi is lacking yet remains the subject of ongoing debate [7–9]. genomes, encode miRNAs. While deciphering the func- Nevertheless, at least some components of the RNAi machinery tions of viral miRNAs has lagged behind their discovery, recent functional studies are bringing into focus these appear to protect mammalian cells against endogenous transposon roles. Some of the best characterized viral miRNA activity [10–12]. functions include subtle roles in prolonging the longevity Prokaryotes also possess a nucleic acid-based defense called of infected cells, evading the immune response, and Clustered Regularly Interspaced Short Palindromic Repeats regulating the switch to lytic infection. Notably, all of (CRISPRs). Like RNAi, CRISPRs can be thought of as a nucleic these functions are particularly important during persis- acid-based adaptive immune response providing protection against tent infections. -
Role of Micrornas in Hepatocarcinogenesis
Role of microRNAs in Hepatocarcinogenesis DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Bo Wang, M.S. Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2012 Dissertation Committee: Dr. Jacob T. Samson, Advisor Dr. Kalpana Ghoshal Dr. SaÏd Sif Dr. Thomas D. Schmittgen Copyright by Bo Wang 2012 Abstract MicroRNAs are conserved, small (20-25 nucleotide) noncoding RNAs that negatively regulate expression of mRNAs at the post-transcriptional level. MicroRNA signature is altered in different disease states including cancer and some microRNAs act as oncogenes or tumor suppressors. To identify microRNAs that may play a causal role in hepatocarcinogenesis we used an animal model in which C57BL/6 mice fed choline deficient and amino acid defined (CDAA) diet develop nonalcoholic steatohepatitis (NASH)-induced hepatocarcinogenesis after 70 weeks. Microarray analysis identified 30 hepatic microRNAs that are significantly (P≤0.01) altered in mice fed CDAA diet for 6, 18, 32 and 65 weeks compared to those fed choline sufficient and amino acid defined diet (CSAA). Real-time RT-PCR analysis demonstrated upregulation of oncogenic miR-155, miR-181b, miR-221/222 and miR-21 and downregulation of the most abundant liver specific miR-122 at early stages of hepatocarcinogenesis. Western blot analysis showed reduced expression of hepatic PTEN, a target of miR-21, and C/EBPβ, a target of miR-155, in these mice at early stages. DNA binding activity of NF-κB that transactivates miR-155 gene was significantly (P=0.002) elevated in the liver nuclear extract of mice fed CDAA diet.