DOCSLIB.ORG

Crystal Structure of the Human Eif4aiii–CWC22 Complex Shows How A

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Crystal Structure of the Human Eif4aiii–CWC22 Complex Shows How A Crystal structure of the human eIF4AIII–CWC22 PNAS PLUS complex shows how a DEAD-box protein is inhibited by a MIF4G domain Gretel Buchwalda, Steffen Schüsslera, Claire Basquina, Hervé Le Hirb,c, and Elena Contia,1 aDepartment of Structural Cell Biology, Max Planck Institute of Biochemistry, D-82152 Martinsried/Munich, Germany; bInstitut de Biologie de l’Ecole Normale Supérieure, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8197, 75005 Paris, France; and cInstitut de Biologie de l’Ecole Normale Supérieure, Institut National de la Santé et de la Recherche Médicale U1024, 75005 Paris, France Edited by Joan A. Steitz, Howard Hughes Medical Institute, New Haven, CT, and approved October 23, 2013 (received for review August 2, 2013) DEAD-box proteins are involved in all aspects of RNA processing. (17, 18). Conformational regulation is often used to modulate the They bind RNA in an ATP-dependent manner and couple ATP ATPase activity of DEAD-box proteins, for example in the hydrolysis to structural and compositional rearrangements of ribo- activation of the translation initiation factor 4AI (eIF4AI) by nucleoprotein particles. Conformational control is a major point of the MIF4G domain of eIF4G (22) and in the inhibition of regulation for DEAD-box proteins to act on appropriate substrates eIF4AI by the MA3 domain of PDCD4 (23, 24). The con- and in a timely manner in vivo. Binding partners containing a middle formational changes of DEAD-box proteins are important not domain of translation initiation factor 4G (MIF4G) are emerging as only for catalysis, but also for protein–protein interactions. In important regulators. Well-known examples are eIF4G and Gle1, the case of the EJC, for example, the closed RNA-bound con- which bind and activate the DEAD-box proteins eIF4A and Dbp5. formation of eIF4AIII is required to form a core complex with Here, we report the mechanism of an inhibiting MIF4G domain. its binding partners MAGO, Y14, and Barentsz (also known as We determined the 2.0-Å resolution structure of the complex of Metastatic Lymph Node 51; MLN51) (8, 17, 18). human eIF4AIII and the MIF4G domain of the splicing factor Com- The EJC core is assembled in an ATP-dependent manner on plexed With Cef1 (CWC22), an essential prerequisite for exon junc- – spliced mRNAs typically, but not exclusively, 20 24 nt upstream BIOCHEMISTRY tion complex assembly by the splicing machinery. The CWC22 MIF4G of exon-exon junctions (25–27). EJC assembly is a complex, domain binds both RecA domains of eIF4AIII. The mode of RecA2 stepwise process that is tightly coupled to the splicing reaction recognition is similar to that observed in the activating complexes, (28–30). Recent studies in human cells have shown that EJC yet is specific for eIF4AIII. The way the CWC22 MIF4G domain latches assembly requires the binding of eIF4AIII to the splicing factor on the eIF4AIII RecA1 domain is markedly different from activating Complexed With Cef1 (CWC22) (31–33). The interaction of complexes. In the CWC22–eIF4AIII complex, the RNA-binding and CWC22 with eIF4AIII is incompatible with the interaction with ATP-binding motifs of the two RecA domains do not face each other, as would be required in the active state, but are in diametrically MAGO and Y14 and is necessary for eIF4AIII recruitment to opposite positions. The binding mode of CWC22 to eIF4AIII reveals the splicing machinery (31, 32). Consistent with its role in EJC a facet of how MIF4G domains use their versatile structural frame- deposition, CWC22 participates to the degradation of an en- works to activate or inhibit DEAD-box proteins. dogenous NMD target in vivo (33). In vitro, CWC22 reduces the ATPase activity of eIF4AIII, suggesting that CWC22 keeps the helicase | mRNP | NMD DEAD-box protein in an inactive state before the EJC assembles at exon ligation (31). CWC22 contains two conserved regions EAD-box proteins are a large family of RNA-dependent fi DATPases involved in many aspects of RNA metabolism, Signi cance including processing, transport, translation, and decay (reviewed in refs. 1 and 2). These proteins generally function to remodel The fate of eukaryotic mRNAs is intimately linked to the com- ribonucleoprotein particles (RNPs), by locally unwinding the plement of proteins that associate with them to form mRNA— nucleic acid or by displacing and/or recruiting other factors to the protein complexes, the so-called messenger ribonucleoprotein nucleic acid they bind to (3–8). Although DEAD-box proteins particles (mRNPs). Transitions in the architecture of an mRNP fi recognize single-stranded RNAs in a sequence-independent man- lead to speci c functional consequences. DEAD-box proteins are ner in vitro, they act with exquisite specificity in vivo. As an example, key players in orchestrating these structural rearrangements: the translation initiation factor 4AI (eIF4AI), unwinds RNA sec- They associate with RNA in response to ATP binding and disso- ondary structure at the 5′ untranslated region (UTR) of mRNAs, ciate from it upon ATP hydrolysis. In this paper, we have eluci- and promotes the recruitment of the small ribosomal subunit (9– dated the molecular mechanisms by which a DEAD-box protein, 11). In contrast, the closely related paralogue eIF4AIII binds tightly which in human cells marks spliced mRNPs for a specialized on spliced mRNAs as part of the exon junction complex (EJC) (8). surveillance pathway, is recognized by the MIF4G domain of The EJC promotes nonsense-mediated mRNA decay (NMD) in a splicing factor. This structure shows how a MIF4G domain can human cells (12–14) and the localization of oskar mRNA in the act as a negative regulator of DEAD-box ATPase activity. Drosophila embryo (14, 15). Author contributions: G.B., H.L.H., and E.C. designed research; G.B., S.S., and C.B. per- DEAD-box proteins have a common architecture based on formed research; G.B., S.S., C.B., and E.C. analyzed data; and G.B. and E.C. wrote the two RecA domains connected by a flexible linker (reviewed in paper. ref. 3). A hallmark of these proteins is the conformational The authors declare no conflict of interest. plasticity with which they cycle between the active and inactive This article is a PNAS Direct Submission. states of the ATPase reaction (reviewed in ref. 4). In the active Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, state, the two RecA domains adopt a characteristic closed con- www.pdb.org (PDB ID code 4C9B). formation that positions the residues responsible for ATP hy- 1To whom correspondence should be addressed. E-mail: [email protected]. – drolysis in the appropriate geometry for catalysis (16 21). In the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. inactive state, the two domains are in more open configurations 1073/pnas.1314684110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1314684110 PNAS Early Edition | 1of8 Downloaded by guest on October 2, 2021 predicted to fold into MIF4G and MA3 domains. Counterintu- proteins to the RecA2 domain of eIF4AIII and to a region of itively with respect to the known activating properties of MIF4G CWC22 containing the MIF4G domain (residues 110–409) (31, domains and inhibiting properties of MA3 domains on other 32). We purified different portions of the molecules and assessed DEAD-box proteins (21–24), CWC22 uses its MIF4G domain to their relative binding affinities quantitatively by using isothermal bind eIF4AIII (32). In this work, we have elucidated the mech- titration calorimetry (ITC). anism with which the MIF4G domain of CWC22 specifically An evolutionary conserved region of CWC22 spanning the recognizes and negatively regulates eIF4AIII. MIF4G and MA3 domains (residues 116–656) bound full-length (f.l.) eIF4AIII with a Kd of 67.5 nM (Fig. 1B and Fig. S1). The Results and Discussion MA3 domain (residues 450–665) showed no detectable binding to Domain Requirements for eIF4AIII–CWC22 Complex Formation. Hu- eIF4AIII (Fig. 1B and Fig. S1). The presence of the MA3 domain man eIF4AIII (residues 1–411) contains an N-terminal RecA1 appeared to even have a small negative contribution to eIF4AIII domain and a C-terminal RecA2 domain (17, 18) (Fig. 1A). binding: A segment lacking the entire MA3 domain (residues 116– – CWC22 (residues 1 656) contains MIF4G and MA3 domains 406) resulted in a Kd of 28.4 nM (Fig. 1 B and C). Remarkably, predicted approximately between residues 145–350 and 450–656, a further 20-residue truncation to the stable structural core of the respectively (using the Phyre (34) and HHpred prediction serv- MIF4G domain identified by limited proteolysis (residues 137– ers; ref. 35) (Fig. 1A). Previous coimmunoprecipitation and pull- 406) decreased binding to eIF4AIII 100-fold (Kd 3.1 μM; Fig. 1 B down experiments have mapped the interaction between the two and C). We concluded that elements both within and outside the Fig. 1. Domain requirements for eIF4AIII–CWC22 recognition. (A) Schematic domain organization of human eIF4AIII and CWC22. The RecA domains of eIF4AIII are in yellow. In the case of CWC22, the MIF4G fold is in blue and the N- and C-terminal extension are in cyan and red, respectively. Arrows highlight the regions of the molecules used for crystallization of the complex. The residue numbers of the domain boundaries are indicated (obtained either fromthe structural analysis in this manuscript or predicted in the case of the MA3 domain). (B) Table summarizing the results of the isothermal calorimetry experiments shown in the manuscript and in Fig. S1. The dissociation constants (Kd) with SDs were calculated with the program Origin. (C) Two representative ITC experiments, showing binding of eIF4AIII full length (f.l.) with different regions of CWC22.
Load more

Recommended publications
  • 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.
    [Show full text]
  • 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
    [Show full text]
  • 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.
    [Show full text]
  • The Exon Junction Complex Core Represses Caner-Specific Mature Mrna Re-Splicing: a Potential Key Role in Terminating Splicing
    bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438154; this version posted April 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Communication The Exon Junction Complex Core Represses Caner-specific Mature mRNA Re-splicing: A Potential Key Role in Terminating Splicing Yuta Otani1,2, Toshiki Kameyama1,3 and Akila Mayeda1,* 1 Division of Gene Expression Mechanism, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan 2 Laboratories of Discovery Research, Nippon Shinyaku Co., Ltd., Kyoto, Kyoto 601-8550, Japan 3 Present address: Department Physiology, Fujita Health University, School of Medicine, Toyoake, Aichi 470-1192, Japan * Correspondence: [email protected] (A.M.) 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438154; this version posted April 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract: Using the TSG101 pre-mRNA, we previously discovered cancer-specific re-splicing of mature mRNA that generates aberrant transcripts/proteins. The fact that mRNA is aberrantly re- spliced in various cancer cells implies there must be an important mechanism to prevent deleterious re-splicing on the spliced mRNA in normal cells. We thus postulated that the mRNA re-splicing is controlled by specific repressors and we searched for repressor candidates by siRNA-based screening for mRNA re-splicing activity.
    [Show full text]
  • 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.
    [Show full text]
  • Exon Junction Complexes Can Have Distinct Functional Flavours To
    www.nature.com/scientificreports OPEN Exon Junction Complexes can have distinct functional favours to regulate specifc splicing events Received: 9 November 2017 Zhen Wang1, Lionel Ballut2, Isabelle Barbosa1 & Hervé Le Hir1 Accepted: 11 June 2018 The exon junction complex (EJC) deposited on spliced mRNAs, plays a central role in the post- Published: xx xx xxxx transcriptional gene regulation and specifc gene expression. The EJC core complex is associated with multiple peripheral factors involved in various post-splicing events. Here, using recombinant complex reconstitution and transcriptome-wide analysis, we showed that the EJC peripheral protein complexes ASAP and PSAP form distinct complexes with the EJC core and can confer to EJCs distinct alternative splicing regulatory activities. This study provides the frst evidence that diferent EJCs can have distinct functions, illuminating EJC-dependent gene regulation. Te Exon Junction Complex (EJC) plays a central role in post-transcriptional gene expression control. EJCs tag mRNA exon junctions following intron removal by spliceosomes and accompany spliced mRNAs from the nucleus to the cytoplasm where they are displaced by the translating ribosomes1,2. Te EJC is organized around a core complex made of the proteins eIF4A3, MAGOH, Y14 and MLN51, and this EJC core serves as platforms for multiple peripheral factors during diferent post-transcriptional steps3,4. Dismantled during translation, EJCs mark a very precise period in mRNA life between nuclear splicing and cytoplasmic translation. In this window, EJCs contribute to alternative splicing5–7, intra-cellular RNA localization8, translation efciency9–11 and mRNA stability control by nonsense-mediated mRNA decay (NMD)12–14. At a physiological level, developmental defects and human pathological disorders due to altered expression of EJC proteins show that the EJC dosage is critical for specifc cell fate determinations, such as specifcation of embryonic body axis in drosophila, or Neural Stem Cells division in the mouse8,15,16.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • The Dynamic Fate of the Exon Junction Complex
    The Dynamic Fate of the Exon Junction Complex Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Robert Dennison Patton, B.S. Graduate Program in Physics The Ohio State University 2020 Dissertation Committee Dr. Ralf Bundschuh, Advisor Dr. Guramrit Singh, Co-Advisor Dr. Michael Poirier Dr. Enam Chowdhury 1 © Copyrighted by Robert Dennison Patton 2020 2 Abstract The Exon Junction Complex, or EJC, is a group of proteins deposited on mRNA upstream of exon-exon junctions during splicing, and which stays with the mRNA up until translation. It consists of a trimeric core made up of EIF4A3, Y14, and MAGOH, and serves as a binding platform for a multitude of peripheral proteins. As a lifelong partner of the mRNA the EJC influences almost every step of post-transcriptional mRNA regulation, including splicing, packaging, transport, translation, and Nonsense-Mediated Decay (NMD). In Chapter 2 I show that the EJC exists in two distinct complexes, one containing CASC3, and the other RNPS1. These complexes are localized to the cytoplasm and nucleus, respectively, and a new model is proposed wherein the EJC begins its life post- splicing bound by RNPS1, which at some point before translation in the cytoplasm is exchanged for CASC3. These alternate complexes also take on distinct roles; RNPS1- EJCs help form a compact mRNA structure for easier transport and make the mRNA more susceptible to NMD. CASC3-EJCs, on the other hand, cause a more open mRNA configuration and stabilize it against NMD. Following the work with the two alternate EJCs, in Chapter 3 I examine why previous research only found the CASC3-EJC variant.
    [Show full text]
  • Understanding Regulation of Mrna by RNA Binding Proteins Alexander
    Understanding Regulation of mRNA by RNA Binding Proteins MA SSACHUSETTS INSTITUTE by OF TECHNOLOGY Alexander De Jong Robertson B.S., Stanford University (2008) LIBRARIES Submitted to the Graduate Program in Computational and Systems Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Computational and Systems Biology at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY February 2014 o Massachusetts Institute of Technology 2014. All rights reserved. A A u th o r .... v ..... ... ................................................ Graduate Program in Computational and Systems Biology December 19th, 2013 C ertified by .............................................. Christopher B. Burge Professor Thesis Supervisor A ccepted by ........ ..... ............................. Christopher B. Burge Computational and Systems Biology Ph.D. Program Director 2 Understanding Regulation of mRNA by RNA Binding Proteins by Alexander De Jong Robertson Submitted to the Graduate Program in Computational and Systems Biology on December 19th, 2013, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Computational and Systems Biology Abstract Posttranscriptional regulation of mRNA by RNA-binding proteins plays key roles in regulating the transcriptome over the course of development, between tissues and in disease states. The specific interactions between mRNA and protein are controlled by the proteins' inherent affinities for different RNA sequences as well as other fea- tures such as translation and RNA structure which affect the accessibility of mRNA. The stabilities of mRNA transcripts are regulated by nonsense-mediated mRNA de- cay (NMD), a quality control degradation pathway. In this thesis, I present a novel method for high throughput characterization of the binding affinities of proteins for mRNA sequences and an integrative analysis of NMD using deep sequencing data.
    [Show full text]
  • UPF1: from Mrna Surveillance to Protein Quality Control
    biomedicines Review UPF1: From mRNA Surveillance to Protein Quality Control Hyun Jung Hwang 1,2, Yeonkyoung Park 1,2 and Yoon Ki Kim 1,2,* 1 Creative Research Initiatives Center for Molecular Biology of Translation, Korea University, Seoul 02841, Korea; [email protected] (H.J.H.); [email protected] (Y.P.) 2 Division of Life Sciences, Korea University, Seoul 02841, Korea * Correspondence: [email protected] Abstract: Selective recognition and removal of faulty transcripts and misfolded polypeptides are crucial for cell viability. In eukaryotic cells, nonsense-mediated mRNA decay (NMD) constitutes an mRNA surveillance pathway for sensing and degrading aberrant transcripts harboring premature termination codons (PTCs). NMD functions also as a post-transcriptional gene regulatory mechanism by downregulating naturally occurring mRNAs. As NMD is activated only after a ribosome reaches a PTC, PTC-containing mRNAs inevitably produce truncated and potentially misfolded polypeptides as byproducts. To cope with the emergence of misfolded polypeptides, eukaryotic cells have evolved sophisticated mechanisms such as chaperone-mediated protein refolding, rapid degradation of misfolded polypeptides through the ubiquitin–proteasome system, and sequestration of misfolded polypeptides to the aggresome for autophagy-mediated degradation. In this review, we discuss how UPF1, a key NMD factor, contributes to the selective removal of faulty transcripts via NMD at the molecular level. We then highlight recent advances on UPF1-mediated communication between mRNA surveillance and protein quality control. Keywords: nonsense-mediated mRNA decay; UPF1; aggresome; CTIF; mRNA surveillance; protein quality control Citation: Hwang, H.J.; Park, Y.; Kim, Y.K. UPF1: From mRNA Surveillance to Protein Quality Control. Biomedicines 2021, 9, 995.
    [Show full text]
  • Transcriptome Analysis of Alternative Splicing-Coupled Nonsense-Mediated Mrna Decay in Human Cells Reveals Broad Regulatory Potential
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.01.183327. this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Transcriptome analysis of alternative splicing-coupled nonsense-mediated mRNA decay in human cells reveals broad regulatory potential Courtney E. French1,#a*, Gang Wei2,#b*, James P. B. Lloyd2,3,#c, Zhiqiang Hu2, Angela N. Brooks1,#d, Steven E. Brenner1,2,3,$ 1 Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA 2 Department of Plant and Microbial Biology, University of California, Berkeley, CA, 94720, USA 3 Center for RNA Systems Biology, University of California, Berkeley, CA, 94720, USA #a Current address: Department of Paediatrics, University of Cambridge, Cambridge, CB2 1TN, UK #b Current address: State Key Laboratory of Genetics Engineering & MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, 200433, China #c Current address: ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Perth, Australia #d Current address: Department of Biomolecular Engineering, University of California, Santa Cruz, CA, USA * These authors contributed equally to this work $ Correspondence: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.01.183327. this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. It is made available under a CC-BY 4.0 International license. Abstract: To explore the regulatory potential of nonsense-mediated mRNA decay (NMD) coupled with alternative splicing, we globally surveyed the transcripts targeted by this pathway via RNA- Seq analysis of HeLa cells in which NMD had been inhibited.
    [Show full text]