DOCSLIB.ORG

Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

Research Collection Doctoral Thesis Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6 Author(s): Voigts-Hoffmann, Felix Publication Date: 2012 Permanent Link: https://doi.org/10.3929/ethz-a-007303759 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library ETH Zurich Dissertation No. 20189 Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6 A dissertation submitted to ETH ZÜRICH for the degree of Doctor of Sciences (Dr. sc. ETH Zurich) presented by Felix Voigts-Hoffmann MSc Molecular Biotechnology, Universität Heidelberg born April 11, 1981 citizen of Göttingen, Germany accepted on recommendation of Prof. Dr. Nenad Ban (Examiner) Prof. Dr. Raimund Dutzler (Co-examiner) Prof. Dr. Rudolf Glockshuber (Co-examiner) 2012 blank page ii Summary Ribosomes are large complexes of several ribosomal RNAs and dozens of proteins, which catalyze the synthesis of proteins according to the sequence encoded in messenger RNA. Over the last decade, prokaryotic ribosome structures have provided the basis for a mechanistic understanding of protein synthesis. While the core functional centers are conserved in all kingdoms, eukaryotic ribosomes are much larger than archaeal or bacterial ribosomes. Eukaryotic ribosomal rRNA and proteins contain extensions or insertions to the prokaryotic core, and many eukaryotic proteins do not have prokaryotic counterparts. Furthermore, translation regulation and ribosome biogenesis is much more complex in eukaryotes, and defects in components of the translation machinery are associated with human diseases and cancer. Atomic resolution structures of eukaryotic ribosomes are required to understand the regulatory aspects of translation in higher organisms at a molecular level, and to elucidate the structural differences to the prokaryotic ribosome that are the basis of antibiotic selectivity. T. We thermophila have determined the crystal structure of the large ribosomal subunit of the ciliate in complex with initiation factor 6 and cycloheximide (CHX), an inhibitor of eukaryotic protein synthesis. At a resolution of 3.5 angstrom (Å), the atomic model reveals the structure and localization of the eukaryotic-­‐specific protein moieties and rRNA expansion segments. While the peptidyl transferase center (PTC), the subunit interface, and the surface around the exit tunnel are conserved, rRNA expansion segments and eukaryotic specific protein moieties form a complex network of interactions on the solvent-­‐exposed side of the large subunit, and on the back of stalk regions. Some proteins also reach deeply into the core of large subunit, suggesting that they may have a role in the stabilization of the rRNA. We observe difference density for the glutarimide antibiotic cycloheximide (CHX) at the tRNA exit site and localize mutations of ribosomal proteins that are known to confer CHX resistance, rationalizing existing biochemical data. Furthermore, differences to the prokaryotic ribosome in the polypeptide exit tunnel may have important consequences for the binding of macrolide antibiotics such as erythromycin. Eukaryotic initiation factor 6 is required to prevent joining of the small subunit to 60S precursors during ribosome biogenesis and nuclear export. It has also been linked to cellular signaling pathways and is known to play an important role during development, in human diseases, and in cancer. The structure of the 60S:eIF6 complex resolves an iii ambiguity about the binding site and facilitates further characterization of the cellular function of eIF6. In eukaryotes, but not in archaea, RPL40 is expressed as a ubiquitin fusion. In analogy to the small ribosomal subunit ubiquitin fusion protein rpS27A, it occupies a functionally important position near the elongation factor binding site, indicating that ubiquitin cleavage is prerequisite T. thermophila for ribosome function. The structure of the large ribosomal subunit from complements the recently published structure of the 40S subunit from the same organism, establishing a structural framework for analyses of the eukaryotic-­‐specific aspects of translation. Furthermore, crystals of individual ribosomal subunits provide the opportunity to study complexes with regulatory factors and proteins required for ribosome biogenesis. Subtle structural differences to the bacterial ribosome in the areas surrounding conserved functional centers may be exploited for the design of antibiotics, and inhibitors of eukaryotic translation may hold therapeutic potential for the treatment of fungal infections and cancer. iv Zusammenfassung Ribosomen sind die Proteinfabriken der Zelle und katalysieren die Polymerisation von Aminosäuren in der durch die Boten RNA (Messenger RNA, mRNA) festgelegten Reihenfolge. Sie bestehen aus mehreren ribosomalen RNAs (rRNAs) und Dutzenden von Proteinen. Basierend auf den Kristallstrukturen prokaryotischer Ribosomen konnte im letzten Jahrzehnt ein detailliertes Verständnis der Proteinbiosynthese in diesen Organismen erarbeitet werden. Während die grundlegenden Funktionen in allen Organismen konserviert sind, gibt es signifikante Abweichungen in Bezug auf die Biogenese und Regulierung. Eukaryotische Ribosomen sind deutlich grösser als die Ribosomen von Bakterien oder Archaen und enthalten zusätzliche Proteine sowie Expansionssegmente, welche den konservierten Kern der ribosomalen RNA erweitern. Genetische Abweichungen in der Proteinbiosynthese Maschinerie führen zu schweren Erkrankungen und Entwicklungsdefekten, welche mit einem erhöhten Krebsrisiko einhergehen. Um die Regulationsmechanismen der eukaryotischen Translation zu verstehen, und die Unterschiede zum bakteriellen System zu eruieren, welche die selektive Wirkung von Antibiotika ermöglichen, sind hochaufgelöste Strukturen des eukaryotischen Ribosoms erforderlich. Wir haben die T. Kristallstruktur thermophila eines Komplexes der großen ribosomalen Untereinheit des Wimperntierchens mit dem eukaryotischen Initiationsfaktor 6 bei einer Auflösung von 3.5 Ångstrom (Å) bestimmt. Das atomare Modell zeigt die Struktur und Lokalisierung der rRNA Expansionssegmente und der zusätzlichen Proteinbestandteile. Der Komplex enthält weiterhin das Antibiotikum Cycloheximid (CHX), welches die Proteinbiosynthese bei Eukaryoten blockiert. Das aktive Zentrum und die Grenzfläche zur kleinen ribosomalen Untereinheit sind im Vergleich zum prokaryotischen Ribosom stark konserviert. Die für eukaryotische Ribosomen spezifischen rRNA Expansionssegmente und Proteinbestandteile bilden ein komplexes Netzwerk von Interaktionen auf der gegenüberliegenden, der kleinen Untereinheit abgewandten Seite. Davon ausgenommen ist die Region um den ribosomalen Tunnel, durch den die synthetisierten Polypeptide die große Untereinheit verlassen. Einige eukaryotische Proteinbestandteile reichen weit in den Kern der großen ribosomalen Untereinheit hinein und könnten die Struktur der ribosomalen RNA stabilisieren. v Anhand von Differenz-­‐Elektronendichte Karten können wir die Bindungsstelle von Cycloheximid in der ribosomalen E-­‐Stelle lokalisieren und biochemische Ergebnisse rationalisieren, nach denen Mutationen in ribosomalen Proteinen die Bindung von Cycloheximid beeinträchtigen. Strukturelle Unterschiede zum prokaryotischen Ribosom im Bereich des Tunnels können signifikante Auswirkungen auf die Bindung von Makrolid-­‐Antibiotika haben, zu denen auch Erythromycin gehört. Der eukaryotische Translations-­‐Initiationsfaktor eIF6 verhindert während der Biogenese von Ribosomen die Assoziation von kleiner und großer Untereinheit. Des Weiteren spielt eIF6 eine Rolle bei menschlichen Erkrankungen und Krebs. Die Kristallstruktur des Komplexes klärt eine Unsicherheit in Bezug auf die Bindungsstelle von eIF6 an der großen Untereinheit und ermöglicht weitere Experimente zur Untersuchung einer möglichen Verbindung zwischen eIF6 und zellulären Signaltransduktionswegen. Im Gegensatz zu Archaen ist das ribosomale Protein RPL40 bei Eukaryoten mit einer Ubiquitin Domäne fusioniert. Wie ein Ubiquitin Fusionsprotein der kleinen ribosomalen Untereinheit, rpS27A, nimmt auch RPL40 eine funktionell wichtige Position in der Nähe der ribosomalen Bindungsstelle für Elongationsfaktoren ein. Daher ist die proteolytische Entfernung von Ubiquitin Domänen für die Funktion des eukaryotischen Ribosoms unerlässlich. In Verbindung mit der kürzlich veröffentlichen Kristallstruktur der T. kleinen ribosomalen Untereinheit thermophila bietet die Struktur der großen Ribosomalen Untereinheit von eine Grundlage für das Verständnis eukaryotischer Aspekte der Proteinbiosynthese auf molekularer Ebene. Insbesondere bieten Kristalle der einzelnen Untereinheiten die Möglichkeit, Komplexe mit Regulations-­‐ und Biogenesefaktoren zu untersuchen. Die strukturellen Unterschiede zum prokaryotischen Ribosom können für die Entwicklung selektiver Antibiotika genutzt werden. Darüber hinaus könnten auch Inhibitoren der eukaryotischen Proteinbiosynthese therapeutisch einsetzbar sein, beispielsweise in der Behandlung von Krebserkrankungen oder Pilzinfektionen. vi Acknowledgements Nenad Ban Sebastian Klinge and Marc André Leibundgut Sofia Arpagaus, Tabitha Bucher, Timm Meier, Julius Rabl and Jan Erzberger The Ban group The SLS Team Family and Friends vii blank page viii Table of Contents Summary ..............................................................................................................................................................
Recommended publications
  • Evolution of Translation EF-Tu: Trna

    Evolution of Translation EF-Tu: Trna

    University of Illinois at Urbana-Champaign Luthey-Schulten Group NIH Resource for Macromolecular Modeling and Bioinformatics Computational Biophysics Workshop Evolution of Translation EF-Tu: tRNA VMD Developer: John Stone MultiSeq Developers Tutorial Authors Elijah Roberts Ke Chen John Eargle John Eargle Dan Wright Zhaleh Ghaemi Jonathan Lai Zan Luthey-Schulten August 2014 A current version of this tutorial is available at http://www.scs.illinois.edu/~schulten/tutorials/ef-tu CONTENTS 2 Contents 1 Introduction 3 1.1 The Elongation Factor Tu . 3 1.2 Getting Started . 4 1.2.1 Requirements . 4 1.2.2 Copying the tutorial files . 4 1.2.3 Working directory . 4 1.2.4 Preferences . 4 1.3 Configuring BLAST for MultiSeq . 5 2 Comparative Analysis of EF-Tu 5 2.1 Finding archaeal EF1A sequences . 6 2.2 Aligning archaeal sequences and removing redundancy . 8 2.3 Finding bacteria EF-Tu sequences . 11 2.4 Performing ClustalW Multiple Sequence and Profile-Profile Align- ments . 12 2.5 Creating Multiple Sequence with MAFFT . 16 2.6 Conservation of EF-Tu among the Bacteria . 16 2.7 Finding conserved residues across the bacterial and archaeal do- mains . 20 2.8 EF-Tu Interface with the Ribosome . 21 3 Computing a Maximum Likelihood Phylogenetic Tree with RAxML 23 3.1 Load the Phylogenetic Tree into MultiSeq . 25 3.2 Reroot and Manipulate the Phylogenetic Tree . 25 4 MultiSeq TCL Scripting: Genomic Context 27 5 Appendix A 30 5.1 Building a BLAST Database . 30 6 Appendix B 31 6.1 Saving QR subset of alignments in PHYLIP and FASTA format 31 6.2 Calculating Maximum Likelihood Trees with RAxML .
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
  • Domain of Escherichia Coli 16S Ribosomal RNA Using Site-Directed Photoaffinity Crosslinking

    Domain of Escherichia Coli 16S Ribosomal RNA Using Site-Directed Photoaffinity Crosslinking

    Downloaded from rnajournal.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press RNA (1998), 4:1455–1466+ Cambridge University Press+ Printed in the USA+ Copyright © 1998 RNA Society+ Analysis of the conformation of the 39 major domain of Escherichia coli 16S ribosomal RNA using site-directed photoaffinity crosslinking ALEXANDRE MONTPETIT,1 CATHERINE PAYANT,1 JAMES M. NOLAN,2 and LÉA BRAKIER-GINGRAS1 1Département de Biochimie, Université de Montréal, Montréal, Québec H3T 1J4, Canada 2Department of Biochemistry, Tulane University Medical Center, New Orleans, Louisiana 70112, USA ABSTRACT The 39 major domain of Escherichia coli 16S rRNA, which occupies the head of the small ribosomal subunit, is involved in several functions of the ribosome. We have used a site-specific crosslinking procedure to gain further insights into the higher-order structure of this domain. Circularly permuted RNAs were used to introduce an azi- dophenacyl group at specific positions within the 39 major domain. Crosslinks were generated in a high-ionic strength buffer that has been used for ribosome reconstitution studies and so enables the RNA to adopt a structure recognized by ribosomal proteins. The crosslinking sites were identified by primer extension and confirmed by assessing the mobility of the crosslinked RNA lariats in denaturing polyacrylamide gels. Eight crosslinks were characterized. Among them, one crosslink demonstrates that helix 28 is proximal to the top of helix 34, and two others show that the 1337 region, located in an internal loop at the junction of helices 29, 30, 41, and 42, is proximal to the center of helix 30 and to a segment connecting helix 28 to helix 29.
  • Effects of Oxidative Stress on Protein Translation

    Effects of Oxidative Stress on Protein Translation

    International Journal of Molecular Sciences Review Effects of Oxidative Stress on Protein Translation: Implications for Cardiovascular Diseases Arnab Ghosh * and Natalia Shcherbik * Department for Cell Biology and Neuroscience, School of Osteopathic Medicine, Rowan University, 2 Medical Center Drive, Stratford, NJ 08084, USA * Correspondence: [email protected] (A.G.); [email protected] (N.S.); Tel.: +1-856-566-6907 (A.G.); +1-856-566-6914 (N.S.) Received: 24 March 2020; Accepted: 9 April 2020; Published: 11 April 2020 Abstract: Cardiovascular diseases (CVDs) are a group of disorders that affect the heart and blood vessels. Due to their multifactorial nature and wide variation, CVDs are the leading cause of death worldwide. Understanding the molecular alterations leading to the development of heart and vessel pathologies is crucial for successfully treating and preventing CVDs. One of the causative factors of CVD etiology and progression is acute oxidative stress, a toxic condition characterized by elevated intracellular levels of reactive oxygen species (ROS). Left unabated, ROS can damage virtually any cellular component and affect essential biological processes, including protein synthesis. Defective or insufficient protein translation results in production of faulty protein products and disturbances of protein homeostasis, thus promoting pathologies. The relationships between translational dysregulation, ROS, and cardiovascular disorders will be examined in this review. Keywords: protein translation; ribosome; RNA; IRES; uORF; miRNA; cardiovascular diseases; reactive oxygen species; oxidative stress; antioxidants 1. Introduction The process of protein synthesis, or protein translation, constitutes the last and final step of the central dogma of molecular biology: assembly of polypeptides based on the information encoded by mRNAs. This complex process employs multiple essential players, including ribosomes, mRNAs, tRNAs, and numerous translational factors, enzymes, and regulatory proteins.
  • Ef-G:Trna Dynamics During the Elongation Cycle of Protein Synthesis

    Ef-G:Trna Dynamics During the Elongation Cycle of Protein Synthesis

    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2015 Ef-G:trna Dynamics During the Elongation Cycle of Protein Synthesis Rong Shen University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons Recommended Citation Shen, Rong, "Ef-G:trna Dynamics During the Elongation Cycle of Protein Synthesis" (2015). Publicly Accessible Penn Dissertations. 1131. https://repository.upenn.edu/edissertations/1131 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1131 For more information, please contact [email protected]. Ef-G:trna Dynamics During the Elongation Cycle of Protein Synthesis Abstract During polypeptide elongation cycle, prokaryotic elongation factor G (EF-G) catalyzes the coupled translocations on the ribosome of mRNA and A- and P-site bound tRNAs. Continued progress has been achieved in understanding this key process, including results of structural, ensemble kinetic and single- molecule studies. However, most of work has been focused on the pre-equilibrium states of this fast process, leaving the real time dynamics, especially how EF-G interacts with the A-site tRNA in the pretranslocation complex, not fully elucidated. In this thesis, the kinetics of EF-G catalyzed translocation is investigated by both ensemble and single molecule fluorescence resonance energy transfer studies to further explore the underlying mechanism. In the ensemble work, EF-G mutants were designed and expressed successfully. The labeled EF-G mutants show good translocation activity in two different assays. In the smFRET work, by attachment of a fluorescent probe at position 693 on EF-G permits monitoring of FRET efficiencies to sites in both ribosomal protein L11 and A-site tRNA.
  • The Ribosomal Peptidyl Transferase Center: Structure, Function, Evolution, Inhibition

    The Ribosomal Peptidyl Transferase Center: Structure, Function, Evolution, Inhibition

    Critical Reviews in Biochemistry and Molecular Biology, 40:285–311, 2005 Copyright c Taylor & Francis Inc. ! ISSN: 1040-9238 print / 1549-7798 online DOI: 10.1080/10409230500326334 The Ribosomal Peptidyl Transferase Center: Structure, Function, Evolution, Inhibition Norbert Polacek Innsbruck Biocenter, Division of ABSTRACT The ribosomal peptidyl transferase center (PTC) resides in the Genomics and RNomics, large ribosomal subunit and catalyzes the two principal chemical reactions of Innsbruck Medical University, protein synthesis: peptide bond formation and peptide release. The catalytic Innsbruck, Austria mechanisms employed and their inhibition by antibiotics have been in the Alexander S. Mankin focus of molecular and structural biologists for decades. With the elucidation Center for Pharmaceutical of atomic structures of the large ribosomal subunit at the dawn of the new Biotechnology, University of millennium, these questions gained a new level of molecular significance. The Illinois at Chicago, Chicago, crystallographic structures compellingly confirmed that peptidyl transferase is IL 60607, USA an RNA enzyme. This places the ribosome on the list of naturally occurring riboyzmes that outlived the transition from the pre-biotic RNA World to con- temporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond for- mation primarily by substrate positioning. At the same time, peptide release should more strongly depend on chemical catalysis likely involving an rRNA group of the PTC. The PTC is characterized by the most pronounced accu- mulation of universally conserved rRNA nucleotides in the entire ribosome. Thus, it came as a surprise that recent findings revealed an unexpected high level of variation in the mode of antibiotic binding to the PTC of ribosomes from different organisms.
  • Mitochondrial Translation and Its Impact on Protein Homeostasis And

    Mitochondrial Translation and Its Impact on Protein Homeostasis And

    Mitochondrial translation and its impact on protein homeostasis and aging Tamara Suhm Academic dissertation for the Degree of Doctor of Philosophy in Biochemistry at Stockholm University to be publicly defended on Friday 15 February 2019 at 09.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B. Abstract Besides their famous role as powerhouse of the cell, mitochondria are also involved in many signaling processes and metabolism. Therefore, it is unsurprising that mitochondria are no isolated organelles but are in constant crosstalk with other parts of the cell. Due to the endosymbiotic origin of mitochondria, they still contain their own genome and gene expression machinery. The mitochondrial genome of yeast encodes eight proteins whereof seven are core subunits of the respiratory chain and ATP synthase. These subunits need to be assembled with subunits imported from the cytosol to ensure energy supply of the cell. Hence, coordination, timing and accuracy of mitochondrial gene expression is crucial for cellular energy production and homeostasis. Despite the central role of mitochondrial translation surprisingly little is known about the molecular mechanisms. In this work, I used baker’s yeast Saccharomyces cerevisiae to study different aspects of mitochondrial translation. Exploiting the unique possibility to make directed modifications in the mitochondrial genome of yeast, I established a mitochondrial encoded GFP reporter. This reporter allows monitoring of mitochondrial translation with different detection methods and enables more detailed studies focusing on timing and regulation of mitochondrial translation. Furthermore, employing insights gained from bacterial translation, we showed that mitochondrial translation efficiency directly impacts on protein homeostasis of the cytoplasm and lifespan by affecting stress handling.
  • Loss of Sbds in Shwachman-Diamond Syndrome Murine Model Leads to Reduction of 80S Ribosomes and Altered Transcript Binding

    Loss of Sbds in Shwachman-Diamond Syndrome Murine Model Leads to Reduction of 80S Ribosomes and Altered Transcript Binding

    Loss of Sbds in Shwachman-Diamond Syndrome Murine Model Leads to Reduction of 80S Ribosomes and Altered Transcript Binding by Hongrui Liu A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Hongrui Liu 2016 Loss of Sbds in Shwachman-Diamond Syndrome Murine Model Leads to Reduction of 80S Ribosomes and Altered Transcript Binding Hongrui Liu Doctor of Philosophy Department of Molecular Genetics University of Toronto 2016 Abstract Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease characterized by growth retardation, exocrine pancreatic dysfunction, skeletal dysplasia, cognitive impairment and bone marrow failure. SDS is caused by mutations in SBDS (Shwachman- Bodian-Diamond syndrome). A recent model proposes that SBDS/Sbds functions together with EFL1/Efl1 to release EIF6/Eif6 from the pre-60S complex, enabling ribosomal subunit joining for translation initiation. To assess the protein synthesis deficiency that has been detected in SDS, I examined ribosomal profiles of murine fetal organs with the SDS-associated missense mutation, R126T (SbdsR126T/R126T). The SDS organ extracts revealed reduced 80S monosomes and preserved polysomes, with no ribosomal subunit imbalance compared to matched controls. Further, Eif6 was found to bind to both the 60S and 80S ribosome complexes in mutants in contrast to only 60S complexes in controls. To investigate these changes and to learn how the SDS translatome is affected, total and polysomal mRNAs of mutant and control samples were studied using cDNA microarray analyses. By comparing individual polysomal transcripts ii to respective total transcript levels, I found 799 transcripts (of 18,936 analyzed) with altered polysome loading in mutant fetal livers, with 634 being increased.
  • The Microbiota-Produced N-Formyl Peptide Fmlf Promotes Obesity-Induced Glucose

    The Microbiota-Produced N-Formyl Peptide Fmlf Promotes Obesity-Induced Glucose

    Page 1 of 230 Diabetes Title: The microbiota-produced N-formyl peptide fMLF promotes obesity-induced glucose intolerance Joshua Wollam1, Matthew Riopel1, Yong-Jiang Xu1,2, Andrew M. F. Johnson1, Jachelle M. Ofrecio1, Wei Ying1, Dalila El Ouarrat1, Luisa S. Chan3, Andrew W. Han3, Nadir A. Mahmood3, Caitlin N. Ryan3, Yun Sok Lee1, Jeramie D. Watrous1,2, Mahendra D. Chordia4, Dongfeng Pan4, Mohit Jain1,2, Jerrold M. Olefsky1 * Affiliations: 1 Division of Endocrinology & Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA. 2 Department of Pharmacology, University of California, San Diego, La Jolla, California, USA. 3 Second Genome, Inc., South San Francisco, California, USA. 4 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, USA. * Correspondence to: 858-534-2230, [email protected] Word Count: 4749 Figures: 6 Supplemental Figures: 11 Supplemental Tables: 5 1 Diabetes Publish Ahead of Print, published online April 22, 2019 Diabetes Page 2 of 230 ABSTRACT The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)- induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon- like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota.
  • History of the Ribosome and the Origin of Translation

    History of the Ribosome and the Origin of Translation

    History of the ribosome and the origin of translation Anton S. Petrova,1, Burak Gulena, Ashlyn M. Norrisa, Nicholas A. Kovacsa, Chad R. Berniera, Kathryn A. Laniera, George E. Foxb, Stephen C. Harveyc, Roger M. Wartellc, Nicholas V. Huda, and Loren Dean Williamsa,1 aSchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332; bDepartment of Biology and Biochemistry, University of Houston, Houston, TX, 77204; and cSchool of Biology, Georgia Institute of Technology, Atlanta, GA 30332 Edited by David M. Hillis, The University of Texas at Austin, Austin, TX, and approved November 6, 2015 (received for review May 18, 2015) We present a molecular-level model for the origin and evolution of building up of the functional centers, proceeds to the establishment the translation system, using a 3D comparative method. In this model, of the common core, and continues to the development of large the ribosome evolved by accretion, recursively adding expansion metazoan rRNAs. segments, iteratively growing, subsuming, and freezing the rRNA. Incremental evolution of function is mapped out by stepwise Functions of expansion segments in the ancestral ribosome are accretion of rRNA. In the extant ribosome, specific segments of assigned by correspondence with their functions in the extant rRNA perform specific functions including peptidyl transfer, ribosome. The model explains the evolution of the large ribosomal subunit association, decoding, and energy-driven translocation subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic (11). The model assumes that the correlations of rRNA segments ribosomes evolved in six phases, sequentially acquiring capabilities with their functions have been reasonably maintained over the for RNA folding, catalysis, subunit association, correlated evolution, broad course of ribosomal evolution.
  • Eif6 Promotes the Malignant Progression of Human

    Eif6 Promotes the Malignant Progression of Human

    Sun et al. J Transl Med (2021) 19:216 https://doi.org/10.1186/s12967-021-02877-4 Journal of Translational Medicine RESEARCH Open Access eIF6 promotes the malignant progression of human hepatocellular carcinoma via the mTOR signaling pathway Liping Sun1†, Shuguang Liu3, Xiaopai Wang4, Xuefeng Zheng5, Ya Chen1 and Hong Shen1,2* Abstract Background: Eukaryotic translation initiation factor 6 (eIF6) has a crucial function in the maturation of 60S ribosomal subunits, and it controls the initiation of protein translation. Although emerging studies indicate that eIF6 is aberrantly expressed in various types of cancers, the functions and underlying molecular mechanisms of eIF6 in the pathological progression of hepatocellular carcinoma (HCC) remain unclear. This study aimed to evaluate the potential diagnostic and prognostic value of eIF6 in patients with HCC. Methods: HCC samples enrolled from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and our cohort were used to explore the role and mechanism of eIF6 in HCC. The diagnostic power of eIF6 was verifed by receiver operating characteristic curve (ROC) analysis and its prognostic value was assessed by Kaplan–Meier analysis, and then related biological functions of eIF6 were determined in vitro and in vivo cancer models. In addition, poten- tial molecular mechanism of eIF6 in HCC was unveiled by the gene set enrichment analysis and western blot assay. Results: We demonstrated that eIF6 expression was markedly increased in HCC, and elevated eIF6 expression cor- related with pathological progression of HCC. Besides, eIF6 served as not only a new diagnostic biomarker but also an independent risk factor for OS in HCC patients.
  • Ribosomal DNA Copy Number Is Coupled with Gene Expression Variation and Mitochondrial Abundance in Humans

    Ribosomal DNA Copy Number Is Coupled with Gene Expression Variation and Mitochondrial Abundance in Humans

    ARTICLE Received 12 Apr 2014 | Accepted 30 Jul 2014 | Published 11 Sep 2014 DOI: 10.1038/ncomms5850 Ribosomal DNA copy number is coupled with gene expression variation and mitochondrial abundance in humans John G. Gibbons1, Alan T. Branco1, Shoukai Yu1 & Bernardo Lemos1 Ribosomes are essential intracellular machines composed of proteins and RNA molecules. The DNA sequences (rDNA) encoding ribosomal RNAs (rRNAs) are tandemly repeated and give origin to the nucleolus. Here we develop a computational method for estimating rDNA dosage (copy number) and mitochondrial DNA abundance using whole-genome short-read DNA sequencing. We estimate these attributes across hundreds of human genomes and their association with global gene expression. The analyses uncover abundant variation in rDNA dosage that is coupled with the expression of hundreds of functionally coherent gene sets. These include associations with genes coding for chromatin components that target the nucleolus, including CTCF and HP1b. Finally, the data show an inverse association between rDNA dosage and mitochondrial DNA abundance that is manifested across genotypes. Our findings uncover a novel and cryptic source of hypervariable genomic diversity with global regulatory consequences (ribosomal eQTL) in humans. The variation provides a mechanism for cellular homeostasis and for rapid and reversible adaptation. 1 Program in Molecular and Integrative Physiological Sciences, Department of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Building 2, Room 219, Boston, Massachusetts 02115, USA. Correspondence and requests for materials should be addressed to B.L. (email: [email protected]). NATURE COMMUNICATIONS | 5:4850 | DOI: 10.1038/ncomms5850 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited.