Rps3/Us3 Promotes Mrna Binding at the 40S Ribosome Entry Channel and Stabilizes Preinitiation Complexes at Start Codons
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Translational Resistance of Late Alphavirus Mrna to Eif2␣ Phosphorylation: a Strategy to Overcome the Antiviral Effect of Protein Kinase PKR
Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Translational resistance of late alphavirus mRNA to eIF2␣ phosphorylation: a strategy to overcome the antiviral effect of protein kinase PKR Iván Ventoso,1,3 Miguel Angel Sanz,2 Susana Molina,2 Juan José Berlanga,2 Luis Carrasco,2 and Mariano Esteban1 1Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología/CSIC, Cantoblanco, E-28049 Madrid, Spain; 2Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Facultad de Ciencias, Cantoblanco, E-28049 Madrid, Spain The double-stranded RNA-dependent protein kinase (PKR) is one of the four mammalian kinases that phosphorylates the translation initiation factor 2␣ in response to virus infection. This kinase is induced by interferon and activated by double-stranded RNA (dsRNA). Phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) blocks translation initiation of both cellular and viral mRNA, inhibiting virus replication. To counteract this effect, most viruses express inhibitors that prevent PKR activation in infected cells. Here we report that PKR is highly activated following infection with alphaviruses Sindbis (SV) and Semliki Forest virus (SFV), leading to the almost complete phosphorylation of eIF2␣. Notably, subgenomic SV 26S mRNA is translated efficiently in the presence of phosphorylated eIF2␣. This modification of eIF2 does not restrict viral replication; SV 26S mRNA initiates translation with canonical methionine in the presence of high levels of phosphorylated eIF2␣. Genetic and biochemical data showed a highly stable RNA hairpin loop located downstream of the AUG initiator codon that is necessary to provide translational resistance to eIF2␣ phosphorylation. This structure can stall the ribosomes on the correct site to initiate translation of SV 26S mRNA, thus bypassing the requirement for a functional eIF2. -
L-Leucine Increases Translation of RPS14 and LARP1 in Erythroblasts
LETTERS TO THE EDITOR Table 1. Top 20 differentially translated known 5’TOP mRNAs in L-leucine increases translation of RPS14 and LARP1 L-leucine treated erythroblasts from del(5q) myelodysplastic syn- in erythroblasts from del(5q) myelodysplastic drome patients. syndrome patients Genes LogFC of TE in patients z score patients Deletion of the long arm of chromosome 5 [del(5q)] is RPS15 3.55 2.46 the most common cytogenetic abnormality found in the RPS27A 3.48 2.40 1 myelodysplastic syndromes (MDS). Patients with the 5q- RPS25 3.47 2.39 syndrome have macrocytic anemia and the del(5q) as the RPS20 3.43 2.35 sole karyotypic abnormality.1 Haploinsufficiency of the ribosomal protein gene RPS14, mapping to the common- RPL12 3.35 2.29 ly deleted region (CDR) on chromosome 5q,2 underlies PABPC4 3.01 2.01 3 the erythroid defect found in the 5q- syndrome, and is RPS24 2.97 1.98 associated with p53 activation,4-6 a block in the process- ing of pre-ribosomal RNA,3 and deregulation of riboso- RPS3 2.95 1.96 mal- and translation-related genes.7 Defective mRNA EEF2 2.83 1.86 translation represents a potential therapeutic target in the RPS18 2.76 1.80 5q- syndrome and other ribosomopathies, such as RPS26 2.75 1.79 Diamond-Blackfan anemia (DBA).8 Evidence suggests that the translation enhancer L- RPS5 2.69 1.74 leucine may have some efficacy in the treatment of the RPS21 2.64 1.70 8 5q- syndrome and DBA. A DBA patient treated with L- RPS9 2.54 1.62 leucine showed a marked improvement in anemia and 8 EIF3E 2.53 1.61 achieved transfusion independence. -
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 . -
Effects of Single Amino Acid Deficiency on Mrna Translation Are Markedly
www.nature.com/scientificreports OPEN Efects of single amino acid defciency on mRNA translation are markedly diferent for methionine Received: 12 December 2016 Accepted: 4 May 2018 versus leucine Published: xx xx xxxx Kevin M. Mazor, Leiming Dong, Yuanhui Mao, Robert V. Swanda, Shu-Bing Qian & Martha H. Stipanuk Although amino acids are known regulators of translation, the unique contributions of specifc amino acids are not well understood. We compared efects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no signifcant efect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E. Leucine starvation suppressed polysome formation and was the only tested condition that caused a signifcant decrease in 4EBP1 phosphorylation or increase in 4EBP1 binding to eIF4E, but efects of leucine starvation were not replicated by overexpressing nonphosphorylatable 4EBP1. This suggests the binding of 4EBP1 to eIF4E may not by itself explain the suppression of mRNA translation under conditions of leucine starvation. Ribosome profling suggested that leucine deprivation may primarily inhibit ribosome loading, whereas methionine deprivation may primarily impair start site recognition. These data underscore our lack of a full -
Evidence for Differential Alternative Splicing in Blood of Young Boys With
Stamova et al. Molecular Autism 2013, 4:30 http://www.molecularautism.com/content/4/1/30 RESEARCH Open Access Evidence for differential alternative splicing in blood of young boys with autism spectrum disorders Boryana S Stamova1,2,5*, Yingfang Tian1,2,4, Christine W Nordahl1,3, Mark D Shen1,3, Sally Rogers1,3, David G Amaral1,3 and Frank R Sharp1,2 Abstract Background: Since RNA expression differences have been reported in autism spectrum disorder (ASD) for blood and brain, and differential alternative splicing (DAS) has been reported in ASD brains, we determined if there was DAS in blood mRNA of ASD subjects compared to typically developing (TD) controls, as well as in ASD subgroups related to cerebral volume. Methods: RNA from blood was processed on whole genome exon arrays for 2-4–year-old ASD and TD boys. An ANCOVA with age and batch as covariates was used to predict DAS for ALL ASD (n=30), ASD with normal total cerebral volumes (NTCV), and ASD with large total cerebral volumes (LTCV) compared to TD controls (n=20). Results: A total of 53 genes were predicted to have DAS for ALL ASD versus TD, 169 genes for ASD_NTCV versus TD, 1 gene for ASD_LTCV versus TD, and 27 genes for ASD_LTCV versus ASD_NTCV. These differences were significant at P <0.05 after false discovery rate corrections for multiple comparisons (FDR <5% false positives). A number of the genes predicted to have DAS in ASD are known to regulate DAS (SFPQ, SRPK1, SRSF11, SRSF2IP, FUS, LSM14A). In addition, a number of genes with predicted DAS are involved in pathways implicated in previous ASD studies, such as ROS monocyte/macrophage, Natural Killer Cell, mTOR, and NGF signaling. -
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. -
Detecting Translational Regulation by Change Point Analysis of Ribosome Profiling Datasets
bioRxiv preprint doi: https://doi.org/10.1101/003210; this version posted March 5, 2014. 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 4.0 International license. Detecting translational regulation by change point analysis of ribosome profiling datasets Zupanic A1, Meplan C2, Grellscheid SN3, Mathers JC1, Kirkwood TBL1, Hesketh JE2, Shanley DP1 1Centre for Integrated Systems Biology of Ageing & Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle-upon-Tyne, NE4 5PL, UK 2Institute for Cell and Molecular Biosciences and Human Nutrition Research Centre, Newcastle University, Newcastle-upon-Tyne, NE2 4HH, UK 3School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, UK Running title: Translational regulation in ribosome profiles Keywords: ribosome profiling, translation regulation, change point, mathematical model Corresponding author: Daryl Shanley Centre for Integrated Systems Biology of Ageing & Nutrition, Institute for Ageing and Health, Newcastle University, NE4 5PL, UK Email: [email protected] Fax: +441912481101 1 bioRxiv preprint doi: https://doi.org/10.1101/003210; this version posted March 5, 2014. 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 4.0 International license. Abstract Ribo-Seq maps the location of translating ribosomes on mature mRNA transcripts. While ribosome density is constant along the length of the mRNA coding region, it can be altered by translational regulatory events. -
YEAST CELLS MAY USE AUC OR AAG AS INITIATION CODON for PROTEIN SYNTHESIS by OLE OLSEN
Carlsberg Res. Commun. Vol. 52, p. 83-90, 1987 YEAST CELLS MAY USE AUC OR AAG AS INITIATION CODON FOR PROTEIN SYNTHESIS by OLE OLSEN Department of Physiology, Carlsberg Laboratory, Gamle Cadsbergvej 10, DK-2500 Copenhagen Valby Keywords: Recombinant DNA, translation initiation, secretion A yeast expression plasmid without an ATG codon for initiation of mouse a-amylase protein synthesis directs the synthesis and secretion of active enzyme indistinguishable from both native mouse a-amylase and amylase synthesized from plasmids with normal AT(3 initiation codons. The initiation of amylase synthesis directed by this plasmid is at either an AUC or an AAG codon. In either case the amino acid sequence of the hydrophobic core and peptidase cleaving region of the signal peptide are normal, and the protein translation remains in frame with the structural gene of the mouse a-amylase. 1. INTRODUCTION mal context for initiation was 6NNAUGGA A where Initiation of protein synthesis in eukaryotes is a purine in position -3 (three nucleotides up- catalysed by a relatively large number of specific stream of the AUG initiator codon) is most eukaryotic initation factors (eIFs) bringing highly conserved. about the formation of the 80S initiation com- Until recently it was generally believed that plex composed ofmRNA, an 80S ribosome and eukaryotic ribosomes initiate exclusively at met-tRNAi (l 3). Eukaryotic met-tRNAi differs AUG codons although it had been shown by from its prokaryotic counterpart by being asso- RAJBHANDARYand GHOSH (24) that yeast met- ciated with the 40S pre-initation complex before tRNAi may function with either AUG or GUG binding to mRNA (13). -
DHX29 Antibody A
Revision 1 C 0 2 - t DHX29 Antibody a e r o t S Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) 9 5 Web: [email protected] 1 www.cellsignal.com 4 # 3 Trask Lane Danvers Massachusetts 01923 USA For Research Use Only. Not For Use In Diagnostic Procedures. Applications: Reactivity: Sensitivity: MW (kDa): Source: UniProt ID: Entrez-Gene Id: WB, IP H M R Endogenous 155 Rabbit Q7Z478 54505 Product Usage Information Application Dilution Western Blotting 1:1000 Immunoprecipitation 1:50 Storage Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody. Specificity / Sensitivity DHX29 Antibody detects endogenous levels of total DHX29 protein. Species Reactivity: Human, Mouse, Rat Source / Purification Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues at the C-terminus of human DHX29. Antibodies were purified by protein A and peptide affinity chromatography. Background DHX29 is an ATP-dependent RNA helicase that belongs to the DEAD-box helicase family (DEAH subfamily). DHX29 contains one central helicase and one helicase at the carboxy-terminal domain (1). Its function has not been fully established but DHX29 was recently shown to facilitate translation initiation on mRNAs with structured 5' untranslated regions (2). DHX29 binds 40S subunits and hydrolyzes ATP, GTP, UTP, and CTP. Hydrolysis of nucleotide triphosphates by DHX29 is strongly stimulated by 43S complexes and is required for DHX29 activity in promoting 48S complex formation (2). 1. de la Cruz, J. -
Structural Insights Into Mrna Reading Frame Regulation by Trna
RESEARCH ARTICLE Structural insights into mRNA reading frame regulation by tRNA modification and slippery codon–anticodon pairing Eric D Hoffer1, Samuel Hong1, S Sunita1, Tatsuya Maehigashi1, Ruben L Gonzalez Jnr2, Paul C Whitford3, Christine M Dunham1* 1Department of Biochemistry, Emory University School of Medicine, Atlanta, United States; 2Department of Chemistry, Columbia University, New York, United States; 3Department of Physics, Northeastern University, Boston, United States Abstract Modifications in the tRNA anticodon loop, adjacent to the three-nucleotide anticodon, influence translation fidelity by stabilizing the tRNA to allow for accurate reading of the mRNA genetic code. One example is the N1-methylguanosine modification at guanine nucleotide 37 (m1G37) located in the anticodon loop andimmediately adjacent to the anticodon nucleotides 34, 35, 36. The absence of m1G37 in tRNAPro causes +1 frameshifting on polynucleotide, slippery codons. Here, we report structures of the bacterial ribosome containing tRNAPro bound to either cognate or slippery codons to determine how the m1G37 modification prevents mRNA frameshifting. The structures reveal that certain codon–anticodon contexts and the lack of m1G37 destabilize interactions of tRNAPro with the P site of the ribosome, causing large conformational changes typically only seen during EF-G-mediated translocation of the mRNA-tRNA pairs. These studies provide molecular insights into how m1G37 stabilizes the interactions of tRNAPro with the ribosome in the context of a slippery mRNA codon. *For correspondence: Introduction [email protected] Post-transcriptionally modified RNAs, including ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA), stabilize RNA tertiary structures during ribonucleoprotein biogenesis, reg- Competing interests: The ulate mRNA metabolism, and influence other facets of gene expression. -
Ten Commandments for a Good Scientist
Unravelling the mechanism of differential biological responses induced by food-borne xeno- and phyto-estrogenic compounds Ana María Sotoca Covaleda Wageningen 2010 Thesis committee Thesis supervisors Prof. dr. ir. Ivonne M.C.M. Rietjens Professor of Toxicology Wageningen University Prof. dr. Albertinka J. Murk Personal chair at the sub-department of Toxicology Wageningen University Thesis co-supervisor Dr. ir. Jacques J.M. Vervoort Associate professor at the Laboratory of Biochemistry Wageningen University Other members Prof. dr. Michael R. Muller, Wageningen University Prof. dr. ir. Huub F.J. Savelkoul, Wageningen University Prof. dr. Everardus J. van Zoelen, Radboud University Nijmegen Dr. ir. Toine F.H. Bovee, RIKILT, Wageningen This research was conducted under the auspices of the Graduate School VLAG Unravelling the mechanism of differential biological responses induced by food-borne xeno- and phyto-estrogenic compounds Ana María Sotoca Covaleda Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Tuesday 14 September 2010 at 4 p.m. in the Aula Unravelling the mechanism of differential biological responses induced by food-borne xeno- and phyto-estrogenic compounds. Ana María Sotoca Covaleda Thesis Wageningen University, Wageningen, The Netherlands, 2010, With references, and with summary in Dutch. ISBN: 978-90-8585-707-5 “Caminante no hay camino, se hace camino al andar. Al andar se hace camino, y al volver la vista atrás se ve la senda que nunca se ha de volver a pisar” - Antonio Machado – A mi madre. -
Circular Code Motifs in the Ribosome: a Missing Link in the Evolution of Translation?
Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Circular code motifs in the ribosome: a missing link in the evolution of translation? Gopal Dila1, Raymond Ripp1, Claudine Mayer1,2,3, Olivier Poch1, Christian J. Michel1,* and Julie D. Thompson1,* 1 Department of Computer Science, ICube, CNRS, University of Strasbourg, Strasbourg, France 2 Unité de Microbiologie Structurale, Institut Pasteur, CNRS, 75724 Paris Cedex 15, France 3 Université Paris Diderot, Sorbonne Paris Cité, 75724 Paris Cedex 15, France * To whom correspondence should be addressed; Email: [email protected] *Corresponding authors: Names: Christian J. Michel, Julie D. Thompson Address: Department of Computer Science, ICube, Strasbourg, France Phone: (33) 0368853296 Email: [email protected], [email protected] Running title: circular code motifs in the ribosome Keywords: origin of life, genetic code, circular code, translation, ribosome evolution 1 Dila et al. Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Abstract The origin of the genetic code remains enigmatic five decades after it was elucidated, although there is growing evidence that the code co-evolved progressively with the ribosome. A number of primordial codes were proposed as ancestors of the modern genetic code, including comma-free codes such as the RRY, RNY or GNC codes (R = G or A, Y = C or T, N = any nucleotide), and the X circular code, an error-correcting code that also allows identification and maintenance of the reading frame. It was demonstrated previously that motifs of the X circular code are significantly enriched in the protein-coding genes of most organisms, from bacteria to eukaryotes.