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Characterization of the Terminal Oligopyrimidine Mrna Binding Properties of La-Related Protein 1

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Characterization of the Terminal Oligopyrimidine Mrna Binding Properties of La-Related Protein 1 Characterization of the Terminal Oligopyrimidine mRNA Binding Properties of La-Related Protein 1 By Jaclyn Hearnden Department of Biochemistry McGill University Montreal, Quebec, Canada April 2016 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Masters of Science. © Jaclyn Hearnden, 2016 0 Abstract The conservation of cellular energy during unfavorable conditions requires efficient deactivation of protein production. When environmental conditions cannot support basic cellular functions, various intracellular signaling modules respond, notably mammalian Target of Rapamycin Complex 1 (mTORC1)- a master regulator of protein synthesis. Although the bulk of mRNA translation is rapidly downregulated following nutrient and oxygen deprivation by mTORC1, a subset of mRNA transcripts featuring Terminal OligoPyrimidine (TOP) sequences in their 5’ UnTranslated Region (5’UTR) are uniquely sensitive to mTORC1 regulation through an ill-defined mechanism. These mTORC1-sensitive transcripts include many ribosomal proteins and some general translation factors. La- Related Protein 1 (LARP1) has been suggested as the missing link between TOP transcript translational regulation and mTORC1; LARP1 is an established mTORC1 phosphorylation target with multiple RNA binding domains. In this thesis, the direct binding of LARP1 to various TOP 5’UTR RNA sequences is shown to be higher affinity than binding to non-specific targets by incubating purified recombinant proteins with various radioactive RNA probes and visualizing complex formation through ElectroMobility Shift Assays (EMSAs). Using such an approach, we also define which domains within LARP1 contribute to TOP RNA binding, and assign a putative functional role to a non-TOP RNA binding domain. Based on homology alignments and structural predictions, we generated point mutations within the domain of interest and map residues relevant to maintaining TOP RNA binding activity. Several conserved features of TOP mRNAs necessary for LARP1 binding are also briefly examined. All together, this thesis expands our understanding of the complex contribution of LARP1 as a regulator of TOP mRNA translation. 1 Résumé “Caractérisation de l’intéraction entre LARP1 et les ARNm TOP” La conservation de l’énergie cellulaire lors de conditions défavorables requiert la désactivation efficace de la production de protéines. Lorsque l’environnement ne permet pas le support des fonctions cellulaires de base, plusieurs cascades de signalisation intracellulaire réagissent, notamment mTORC1 (de l’anglais mammalian Target of Rapamycin Complex 1), un régulateur important de la synthèse protéique. Bien que la majorité de la traduction d’ARNm soit arrêtée par mTORC1 dès la détection d’un manque d’oxygène ou de nutriments, un groupe d’ARNm comportant une séquence riche en pyrimidines ou TOP (de l’anglais Terminal OligoPyrimidine) dans leur région 5’ non-traduite ou 5’UTR (de l’anglais 5’ UnTranslated Region) sont particulièrement sensible à l’action de mTORC1, mais le mécanisme responsable de cet effet demeure mal compris. Ces transcrits mTORC1-sensibles en incluent de nombreux encodant des protéines ribosomal ainsi que des facteurs généraux d’initiation de la traduction. Il a été suggéré que LARP1 (de l’anglais La- Related Protein 1) pourrait représenter le chaînon manquant entre la régulation traductionnelle des transcrits TOP et l’activité de mTORC1, LARP1 étant une cible bien établi de phosphorylation par mTORC1 qui possède plusieurs domaine d’interaction à l’ARN. Dans la présente thèse, il est démontré que LARP1 lie certaines séquences TOP avec une affinité supérieure aux séquences d’ARN non-spécifiques en incubant des proteins recombinantes purifiées avec différentes sondes d’ARN radioactives et en visualisant la formation d’un complexe par essai de décalage de mobilité électrophorétique (EMSA, de l’anglais electromobility shift assay). Grâce à cette approche, nous avons identifié les domaines de 2 LARP1 contribuant à l’interaction avec l’ARN TOP et nous assignons une fonction putative à un domaine liant l’ARN non-TOP. En nous basant sur l’alignement d’homologues de LARP1 et des prédictions sur sa structure, nous avons généré des mutations ponctuelles dans le domaine d’intérêt et cartographié les résidus maintenant la capacité de LARP1 à lier l’ARN TOP. Un survol de plusieurs caractéristiques de LARP1 maintenues à travers les espèces nécessaires à cette interaction est aussi inclus. Prise dans son ensemble, cette thèse élargie notre compréhension de la complexité avec laquelle LARP1 contribue à la régulation de la traduction des ARNm TOP. 3 Acknowledgements Endless thanks to Dr. Edna Matta-Camacho, who was responsible for my training and guidance. Dr. Matta-Camacho made great contributions to the experimental design involved in this project and the execution of some experiments (ITC, NMR, crystallization screenings, certain computational analyses). Thank you to Dr. Nahum Sonenberg for the opportunity. Thank you to Nathaniel Robichaud for the translation of my abstract. Thank you to Dr. Abba Malina and Dr. Edna Matta-Camacho for thesis corrections. Thank you to Isabelle Harvey, Annamaria Kiss, Sandra Perreault, and Christine Sgherri for invaluable administrative assistance. Thank you to all members of the Sonenberg lab but especially: Soroush Tahmasebi, Nathaniel Robichaud, Chadi Zakaria, Yuri Svitkin, Nadeem Siddiqui, Tommy Alain, and Bruno Fonseca. Special thanks to Meena Vipparti. Thank you to Défi Canderel and the Canadian Institute of Health Research (CIHR) for financial support. 4 Table of Contents Abstract………………………………………………………………………………………………………………….…………………1 Resume……………………………………………………………………………………………………………………….…………….2 Acknowledgements……………………………………………………………………………………………………….………….4 Table of Contents……………………………………………………………………………………………………………….……..5 1. Introduction ........................................................................................................................ 7 1.1 An overview of translation……………………………………………………………………………………….7 1.2 Characteristics of ribosomal protein transcripts……………………………………………………….8 1.3 mTORC1 and TOP mRNA translation .......................................................................... 10 1.4 The increasing interest in La-Related Protein 1 ....................................................... ….11 1.5 The La-Related Family ................................................................................................ .14 1.6 Functions for LARP1 in Model Organisms.................................................................. .16 1.7 LARP1 and TOP Transcripts ........................................................................................ .19 1.8 LARP1 in Cancer………………………………………………………………………………………................23 1.9 Research Question, Hypothesis, Aims…………………………………………………………………….23 2. Results……………………………………………………………………………………………………………………..25 2.1 Recombinant LARP1……………………………………………………………………………………………….25 2.2 Direct binding of TOP 5’UTR by LARP1……………………………………………………………………29 2.3 LARP1 species and their RNA shift patterns…………………………………………………………….30 2.4 LARP1 TOP binding and sequence specificity………………………………………………………….33 5 2.5 TOP transcript binding by LARP1 domain fragments……………………………………………….37 2.6 The La domain of LARP1…………………………………………………………………………………………40 2.7 LARP1 C-terminal aromatic residues contribute to TOP binding by LARP1………………43 2.8 Truncation of a TOP sequence from the 3’ end abolishes binding by LARP1……………46 2.9 LARP1 transcript levels in human breast tumors…………………………………………………….48 3. Discussion ......................................................................................................................... 51 4. Materials and Methods .................................................................................................... 55 4.1 Cloning……………………………………………………………………………………………………………………55 4.2 Site-Directed Mutagenesis .......................................................................................... 56 4.3 Protein Expression and Purification ............................................................................ 56 4.4 Electrophoretic Mobility Shift Assay ........................................................................... 57 4.5 Coomassie Protein Gels………………………………………………………………..………………………..57 4.6 Western Blot…………………………………………………………………………………..………………………57 4.7 RNA Sequences………………………………………………………………………………………………………58 4.8 Isothermal Titration Calorimetry…………………………………………………………………………….58 4.9 Nuclear Magnetic Resonance………………………………………………………………………………….58 4.10 Quantitative Real Time Polymerase Chain Reaction……………………………………………..59 5. References……………………………………………………………………………….………………………………….60 6 Introduction 1.1 An overview of translation Translation concludes the conversion of genetic information into proteins capable of carrying out cellular functions. Each mRNA transcript generated from genomic DNA is soon modified and joined by various RNA-binding factors that work to ensure efficient translation into protein. Transcripts are typically modified at the 5’ end with a methyl 7’guanosine cap and at the 3’ end with the addition of a string of adenosine residues, referred to as the polyA tail. The 5’ cap forms the binding site for eukaryotic Initiation Factor 4E (eIF4E), and along with eIF4E, additional translation initiation factors eIF4A and eIF4G form the eIF4F complex, which acts to recruit the ribosomal pre-initiation complex to the 5’ end of the message. Transcript circularization
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