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RNA and Protein Interactions by Eif4b During Translation Initiation

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RNA and Protein Interactions by Eif4b During Translation Initiation • RNA and protein interactions by eIF4B during translation initiation by Nathalie Méthot Department of lliochemistry McGiU University Montréal. Québec, Canada May 1996 A Thesis submitted to the Faculty ofGraduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy e Nathalie Méthot, 1996 • i National Llbrary Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Direction des acquisitions et BlblltJgraphlc Services Branch des services bibliographiques 395 Welhnglon Streel 395. rue Wellington Ottawa. Onl:;mo Ollawa (Onlanoj KIA ON4 K1AON4 The duthor has granted an L'auteur a accordé une licence irrevocable non-exclusive licence irrévocable et non exclusive allowing the National Library of permettant à la Bibliothèque Canada to reproduce, loan, nationale du Canada de distribute or sell copies of reproduire, prêter, distribuer ou his/her thesis by any means and vendre des copies de sa thèse in any form or format, making de quelque manière et sous this thesis available to interested quelque forme que ce soit pour persons. mettre des exemplaires de cette thèse à la disposition des personnes intéressées. The author retains ownership of L'auteur conserve la propriété du the copyright in his/her thesis. droit d'auteur qui protège sa Neither the thesis nor substantial thèse. Ni la thèse ni des extraits extracts from it may be printed or substantiels de celle-ci ne otherwise reproduced without doivent être imprimés ou his/her permission. autrement reproduits sans son autorisation. 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Contributors should note thill certain rishts ~ ~tained: as nmcd ca the ~rer 3Jrcemcnl. iurur~ U~ t'l(fhe U/Qrk by the oricin;lI 3urhonl cOpyrighl h.ldm i5 .Uowed, ~rovided u..l ' ....dord a<:knowledgmenllo lh: juum3ll, :;'·.n. ûmbrid&. will provide leUen of gr..., penn1.ssiun lO al:thcrs ~hnllld th~ir NtuR puhlilher require wrincn doc:umcnt3rion. Thini p:l.."ty r:quests co republi:ih all or r-Ut ur .A Cambricf~e journal ilI1iclc are gtilnted nnly upon the condiIlon rh:u rhe 3Ulho,,' pcmunion il JJsa .liought (~·hich l'Dwt.bc anumcd to lx­ ll'In1ed iflhe ."tho", do nol respond wilhin fourwed<,): and SO~ ohny proa:.d. from penNuion (...will Le asslgned ID lhe eonrribuIOl>. PJt:lse: he ~ure {hot you lubmit the Transfcr oC Copyright Ag~mcnt 10 the t::dirnr al(ln~ \l'iLh ~Qur mJ.nu ..cripr or ol:i. soon aJ possible lhe~fle~ in ordlr m prevent any dcby in pltlCe.5sing your contribution for publication. • A Monique et Gilles, qui m'ont donné de nombreux encouragements, et qui ont toujours su rétablir, au besoin, l'équilibre si nécéssaire à l'accomplissement d'un travail de cene envergure, et à mon frère Michel, dont le courage et les succès ont été et sont toujours une source d'inspiration. • ü Abstract • One of the most enduring questions pcnaining to eukaryotic translation initiation is how the 40S ribosomal subunit recognires and binds at or ncar the Cap-slrUcture of mRNAs. Eukaryotic initiation factor 4B (eIF4B) is one of the factors that an: required for this Step of protein synthesis. eIF4B stimulates the RNA helicasc activity of elF4A and eIF4F. melting RNA sccondary sU1lcture in the 5' untranslalCd region (UTR). and is thus believed 10 contribute to ribosome bil'ding by creating an an:a of single-stf".U1dcd RNA accessible to the 40S ribosomal subunit We studied the mcchanism of action of eIF4B by initiating a slrUcture-function analysis of this factor. An RNA binding site. located in the carboxy-terminal end bctwecn amino acids 367 and 423. was found essential for non-specific RNA binding and eIF4A helicase stimulation. This region is distinct and independent l'rom the canonical RNA Recognition Motif (RRM) locatcd ncar the amino-terminus. The latter plays no role in non-specific RNA binding and has littie impact on the eIF4A helicase stimulation. Aself-association region located bctwecn residues 213-312 was identified. This segment is rich in aspartic acid. arginine. tyrosine and glycine (DRYG) residues. and can seif-associate indepcndcntly l'rom other regions ofeIF4B. The DRYG domain also interaclS dircctly with the pl70 subunit ofeIF3. Finally. iterative in vitro RNA selection demonstrated that the elF4B RRM is functional and hinds specifically to RNA stem-loop structures. The RRM also associates with 18S rRNA. eIF4B possesses two independent RNA binding sites and associates with two different RNA molecules simultaneously. We conclude that elF4B is organized into threc distinct domains: the carboxy-terminai eIF4A RNA helicase stimuiatory domain. the DRYG dimerization and elF3 pl70 interaction domain. and the RRM. Furthermore. two additionai mechanisms by which eIF4B could stimulate ribosome binding to the mRNA are now apparent eIF4B may target the 40S ribosomal subunit to the 5'lITR ofa mRNA by binding simultaneously to 18S rRNA and the • mRNA, and by interacting directly with elFJ. Résumé • Le mécanisme par lcquella sous-unité ribosomale 40S se lie à la structure coiffe en 5' d'un ARNm demeure une question épineuse du domaine de la traduction chez les eukaryotes. Le facleur eukaryotique d'initiation de traduction eIF4B est l'une des protéines essentielles àce processdS. eIF4B contribue à la liaison du ribosome à l'ARNm en stimulant l'activité ARN-hélicase du facleur de traduction eIF4A Le déroulement de structures secondaires d'ARN par le tandem eIF4NeIF4B crée une aire d'ARN simple-brin dans la partie 5' non-codanle de l'ARNm, accessible au ribosome. Nous avons étudié le mode d'action d'eIF4B par le biais d'une analyse structurelle et fonctionelle de ce facleur. L'extrémité carboxy d'eIF4B possède un site d'adhésion non-spécifique à l'ARN, qui est absolument nécéssaire à la stimulation de l'activité hélicase d'eIF4A. Le motif de reconnai.-sance d'ARN (RRM) d'eIF4B ne joue pas ou peu de rôle à cet effeL Une région médiant l'homodimérisation d'eIF4B, située entre les acides-aminés 213 et 312 de la protéi.,e, a été identifiée. Ce segment promouvoie la dimérisation indépendamment des autres région.~ d'eIF4B et est capable d'interagir directement avec la sous-unité pl70 d'eIF3. Enfin, le RRM d'eIF4B se lie spécifiquement à des ARNs possédant une tige, une boucle et une adénosine non­ conforme dans la tige et s'associe à l'ARNr 18S. eIF4B est capable de se lier simultanément à deux molécules d'ARN différentes. Ainsi, eIF4B possède trois domaines distincts et indépendants: l'extrémité carboxy, qui stimule l'activité hélicase d'eIF4A,le domaine DRYG, qui est responsable de l'homodimérisation et de l'interaction avec la sous-unité pl70 d'eIFJ, et le RRM, qui s'associe a l'ARNr 18S. Deux mécanismes supplémentaires par lesquels eIF4B stimule la liaison de la sous­ unité ribosomale 40S à l'ARNm peuvent être envisagés. eIF4B se lierait simultanément à l'ARNr 18S et à l'ARNm, et eIF4B inte."llgirait directement avec eIF3, déja present sur le ribosome. Ainsi, eIF4B établit un pont entre l'ARNm et le ribosome. • iv Foreword • Guidelines Concernlng Thesis Preparation: Manuscript and Authorshlp This thesis was organized according ID the Faculty of Graduate Stuilles and Rescarch "Guidelines Conceming Thesis Preparation". following a fonnat approved by the Dcpartment ofBiochcmistry. Part of Ibis thesis includes texts from original reports published or submitted for publication. and thus. as stipulated in the Faculty of Graduate Studies and Rescarch "guidelines Conceming Thesis Preparation". 1herein cite the text of the "Manuscript and Authorship" section: Candidates have the option of including. as part of the thesis. the text of a paper(s) submitted or to he submitted for publication. or the clearly-duplicated text of a published pcper(s). These texts must he hound as an inl<:gral part of the thesis. If this option is chosen. connecting lexts that provide logical bridges between the different papers are mandatory. The thesis must be written in such a way that il is more than a mere collection of manuscripts; in other words. results ofa series of papers must he integrated. The thesis must still confonn ta ail other requirements ofthe "Guidelines for Thesis Preparation". The thesis must indude: A Table of Contents. an abstracl in English and in French. an introduction which clearly states the rationale and the objective of the study. a comprehensive review of the literature. a final conclusion and summary and a thorough bibliography or reference IisL • v Additional material must he provided where appropriate (e.g. in appendices) and in suflicient detail te a1low a clear and precise judgmcnt te he made of importance and • originality of the research reported in the thesis.
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