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“The impact of mRNA 5’TL heterogeneity: what we can learn from the 53BP1 and EIF4E3 genes”

English title “The impact of mRNA 5’TL heterogeneity: what we can learn from the 53BP1 and EIF4E3 genes”
Applicant Curran Joseph
Number 175560
Funding scheme Project funding (Div. I-III)
Research institution Dépt Microbiologie et Médecine Moléculaire Faculté de Médecine Université de Genève
Institution of higher education University of Geneva - GE
Main discipline Molecular Biology
Start/End 01.10.2017 - 30.09.2021
Approved amount 496'000.00
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All Disciplines (3)

Discipline
Molecular Biology
Biochemistry
Genetics

Keywords (3)

5'TL complexity; mammalian translation initiation; protein function

Lay Summary (French)

Lead
Lead Le contrôle traductionnel est une étape critique de la régulation de l’expression des gènes impliqués dans le développement embryonnaire et de nombreux processus cellulaires comme la croissance et la prolifération. La régulation de ce processus se fait majoritairement lors de l'étape d'initiation pendant laquelle les ribosomes vont sélectionner l’ARNm (la matrice pour le décodage) et trouver le codon pour démarrer l’expression protéique.
Lay summary

Titre du Projet de Recherche 

Régulation traductionnelle dans les cellules de mammifères : Les conséquences de l’hétérogénéité transcriptionelle pour le phénotype tumoral.

Contenu et Objectifs du Travail de Recherche

Chez les eucaryotes les ARNm, transcrits à partir de l’ADN génomique, possèdent une région régulatrice en amont de la séquence codant pour la protéine : le 5’UTR (untranslated region ou région non-traduite). La séquence de cette région peut modifier l’expression protéique  quantitativement (la quantité  de protéines exprimées) et qualitativement (la nature des protéines exprimées : certains ARNm mammifères permettent l’expression de différentes chaines polypeptidiques). Il a été montré que certains gènes mammifères expriment plusieurs transcrits d’ARNm avec des 5’UTR différents en utilisant des sites d’initiation transcriptionelle différents (les promoteurs alternatifs). Nous avons récemment observé que l’activité traductionnelle de ces variants est différentes dans les cellules tumorales et non-tumorales. Il s’agit maintenant comprendre les implications de cette observation pour le phénotype tumoral en ciblant deux gènes connus pour être  impliqués dans la régulation de la prolifération cellulaire, 53BP1 et EIF4E3.

Contexte Scientifique et Social du Projet de Recherche.

Les dysfonctionnements dans la régulation traductionnelle sont associés avec plusieurs tumeurs humaines. Mais les implications de l’hétérogénéité observée dans les régions 5’ non-codante pour l’expression protéique  et le phénotype tumoral restent mal définies

Mots-clés

Régulation traductionnelle, hétérogénéité transcriptionelle, phénotype tumorale, les gènes 53BP1 et EIF4E3.

Direct link to Lay Summary Last update: 03.10.2017

Responsible applicant and co-applicants

Employees

Project partner

Collaboration

Group / person Country
Types of collaboration
Prof. Maurizio Molinari, EPFL Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Serge Rudaz, EPGL Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure

Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
Translational Control Meeting CSHL Poster 04.09.2019 Cold Spring Harbour, United States of America Curran Joseph;


Abstract

1.1 Background: The mammalian 5’TL (transcript leader or 5’UTR) carries a mosaic of cis-acting elements (e.g. RNA structure, uAUG/uORFs, IRESes) that regulate the protein readout. Furthermore, the 5’TL can be modified by alternative splicing and promoter switches. Superimposed on the latter is the intrinsic transcriptional start site (TSS) heterogeneity characteristic of PolII. These parameters are regulated in a cell-type specific manner, can be altered in response to changes in the physiological environment and are deregulated in pathologies such as cancer. Collectively, this suggests that the TSS fingerprint (the relative abundance of TSS variants) is a genetic marker for cellular type. However, the impact of this 5’TL complexity with regards to the proteome remains unclear, and this is compounded by the fact that the interplay between the multiple cis-acting elements is cell specific. Our lab has been studying 5’ TL heterogeneity using ELK1 and MDM2 as models. Recently we reported an RNAseq study that characterised 5’ TL complexity within the transcriptome and translatome of the tumoural MCF7 and non-tumoural MCF10A cell lines. We observed 138 genes with TSS variants exhibiting cell-type specific loading onto polysomes.1.2 Proposal-Where we are: High-throughput studies permit us to identify genes expressing cell-type specific TSS variants within the transcriptome and translatome. However, if this is to impact on the cellular phenotype such changes must modify the protein readout. This information cannot be extracted from the datamining. Rather it requires that gene-specific TL variants are experimentally analysed in each cellular context. With this in mind we targeted two genes from our study (53BP1 and EIF4E3). The main protein product of 53BP1 plays a role in DNA repair and has been implicated in breast, ovarian and cervical cancers. It uses two promoters giving rise to three transcript variants. Curiously, the 5’TL variant V3, which has a short uORF, was polysome-associated in MCF7 (tumoural) but non-polysomal in MCF10A (non-tumoural) cells. We have demonstrated that this uORF permits reinitiation events at an internal overlapping ORF (ioORF53BP1). The second selected gene, EIF4E3, is a member of the cap binding protein family. Little is known about its function. It has four major transcript variants arising from independent promoters, and two protein isoforms, namely, isoA and the N-terminally truncated isoB. The RefSeq annotation for the major V1 variant indicated a short 5’TL of only 8 nts. This led us to examine the impact of short TLs on the translational readout. We demonstrated that AUGs within 3nts of the cap can be efficiently used for initiation. Results also confirm other studies indicating that isoA binds the 5’ cap but does not interact with 4EBP1, the negative regulator of the main cap binding protein eIF4E.1.3 Proposal-Where we want to go: To validate that the selective polysomal recruitment of TSS variants impacts on the cellular phenotype one must demonstrate that quantitative and qualitative changes on the protein readout occur and that they have biological consequences. In this light, the selection of EIF4E3 and 53BP1 was not arbitrary. Both express 5’TL variants that markedly alter translational expression with the additional caveat that the 53BP1 V3 TL is uniquely polysomal in the MCF7 cellular context. The onus is now to understand the molecular mechanism underlying this recruitment, dissect the biological function of the ioORF53BP1polypeptide and analyse the implications of quantitative changes in the ratio of the eIF4E3 isoA and isoB with regards to translational control. In parallel we will extend our high-throughput studies on TSS complexity using a modified 5’CAGE protocol in a novel experimental model: tamoxifen drug resistance in MCF7 cells.
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