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The elk-1 gene: A model for how alternative splicing in the 5'UTR can modulate translational reinitiation in mammalian cells.

English title The elk-1 gene: A model for how alternative splicing in the 5'UTR can modulate translational reinitiation in mammalian cells.
Applicant Curran Joseph
Number 143772
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 01.10.2012 - 30.09.2015
Approved amount 265'000.00
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All Disciplines (2)

Discipline
Molecular Biology
Biochemistry

Keywords (4)

translation; reinitiation; splicing; mammalian

Lay Summary (English)

Lead
Lay summary
Protein synthesis on cytoplasmic ribosomes, in which the information encoded on the mRNA transcript is translated to form a polypeptide chain, represents a key event in the expression of the genome. As such, it is at the heart of events controlling cellular proliferation and differentiation. This is highlighted by the fact that its deregulation is a recurrent observation in human cancer. One key feature on the cellular mRNA that regulates the translational readout is the 5’ untranslated region (UTR). Features within this region can influence ribosome recruitment to the mRNA, limit ribosomal access to the AUG initiation codon and even modulate the site at which protein synthesis begins and, as a consequence, change the nature of the protein(s) expressed. These events are “tuned-in” to multiple intracellular signalling pathways that dictate the physiological status of the cell. Furthermore, the nature of the 5’UTR can change. This heterogeneity arises due to the use of alternative promoters and/or alternative splicing of the primary transcript and serves to couple regulatory events in the nucleus and cytoplasm. These changes can impact on the readout quantitatively in terms of the amount of polypeptide synthesised, but also qualitatively in terms of start site selection and the nature of the translational product(s). Intriguingly, heterogeneity is more frequent in genes regulating cell growth. Our research focuses on how 5’UTR heterogeneity modulates the translational readout and seeks to understand how this is integrated into pathways regulating cell growth and differentiation. As a model we have worked on the mammalian ELK1 gene in which 5’UTR heterogeneity arises due to alternative splicing. The Elk1 protein functions as a transcription factor whose principle target is the promoter of the c-FOS proto-oncogene. As such it plays an important role in cell growth control. In addition, a shorter form of Elk-1, arising due to the utilisation of an internal translational start site, is observed in neuronal cells and potentiates neurite growth. Our published work indicates that these initiation events are being “fine-tuned” by the alternative splicing in the 5’UTR. Insights gained from this model system will then be extrapolated to a genome-wide analysis of how 5’UTR heterogeneity impacts on pathologies such as cancer.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
What Is the Impact of mRNA 5' TL Heterogeneity on Translational Start Site Selection and the Mammalian Cellular Phenotype?
Joseph A. Curran and Benjamin Weiss (2016), What Is the Impact of mRNA 5' TL Heterogeneity on Translational Start Site Selection and the Mammalian Cellular Phenotype?, in Frontiers in Genetics (RNA), 7, 156.
CAP+ selection: A combined chemical-enzymatic strategy for efficient eukaryotic mRNA enrichment via the 5’ cap.
Weiss B and Curran J. (2015), CAP+ selection: A combined chemical-enzymatic strategy for efficient eukaryotic mRNA enrichment via the 5’ cap., in Analytical Biochemistry, 484, 72-74.
An AUG codon conserved for protein function rather than translational initiation: the story of the protein sElk1.
Legrand N. Araud T. Conne B. Kuijpers O. Jaquier-Gubler P and Curran J. (2014), An AUG codon conserved for protein function rather than translational initiation: the story of the protein sElk1., in PLoS ONE , 9, e102890.
Alternative Splicing within the elk-1 5' Untranslated Region Serves To Modulate Initiation Events Downstream of the Highly Conserved Upstream Open Reading Frame 2.
Rahim G. Araud T. Jaquier-Gubler P and Curran J. (2012), Alternative Splicing within the elk-1 5' Untranslated Region Serves To Modulate Initiation Events Downstream of the Highly Conserved Upstream Open Reading Frame 2., in Molecular and Cellular Biology , 32, 1745-1756.
The Effect of Heterogeneous Transcription Start Sites (TSS) on the Translatome: Implications for the Mammalian Cellular Phenotype.
Dieudonné Francois-Xavier, O’Connor Patrick B.F, Gubler-Jaquier Pascale, Yasrebi Haleh, Conne Beatrice, Nikolaev Sergey, Antonarakis Stylianos, Baranov Pavel V, Curran Joseph, The Effect of Heterogeneous Transcription Start Sites (TSS) on the Translatome: Implications for the Mammalian Cellular Phenotype., in BMC Genomics.
The impact of the phosphomimetic eIF2αS/D on global translation, reinitiation and the integrated stress response is attenuated in N2a cells.
Legrand N. Jaquier-Gubler P and Curran J., The impact of the phosphomimetic eIF2αS/D on global translation, reinitiation and the integrated stress response is attenuated in N2a cells., in Nucleic Acids Research.

Collaboration

Group / person Country
Types of collaboration
Dr Euphemia Leung, Auckland Cancer Society Research Centre New Zealand (Oceania)
- in-depth/constructive exchanges on approaches, methods or results
Prof. S Antonarakis, Departement de génétique médicale et de développement, UNIGE Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. P Baranov, Department of Biochemistry, University College Cork Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Protein Synthesis and Translational Control Talk given at a conference The impact of the phosphomimetic eIF2αS/D on global translation, reinitiation and the integrated stress response is attenuated in N2a cells. 09.09.2015 Heidelberg, EMBO, Germany Curran Joseph;
EMBO Conference "Protein Synthesis and Translational Control" Poster RNAseq analysis of total and polysomal transcript populations in the MCF7/10A cells lines demonstrates extensive mRNA 5' leader heterogeneity. 08.09.2013 EMBL Heidelberg, Germany Curran Joseph;


Associated projects

Number Title Start Funding scheme
116170 Mammalian Translation initiation and cellular Growth Control: (a). Translational Regulation of the Transcription Factor ELK-1. (b). Studies on Global Regulation. (c). Translational Profiling of Chromosome 21 01.04.2007 Project funding (Div. I-III)

Abstract

The proposed project focuses on the regulation of protein synthesis and how this impacts on cellular physiology. It is very much a continuation and extension of the labs previous FNS grant. Recently, we have begun to focus on 5’UTR heterogeneity and its role in modulating the protein readout. Such variation arises as a result of alternative splicing and/or alternative promoter usage and serves to couple nuclear events to protein expression. These changes can have both quantitative (in terms of the amount of primary protein product synthesised) and qualitative (with regards to the types of primary protein products expressed) consequences. This heterogeneity may also “fine-tune” the expression of a particular gene to rapid intracellular changes. Despite the fact that as much as 22% of mammalian genes have alternative 5’UTRs, many of which are highly conserved, little is known about how this impacts on the cellular proteome. Furthermore, it remains unclear as to what extent non-conserved elements in the 5’UTR serve to modulate the translational readout in a species specific manner. Our work addresses these problems by focusing on a particular cellular gene that exhibits 5’ heterogeneity, namely, elk1. ELK-1 belongs to the subfamily of TCF transcription factors (Ternary Complex Factor) whose activation triggers the immediate early transcriptional response and the activation of genes such as c-fos. In rat neuronal tissue, a shorter N-terminally truncated protein, referred to as sELK-1, was reported. It is initiated from the 7th AUG start site on the mRNA and its expression correlated with NGF-driven neuronal differentiation and neurite outgrowth. Our lab has previously characterised two alternatively spliced 5’ UTR isoforms (5’UTRL and 5’UTRS) of the human gene. Both contained elements that play a role in translational regulation including two uAUGs (the second starting an ORF of only 2 codons) and stable RNA structures. The alternatively spliced exon is positioned just upstream of uORF2. Our published work indicated that leaky scanning, shunting and uORF2-mediated reinitiation all served to regulate ribosomal access to the AUGELK-1 (the 3rd on the mRNA). It also demonstrated that in human cells all ribosomes detected downstream of the AUGELK-1 were in a delayed reinitiation mode. These ribosomes were sequestered by a series of highly conserved, out-of-frame, iAUGs that served as repressors for sELK-1 expression in this cellular context. Recently, we have recently characterised the 5’UTRs from rat (PC12 cell line) and mouse (the N2a neuroblastoma cell line) and observed that the alternatively spliced exonII is species-specific. Furthermore, despite conservation of the iAUGs in these species we readily observed sELK-1 protein expression in N2a and differentiated PC12 cells. Our results, based upon the human system, suggests that this may arise due to a de-repression of negative structural elements within the 5’UTRs (possibly associated with the expression of RNA helicases) coupled to changes in ternary complex levels. They also point to differences in the behaviour of the alternatively spliced transcripts. In this proposal we aim to test this model.
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