Upstream ORF affects MYCN translation depending on exon 1b alternative splicing

Roger Besançon,Elisabeth Garin,Christophe Bertrand,Céline Delloye-Bourgeois,Anne-Catherine Jallas,Sandrine Valsesia-Wittmann,Giovani Tutrone,Clara Locher,Lydie Furhman,Alain Puisieux

doi:10.1186/1471-2407-9-445

Roger Besançon, Elisabeth Garin + Show 8 more

Open Access

https://doi.org/10.1186/1471-2407-9-445

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Abstract

BackgroundThe MYCN gene is transcribed into two major mRNAs: one full-length (MYCN) and one exon 1b-spliced (MYCNΔ1b) mRNA. But nothing is known about their respective ability to translate the MYCN protein.MethodsPlasmids were prepared to enable translation from the upstream (uORF) and major ORF of the two MYCN transcripts. Translation was studied after transfection in neuroblastoma SH-EP cell line. Impact of the upstream AUG on translation was evaluated after directed mutagenesis. Functional study with the two MYCN mRNAs was conducted by a cell viability assay. Existence of a new protein encoded by the MYCNΔ1b uORF was explored by designing a rabbit polyclonal antibody against a specific epitope of this protein.ResultsBoth are translated, but higher levels of protein were seen with MYCNΔ1b mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from MYCN but not from MYCNΔ1b mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained with MYCNΔ1b mRNA translation induces an antiapoptotic effect after serum deprivation that was not observed with low MYCN expression obtained with MYCN mRNA. Here, we showed that MYCNOT: MYCN Overlap Transcript, a new protein of unknown function is translated from the upstream AUG of MYCNΔ1b mRNA.ConclusionsExistence of upstream ORF in MYCN transcripts leads to a new level of MYCN regulation. The resulting MYCN dosage has a weak but significant anti-apoptotic activity after intrinsic apoptosis induction.

Highlights

The MYCN gene is transcribed into two major mRNAs: one full-length (MYCN) and one exon 1b-spliced (MYCNΔ1b) mRNA
MYCN and MYCN 1b mRNAs are differently translated Since alternative splicing of exon 1b affects a potential upstream open reading frame, we speculated that it could influence the translation of MYCN protein
In SH-EP neuroblastoma cells, transfection assays followed by Western blot showed no MYCN expression in presence of the control plasmid, weak but detectable translation from p-MYCN, whereas a very high amount of protein was obtained with pMYCNΔ1b (Fig. 2a)

Summary

Introduction

The MYCN gene is transcribed into two major mRNAs: one full-length (MYCN) and one exon 1b-spliced (MYCNΔ1b) mRNA. Several transcripts have been described; one full length: MYCN mRNA and two alternatively spliced mRNAs; one containing exons 1a, 2 and 3: MYCNΔ1b mRNA [2,3] and the other containing exons 1a and 3: MYCNΔ1b,2 mRNA [4]. MYCN and MYCNΔ1b transcripts might translate the MYCN protein. It was previously hypothesized but neither proven nor explored that an upstream AUG (uAUG) located in +1894 (according to GenBank: Y00664) of MYCNΔ1b mRNA might translate a protein (GenBank: AAG40001.1) [5]. As proposed by van Bokhoven et al, MYCNΔ1b,2 mRNA could be able to initiate translation of a new protein (ΔMYCN) from the same uAUG [4]. Most studies have shown a negative cisregulatory function of the major ORF [6,7,8]

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