Open Access Research article

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

Roger Besançon12*, Sandrine Valsesia-Wittmann3, Clara Locher1, Céline Delloye-Bourgeois1, Lydie Furhman1, Giovani Tutrone1, Christophe Bertrand1, Anne-Catherine Jallas3, Elisabeth Garin3 and Alain Puisieux123

Author Affiliations

1 INSERM UMR590, 28 rue Laënnec, Lyon, France

2 Université de Lyon, université Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, 8 avenue Rockefeller, Lyon, 69008, France

3 Centre Léon Bérard, Laboratoire de Recherche Translationnelle, 28 rue Laënnec, Lyon, France

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BMC Cancer 2009, 9:445  doi:10.1186/1471-2407-9-445

Published: 17 December 2009

Abstract

Background

The 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.

Methods

Plasmids 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.

Results

Both 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.

Conclusions

Existence 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.