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

• Published in: BMC cancer 2009-12, Vol.9 (1), p.445-445, Article 445
• Main Authors: Besançon, Roger, Valsesia-Wittmann, Sandrine, Locher, Clara, Delloye-Bourgeois, Céline, Furhman, Lydie, Tutrone, Giovani, Bertrand, Christophe, Jallas, Anne-Catherine, Garin, Elisabeth, Puisieux, Alain
• Format: Article
• Language: English
• Subjects: Adult; Alternative Splicing; Alternative Splicing - genetics; Apoptosis; Base Sequence; Cancer; Cell Survival; Cell Survival - genetics; Cells, Cultured; Embryos; Exon (Molecular genetics); Exons; Exons - genetics; Experiments; Fetus; Fetus - metabolism; Gene Dosage; Gene Dosage - physiology; Gene Expression; Gene Expression - physiology; Genes; Genetic translation; Humans; Life Sciences; N-Myc Proto-Oncogene Protein; Nuclear Proteins; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Oncogene Proteins; Oncogene Proteins - genetics; Oncogene Proteins - metabolism; Open Reading Frames; Open Reading Frames - genetics; Open Reading Frames - physiology; Physiological aspects; Plasmids; Protein Biosynthesis; Protein Isoforms; Protein Isoforms - genetics; Proteins; RNA Splice Sites; RNA Splice Sites - genetics; RNA, Messenger; RNA, Messenger - genetics; RNA, Messenger - metabolism
• ISSN: 1471-2407; 1471-2407
• DOI: 10.1186/1471-2407-9-445

The MYCN gene is transcribed into two major mRNAs: one full-length (MYCN) and one exon 1b-spliced (MYCNDelta1b) mRNA. But nothing is known about their respective ability to translate the MYCN protein. 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 MYCNDelta1b uORF was explored by designing a rabbit polyclonal antibody against a specific epitope of this protein. Both are translated, but higher levels of protein were seen with MYCNDelta1b mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from MYCN but not from MYCNDelta1b mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained with MYCNDelta1b 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 MYCNDelta1b mRNA. 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.