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

Background: The MYCN gene is transcribed into two major mRNAs: one full-length (MYCN) and one exon 1b-spliced (MYCN Delta 1b) 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 SHEP 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 MYCN Delta 1b 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 MYCN Delta 1b mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from MYCN but not from MYCN Delta 1b mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained with MYCN Delta 1b 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 MYCN Delta 1b 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.