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 (MYCNDelta1b) 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 MYCNDelta1b 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 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.


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.
Besançon
Roger
R
INSERM UMR590, 28 rue Laënnec, Lyon, France. roger.besancon@recherche.univ-lyon1.fr
Valsesia-Wittmann
Sandrine
S
Locher
Clara
C
Delloye-Bourgeois
Céline
C
Furhman
Lydie
L
Tutrone
Giovani
G
Bertrand
Christophe
C
Jallas
Anne-Catherine
AC
Garin
Elisabeth
E
Puisieux
Alain
A
D000328:Adult; D017398:Alternative Splicing / Q000235:genetics*; D001483:Base Sequence; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D005091:Exons / Q000235:genetics; D005333:Fetus / Q000378:metabolism; D018628:Gene Dosage / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D015513:Oncogene Proteins / Q000235:genetics* / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics* / Q000502:physiology; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D020033:Protein Isoforms; D022821:RNA Splice Sites; D012333:RNA, Messenger