mRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons.
mRNA for neuronal Bak (N-Bak), a splice variant of pro-apoptotic Bcl-2 family member Bak is expressed in the neurons. Surprisingly the endogeneous N-Bak protein cannot be demonstrated in the neurons, although the antibodies recognize N-Bak protein from in vitro translation or transiently transfected cells. As N-Bak mRNA contains premature termination codon (PTC) at 89 nucleotides upstream from the last exon-exon junction, it could be degraded by nonsense-mediated decay (NMD) during the pioneer round of translation thus explaining the absence of the protein. We show here that the endogeneous neuronal N-Bak mRNA is not the NMD substrate, as it is not accumulating by cycloheximide treatment, it has a long lifetime, and even prevention of PTC by interfering with the alternative splicing did not lead to translation of the Bak mRNA. N-Bak protein is also not revealed by proteasome inhibitors. Our data suggest strong translational arrest of N-Bak mRNA in the neurons. We show that this arrest is partially mediated by 5'-untranslated region of Bak mRNA and it is not released during mitochondrial apoptosis.
Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland.
M
M
Jakobson
A
A
Lintulahti
U
U
Arumäe
D024506:5' Flanking Region / Q000235:genetics*; D017398:Alternative Splicing; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D018389:Codon, Nonsense; D003513:Cycloheximide / Q000494:pharmacology; D004622:Embryo, Mammalian; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D061251:Primary Cell Culture; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis*; D014162:Transfection; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics* / Q000378:metabolism
D018389:Codon, Nonsense; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D050998:bcl-2 Homologous Antagonist-Killer Protein; D003513:Cycloheximide