Abstract
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.
Highlights
We have described a splice variant of pro-apoptotic BH1-3domain Bcl-2 family member Bak that we named N-Bak for ‘neuronal Bak’ because it is expressed only in the neurons and not in any other tested primary cells or tissues,[6] as reported by others.[7]
The main aim of this study was to address whether the N-Bak mRNA is the substrate for Nonsense-mediated mRNA decay (NMD) that could explain the absence of the encoded protein in the neurons
Inclusion of a 20-nucleotide exon N to N-Bak mRNA causes a translational frameshift and premature translation-termination codon (PTC) that corresponds to the 55-nt rule of NMD
Summary
We have described a splice variant of pro-apoptotic BH1-3domain Bcl-2 family member Bak that we named N-Bak for ‘neuronal Bak’ because it is expressed only in the neurons and not in any other tested primary cells or tissues,[6] as reported by others.[7]. The PTC on the N-Bak mRNA locates at 89 nucleotides upstream from the last exon–exon junction (Figure 1c) corresponding to the 55-nt NMD rule. The N-Bak mRNA could be degraded during the pioneer round of translation, explaining the absence of the protein. In this study we set up to test the hypothesis that N-Bak mRNA is degraded in the neurons by NMD that causes absence of the protein. Our results suggest that N-Bak mRNA is not the NMD substrate in the neurons and belongs to the mRNAs that escape the NMD despite the correspondence to the 55-nt NMD rule. Our data suggest that N-Bak mRNA is translationally arrested and its 50-untranslated region (UTR) is partially responsible for this arrest
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