Institute of Biotechnology, University of Helsinki, FIN-00014, Helsinki, Finland.
Cell Death Dis. 2013 Aug 22;4(8):e777. doi: 10.1038/cddis.2013.297.
N-Bak is a neuron-specific BH3-only splice variant of pro-apoptotic Bcl-2 family member Bak. We have shown that its mRNA is stable in the neurons, whereas the protein cannot be detected by antibodies, suggesting a strong translational arrest of the mRNA. Here we identify two regulatory elements in the N-Bak mRNA that significantly repress translation in the luciferase reporter assay: an upstream open reading frame in the 5'-untranslated region (UTR) and naturally spliced exon-exon junction downstream of the premature translation termination codon in the 3'UTR. We also show that N-Bak mRNA is stored in granular structures in the sympathetic neurons and stays in these granules during intrinsic apoptosis. Finally, we confirm the absence of N-Bak protein by quantitative mass spectrometry analysis in the healthy, apoptotic or stressed sympathetic and cortical neurons. We conclude that N-Bak mRNA is translationally repressed by multiple mechanisms, and the protein does not participate in the classical apoptosis or cellular stress response.
N-Bak 是一种神经元特异性的 Bcl-2 家族促凋亡蛋白 Bak 的 BH3 结构域缺失的剪接变异体。我们已经表明，其 mRNA 在神经元中是稳定的，而蛋白质不能被抗体检测到，这表明该 mRNA 存在强烈的翻译阻断。在这里，我们在 N-Bak mRNA 中鉴定了两个显著抑制荧光素酶报告基因检测中翻译的调节元件：5'非翻译区（UTR）中的上游开放阅读框和 3'UTR 中过早翻译终止密码子下游的天然剪接外显子-外显子连接。我们还表明，N-Bak mRNA 储存在交感神经元的颗粒结构中，并在内在凋亡过程中保持在这些颗粒中。最后，我们通过定量质谱分析在健康、凋亡或应激的交感和皮质神经元中证实了 N-Bak 蛋白的缺失。我们得出结论，N-Bak mRNA 通过多种机制被翻译抑制，该蛋白不参与经典的细胞凋亡或应激反应。
