Department of Cellular Biochemistry, University Medical Center Göttingen, 37073 Göttingen, Germany.
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; Department of Clinical Chemistry, University Medical Center Göttingen, 37073 Göttingen, Germany.
Cell. 2021 Nov 11;184(23):5824-5837.e15. doi: 10.1016/j.cell.2021.09.033. Epub 2021 Oct 20.
The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an in vitro system to silence translation in purified mitochondria. In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.
人类线粒体基因组编码氧化磷酸化系统的十三个核心亚基，线粒体基因表达的缺陷导致严重的神经肌肉疾病。然而，由于缺乏分析这些过程的实验方法，线粒体基因表达的机制仍不清楚。在这里，我们提出了一种在纯化的线粒体中沉默翻译的体外系统。通过化学合成的前体-吗啉代杂交物的体外导入，我们可以靶向单个线粒体 mRNA 的翻译。通过应用这种方法，我们得出结论，双顺反子、重叠的 ATP8/ATP6 转录本通过单个核糖体/mRNA 结合进行翻译。我们表明，COX1 组装因子向翻译核糖体的募集取决于新生链的形成。通过定义 COX1 和 COX2 的 mRNA 特异性相互作用组，我们揭示了细胞质癌胎儿 IGF2BP1（一种 RNA 结合蛋白）在线粒体翻译中的一个意外功能。我们的数据为线粒体翻译和研究线粒体基因表达的创新策略提供了深入的见解。
