Dalla Rosa Ilaria, Zhang Hongliang, Khiati Salim, Wu Xiaolin, Pommier Yves
Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
Laboratory of Molecular Technology, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702.
J Biol Chem. 2017 Dec 8;292(49):20162-20172. doi: 10.1074/jbc.M117.815241. Epub 2017 Oct 11.
Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.
线粒体DNA（mtDNA）对细胞活力至关重要，因为它编码呼吸链复合物的亚基。线粒体拓扑异构酶IB（TOP1MT）通过消除mtDNA转录过程中产生的DNA拓扑张力来促进mtDNA复制，但它似乎并非不可或缺。为了测试缺乏TOP1MT的细胞是否具有异常的mtDNA转录，我们进行了线粒体转录组分析。为此，我们设计并实施了一种定制化平铺阵列，该阵列能够在全基因组范围内、链特异性地同时检测所有线粒体转录本。我们的技术表明，敲除（KO）小鼠细胞能够正常处理线粒体转录本，但蛋白质编码的线粒体转录本有所增加。此外，我们发现由H链转录产生的离散长链非编码RNA，其包含人类和小鼠细胞及组织中线粒体DNA的非编码调控区域。值得注意的是，在缺乏TOP1MT的情况下，这些非编码RNA强烈上调。相反，由mtDNA复制产生的7S DNA在KO细胞中减少。我们提出，D环区域中的长链非编码RNA物种是由H链转录本延伸超过其经典终止位点产生的，并且TOP1MT作为mtDNA转录和D环形成的拓扑屏障和调节器发挥作用。
