Initiation of mtDNA transcription is followed by pausing, and diverges across human cell types and during evolution
Initiation of mtDNA transcription is followed by pausing, and diverges across human cell types and during evolution
复制标题
DOI：
10.1101/gr.209924.116
复制
发表时间：
2017-03-01
期刊：
影响因子：
7
通讯作者：
Mishmar, Dan
中科院分区：
文献类型：
--
作者：
Blumberg, Amit;Rice, Edward J.;Mishmar, Dan
线粒体DNA（mtDNA）基因在多顺反子中共转录，但利用现有的工具在体内定量研究新生mtDNA转录本仍然是不可能的。为此，我们使用深度测序（GRO-seq和PRO-seq）并分析了不同人类细胞系和后生动物生物体中新生mtDNA编码的RNA转录本。令人惊讶的是，在重链和轻链的人类mtDNA转录起始位点（TIS）的准确检测揭示了一个新的保守的轻链TIS附近的转录暂停位点。该暂停位点与细菌暂停序列基序的存在相关，具有降低的SNP密度，并且在所有测试的细胞中具有DNA酶足迹信号。它的位置在保守序列块3（CSBIII），上游已知的转录-复制转换点，表明参与这种转换。非人类生物体的分析使从头mtDNA序列组装，以及检测以前未知的mtDNA TIS，暂停和转录终止位点前所未有的准确性。哺乳动物（黑猩猩、猕猴、褐家鼠和小家鼠）表现出类似人类的mtDNA转录模式，而无脊椎动物（黑腹果蝇和秀丽隐杆线虫）的模式则截然不同。我们的方法为所有真核生物中mtDNA转录的体内定量、无参考序列分析铺平了道路。
Mitochondrial DNA (mtDNA) genes are long known to be cotranscribed in polycistrones, yet it remains impossible to study nascent mtDNA transcripts quantitatively in vivo using existing tools. To this end, we used deep sequencing (GRO-seq and PRO-seq) and analyzed nascent mtDNA-encoded RNA transcripts in diverse human cell lines and metazoan organisms. Surprisingly, accurate detection of human mtDNA transcription initiation sites (TISs) in the heavy and light strands revealed a novel conserved transcription pausing site near the light-strand TIS. This pausing site correlated with the presence of a bacterial pausing sequence motif, with reduced SNP density, and with a DNase footprinting signal in all tested cells. Its location within conserved sequence block 3 (CSBIII), just upstream of the known transcription-replication transition point, suggests involvement in such transition. Analysis of nonhuman organisms enabled de novo mtDNA sequence assembly, as well as detection of previously unknown mtDNA TIS, pausing, and transcription termination sites with unprecedented accuracy. Whereas mammals (Pan troglodytes, Macaca mulatta, Rattus norvegicus, and Mus musculus) showed a human-like mtDNA transcription pattern, the invertebrate pattern (Drosophila melanogaster and Caenorhabditis elegans) profoundly diverged. Our approach paves the path toward in vivo, quantitative, reference sequence-free analysis of mtDNA transcription in all eukaryotes.