POLRMT regulates the switch between replication-primer formation and gene expression of mammalian mtDNA

Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it remains highly controversial if POLRMT also serves as the primase for initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion and TFAM is thus protected from degradation of the AAA+ Lon protease in absence of POLRMT. Lastly, mitochondrial transcription elongation factor (TEFM) can compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role for this factor in transcription. In conclusion, we present here the first in vivo evidence that POLRMT has a key regulatory role in replication of mammalian mtDNA and is part of a mechanism that provides a switch between RNA primer formation for mtDNA replication and mtDNA expression. Overall design: Isolated heart mitochondria from three control mice (L/L) and three Polrmt knockout mice (L/L, cre), aged 3-4 weeks, were sequenced and analyzed for differential expression.

线粒体通过自身的氧化磷酸化系统为细胞提供能量，该系统依赖核基因组与线粒体DNA（mitochondrial DNA, mtDNA）编码基因的协同表达。环状哺乳动物mtDNA的转录依赖于单一的线粒体RNA聚合酶（POLRMT）。尽管转录起始过程已被充分解析，但POLRMT是否同时作为线粒体DNA复制起始的引物酶，这一问题仍存在极大争议。在细胞核中，负责基因表达的RNA聚合酶并无此类功能。在年轻小鼠心脏中条件性敲除Polrmt，会导致严重的线粒体功能障碍，进而引发扩张型心肌病。本研究进一步探究了POLRMT不同表达水平带来的分子效应，并证实POLRMT是体内合成引物以启动线粒体DNA复制的必需因子。此外，用于引物合成的转录起始过程优先于基因表达过程。令人意外的是，在Polrmt敲除小鼠中，线粒体转录因子A（TFAM）以不结合mtDNA的游离池形式存在。尽管mtDNA发生大量耗竭，但TFAM的表达水平保持不变；因此在POLRMT缺失的情况下，TFAM可免于被AAA+家族Lon蛋白酶降解。最后，线粒体转录延伸因子（TEFM）可在杂合子Polrmt敲除小鼠中部分弥补POLRMT的表达不足，这表明该因子在转录过程中发挥直接的调控作用。综上，本研究首次提供了体内证据，证实POLRMT在哺乳动物mtDNA复制中发挥关键调控作用，且其参与构成了调控mtDNA复制所需RNA引物合成与mtDNA表达之间切换的机制。整体实验设计：从3只3~4周龄的对照小鼠（L/L）与3只Polrmt敲除小鼠（L/L, cre）中分离心脏线粒体，对其进行测序并分析差异表达情况。