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Activation of mtDNA transcription by mitochondrial RNA polymerase overexpression enhances stress-induced exercise capacity

ABSTRACT: POLRMT is the sole RNA polymerase in human mitochondria, where it generates primers for mitochondrial DNA (mtDNA) replication and transcribes the mtDNA to express genes encoding essential components of the oxidative phosphorylation (OXPHOS) system. Elevated POLRMT levels are found in several cancers and in mouse models with severe mitochondrial dysfunction. Here, we generated and characterized mice over-expressing Polrmt to investigate the physiological and molecular consequences of elevated POLRMT levels. Increasing POLRMT levels did not result in any pathological phenotype but led to increased exercise performance in male mice under stress conditions. Polrmt overexpression increased de novo transcription initiation, resulting in higher steady-state levels of the promoter-proximal L-strand transcript 7S RNA. Surprisingly, the abundance of mature mitochondrial RNAs was not affected by the elevated POLRMT levels. Furthermore, ubiquitous simultaneous overexpression of Polrmt and Lrpprc, which stabilizes mitochondrial messenger RNAs, did not increase steady-state levels of mitochondrial transcripts in the mouse. Our data show that POLRMT levels regulate transcription initiation, but additional regulatory steps downstream of transcription initiation and transcript stability limit OXPHOS biogenesis.

ORGANISM(S): Mus musculus

PROVIDER: GSE307758 | GEO | 2025/09/11

REPOSITORIES: GEO

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

Project description: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. 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.

2016-08-05 | E-GEOD-83368 | biostudies-arrayexpress

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

2016-08-05 | GSE83368 | GEO

ChIP analysis trial of human TFB2M and POLRMT interaction with mtDNA

Project description:In many mammalian tissues and cells, two classes of mitochondrial DNA (mtDNA) replication intermediates have been observed. One involves leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Surprisingly, removal of either protein resulted in the complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for human mtDNA maintenance. Moreover, the lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of strand-coupled replication initiation.

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High levels of TFAM repress in vivo transcription of mitochondrial DNA

Project description:Mammalian mitochondrial DNA (mtDNA) is coated with mitochondrial transcription factor A (TFAM) and compacted into nucleoids. TFAM is not only the main component of mitochondrial nucleoids but its levels can also control mtDNA copy number. Here we show that the TFAM-to-mtDNA ratio is critical for maintaining normal mtDNA expression in different tissues of the mouse. BAC transgenic mice with a 1.5-fold increase in TFAM protein levels maintain a normal TFAM-to-mtDNA ratio in different tissues and as a consequence mitochondrial gene expression, nucleoid distribution and whole animal metabolism are all unaltered. In contrast, mice expressing TFAM from the CAG promoter in the ROSA26 locus have 4.5-fold increase of TFAM protein levels in heart and skeletal muscle and develop pathology leading to early postnatal lethality. The TFAM-to-mtDNA ratio varies widely between tissues in these mice and is very high in skeletal muscle where it causes strong repression of mtDNA expression and deficient oxidative phosphorylation (OXPHOS) despite normal mtDNA levels. In heart, mtDNA copy number is increased leading to a near normal TFAM-to-mtDNA ratio and maintained OXPHOS capacity. In the liver, mtDNA expression is maintained despite increased TFAM levels and normal mtDNA levels. Here, tissue-specific induction of the LONP1 protease and mitochondrial RNA polymerase (POLRMT) expression counteracts the silencing effect of high TFAM levels. We conclude that the TFAM-to-mtDNA ratio has an important role in maintaining mtDNA expression in vivo. TFAM acts as a general repressor of mtDNA expression and this effect can be counterbalance by tissue-specific expression of regulatory factors.

2021-08-18 | PXD023050 | Pride

Chemical inhibition of mtDNA transcription for treatment of cancer

Project description:Mitochondrial oxidative phosphorylation (OXPHOS) is important for cancer cell growth and the persistence of therapy-resistant cancer stem cells. Here, we have developed novel first-in-class inhibitors of mitochondrial transcription (IMTs) targeting mtDNA gene expression. We show that IMTs specifically target mitochondrial RNA polymerase (POLRMT) and we defined the IMT binding site using exome sequencing of cells rendered resistant to IMTs by chemical mutagenesis and cryo-EM. IMTs act as allosteric inhibitors of POLRMT, impairing binding of the DNA-RNA hybrid and translocation of the nascent RNA. IMT treatment impairs OXPHOS in a dose-dependent way and decreases cell viability in various tumour cells. Surprisingly, several weeks of per oral IMT treatment is well tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in human cancer xenografts. We thus show that the IMTs represent a novel promising class of drugs for tumour treatment acting by inhibition of mtDNA gene expression.

2021-12-03 | PXD018426 | Pride

Demethylzeylasteral inhibits oxidative phosphorylation complex biogenesis by targeting LRPPRC in lung cancer

Project description:Targeted inhibition of mitochondrial oxidative phosphorylation (OXPHOS) complex generation is an emerging and promising cancer treatment strategy, but limited targets and specific inhibitors have been reported. Leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) is an atypical RNA-binding protein that regulates the stability of all 13 mitochondrial DNA-encoded mRNA (mt-mRNA) and thus participates in the synthesis of the OXPHOS complex. LRPPRC is also a prospective therapeutic target for lung adenocarcinoma, serving as a promising target for OXPHOS inhibition. In this study, we identified Demethylzeylasteral (T-96), a small molecule extracted from the Chinese herb Tripterygium wilfordii Hook. f., as a novel inhibitor of LRPPRC. T-96 directly bound to the RNA-binding domain of LRPPRC, inhibiting its interaction with mt-mRNA. This led to instability in both mt-mRNA and LRPPRC protein. Treatment with T-96 significantly reduced the mRNA and protein levels of the OXPHOS complex. As a consequence of LRPPRC inhibition, T-96 treatment induced a defect in the synthesis of the OXPHOS complex, inhibiting mitochondrial aerobic respiration and ATP synthesis. Moreover, T-96 exhibited potent antitumor activity for lung adenocarcinoma in vitro and in vivo, and the antitumor effect of T-96 was dependent on LRPPRC expression. In conclusion, this study not only identified the first traditional Chinese medicine monomer inhibitor against OXPHOS complex biosynthesis as well as a novel target of Demethylzeylasteral, but also shed light on the unique antitumor mechanism of bioactive compounds derived from traditional Chinese medicine.

2024-10-09 | GSE279114 | GEO

Mitochondrial and nuclear genomic response to loss of LRPPRC expression

Project description:Rapid advances in genotyping and sequencing technology have dramatically accelerated the discovery of genes underlying human disease. Elucidating the function of such genes and understanding their role in pathogenesis, however, remains challenging. Here, we introduce a genomic strategy to functionally characterize such genes, and apply it to LRPPRC (leucine-rich PPR-motif containing), a poorly studied gene that is mutated in Leigh Syndrome, French Canadian type (LSFC). We utilize RNAi to engineer an allelic series of cellular models in which LRPPRC has been stably silenced to different levels of knockdown efficiency. Using expression profiling, we discovered a specific role for LRPPRC in the expression of all mitochondrial DNA (mtDNA)-encoded mRNAs, but not the rRNAs, without affecting nuclear genes encoding mitochondrial proteins. We designed seven shRNAs targeting the LRPPRC cDNA sequence to silence its expression in MCH58 immortalized human fibroblasts. The LRPPRC expression level in these cells ranged from 9% to 100%. We demonstrated that knockdown cells carried stable silencing of the target gene and associated biochemical phenotypes. Our goal was to engineer stable knockdown cells which recapitulate the LSFC disease phenotype and subject them to expression profiling using Affymetric microarrays to identify genesets and biochemical pathways that are altered.

2010-03-23 | E-GEOD-20847 | biostudies-arrayexpress

Regulatory principles of human mitochondrial gene expression revealed by kinetic analysis of the RNA life cycle

Project description:Mitochondria play a critical role in cellular metabolism primarily through hosting the oxidative phosphorylation (OXPHOS) machinery that is encoded by mitochondrial DNA (mtDNA) and nuclear DNA, with each gene expression system being regulated by a distinct set of mechanisms. In this study, we performed a quantitative analysis of the mitochondrial messenger RNA (mt-mRNA) life cycle to gain insights into the principles underlying the regulation of mtDNA-encoded protein abundance. Our analysis revealed a unique balance between the rapid turnover and high accumulation of mt-mRNA, leading to a 700-fold higher transcriptional output compared to nuclear-encoded OXPHOS genes. Additionally, we observed that mt-mRNA processing and its association to the mitochondrial ribosome occur rapidly and that these processes are linked mechanistically. These data resulted in a model of mtDNA expression that is predictive across cell lines, revealing that differential turnover and translation efficiencies are the major contributors to mitochondrial-encoded protein synthesis. Applying this framework to a disease model of Leigh syndrome, French–Canadian type, we found that the responsible nuclear-encoded gene, LRPPRC, disrupts OXPHOS biogenesis predominantly through altering mt-mRNA stability. Our findings provide a comprehensive view of the intricate regulatory mechanisms governing mtDNA-encoded protein synthesis, highlighting the importance of quantitatively analyzing the mitochondrial RNA life cycle for decoding the regulatory principles of mtDNA expression.

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MitoExome Sequencing of Mitochondrial OXPHOS Diseases (MGH)

Project description: Human disorders of mitochondrial oxidative phosphorylation (OXPHOS) represent a devastating collection of inherited diseases. These disorders impact at least 1:5000 live births, and are characterized by multi-organ system involvement. They are characterized by remarkable locus heterogeneity, with mutations in the mtDNA as well as in over 77 nuclear genes identified to date. It is estimated that additional genes may be mutated in these disorders. To discover the genetic causes of mitochondrial OXPHOS diseases, we performed targeted, deep sequencing of the entire mitochondrial genome (mtDNA) and the coding exons of over 1000 nuclear genes encoding the mitochondrial proteome. We applied this 'MitoExome' sequencing to 124 unrelated patients with a wide range of OXPHOS disease presentations from the Massachusetts General Hospital Mitochondrial Disorders Clinic. The 2.3Mb targeted region was captured by hybrid selection and Illumina sequenced with paired 76bp reads. The total set of 1605 targeted nuclear genes included 1013 genes with strong evidence of mitochondrial localization from the MitoCarta database, 377 genes with weaker evidence of mitochondrial localization from the MitoP2 database and other sources, and 215 genes known to cause other inborn errors of metabolism. Approximately 88% of targeted bases were well-covered (>20X), with mean 200X coverage per targeted base.

| phs000339 | dbGaP

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