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Mitochondrial oxidative phosphorylation (OXPHOS) is essential for ATP production to maintain sperm linear motility during migration from the uterus to the oviduct. However, ROS are generated as by-products of OXPHOS, causing stress and damaging the sperm quality. This study aimed to clarify the ROS targets in sperm mitochondria that decrease linear motility and to investigate whether mitochondria-target antioxidants (PQQ and CoQ10) affect mitochondrial activity and sperm motility. Sperm linear motility pattern, ATP production, and mitochondrial activity were decreased with increasing ROS levels during incubation in the low-glucose medium. However, sperm motility patterns and ROS levels were not significantly changed in the high-glucose medium. Moreover, the gene expression system (mt-DNA, mitochondrial transcription factor-A (TFAM) and RNA polymerase (POLRMT)) in sperm mitochondria was damaged during incubation in the low-glucose medium. Interestingly, PQQ treatment increased the mt-DNA stability and decreased the damage to TFAM and POLRMT, which resulted in high expression of mitochondrial genes. Furthermore, the antioxidants increased mitochondrial activity and maintained sperm linear motility under the low glucose condition. These results revealed that both ATP production and the mitochondrial transcription system are damaged with increasing ROS levels in sperm that show a linear motility pattern. Treatment with antioxidants, such as PQQ and CoQ10, is beneficial tool to maintain sperm linear motility.

The bacterial heritage of mitochondria, as well as its independent genome [mitochondrial DNA (mtDNA)] and polycistronic transcripts, led to the view that mitochondrial transcriptional regulation relies on an evolutionarily conserved, prokaryotic-like system that is separated from the rest of the cell. Indeed, mtDNA transcription was previously thought to be governed by a few dedicated direct regulators, namely, the mitochondrial RNA polymerase (POLRMT), two transcription factors (TFAM and TF2BM), one transcription elongation (TEFM), and one known transcription termination factor (mTERF1). Recent findings have, however, revealed that known nuclear gene expression regulators are also involved in mtDNA transcription and have identified novel transcriptional features consistent with adaptation of the mitochondria to the regulatory environment of the precursor of the eukaryotic cell. Finally, whereas mammals follow the human mtDNA transcription pattern, other organisms notably diverge in terms of mtDNA transcriptional regulation. Hence, mtDNA transcriptional regulation is likely more evolutionary diverse than once thought.

Mitochondria play fundamental roles in the regulation of life and death of eukaryotic cells. They mediate aerobic energy conversion through the oxidative phosphorylation (OXPHOS) system, and harbor and control the intrinsic pathway of apoptosis. As a descendant of a bacterial endosymbiont, mitochondria retain a vestige of their original genome (mtDNA), and its corresponding full gene expression machinery. Proteins encoded in the mtDNA, all components of the multimeric OXPHOS enzymes, are synthesized in specialized mitochondrial ribosomes (mitoribosomes). Mitoribosomes are therefore essential in the regulation of cellular respiration. Additionally, an increasing body of literature has been reporting an alternative role for several mitochondrial ribosomal proteins as apoptosis-inducing factors. No surprisingly, the expression of genes encoding for mitoribosomal proteins, mitoribosome assembly factors and mitochondrial translation factors is modified in numerous cancers, a trait that has been linked to tumorigenesis and metastasis. In this article, we will review the current knowledge regarding the dual function of mitoribosome components in protein synthesis and apoptosis and their association with cancer susceptibility and development. We will also highlight recent developments in targeting mitochondrial ribosomes for the treatment of cancer.

Background The recent genome-wide association studies (GWAS) of Type 2 Diabetes (T2D) have identified the pancreatic β-cell as the culprit in the pathogenesis of the disease. Mitochondrial metabolism plays a crucial role in the processes controlling release of insulin and β-cell mass. This notion implies that mechanisms controlling mitochondrial function have the potential to play a decisive pathogenetic role in T2D. Scope of the review This article reviews studies demonstrating that there is indeed mitochondrial dysfunction in islets in T2D, and that GWAS have identified a variant in the gene encoding transcription factor B1 mitochondrial ( TFB1M ), predisposing to T2D due to mitochondrial dysfunction and impaired insulin secretion. Mechanistic studies of the nature of this pathogenetic link, as well as of other mitochondrial transcription factors, are described. Major conclusions Based on this, it is argued that transcription and translation in mitochondria are critical processes determining mitochondrial function in β-cells in health and disease.

Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3’-deoxy or 2′-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2′ or 4′ mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.

Mitochondria harbor the oxidative phosphorylation (OXPHOS) system, which under aerobic conditions produces the bulk of cellular adenosine triphosphate (ATP). The mitochondrial genome encodes key components of the OXPHOS system, and it is transcribed by the mitochondrial RNA polymerase, POLRMT. The levels of mitochondrial transcription correlate with the respiratory activity of the cell. Therefore, compounds that can increase or decrease mitochondrial gene transcription may be useful for fine-tuning metabolism and could be used to treat metabolic diseases or certain forms of cancer. We here report the establishment of a novel high-throughput assay technology that has allowed us to screen a library of 430,000 diverse compounds for effects on mitochondrial transcription in vitro. Following secondary screens facilitated by the same assay principle, we identified 55 compounds that efficiently and selectively inhibit mitochondrial transcription and that are active also in cell culture. Our method is easily adaptable to other RNA or DNA polymerases and varying spectroscopic detection technologies.

The mitochondrial transcription initiation machinery in humans consists of three proteins: the RNA polymerase (POLRMT) and two accessory factors, transcription factors A and B2 (TFAM and TFB2M, respectively). This machinery is required for the expression of mitochondrial DNA and the biogenesis of the oxidative phosphorylation system. Previous experiments suggested that TFB2M is required for promoter melting, but conclusive experimental proof for this effect has not been presented. Moreover, the role of TFB2M in promoter unwinding has not been discriminated from that of TFAM. Here we used potassium permanganate footprinting, DNase I footprinting, and in vitro transcription from the mitochondrial light-strand promoter to study the role of TFB2M in transcription initiation. We demonstrate that a complex composed of TFAM and POLRMT was readily formed at the promoter but alone was insufficient for promoter melting, which only occurred when TFB2M joined the complex. We also show that mismatch bubble templates could circumvent the requirement of TFB2M, but TFAM was still required for efficient initiation. Our findings support a model in which TFAM first recruits POLRMT to the promoter, followed by TFB2M binding and induction of promoter melting.

To translate the 13 mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease and a second inefficient or regulated cleavage by mitochondrial intermediate protease. We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation, and longevity pathways to which MRPL12 has been linked.

Enzymes responsible for mitochondrial (mt) DNA synthesis and transcription are encoded by nuclear genome and inherited mutations in these genes may play important roles in enhancing risk of precancer and cancer. Here, genetic variations in 23 functionally relevant tagSNPs in 6 genes responsible for mtDNA synthesis and transcription were studied in 522 cancer and 241 precancer (i.e. leukoplakia) patients and 525 healthy controls using Illumina Golden Gate assay to explore association with risk of oral precancer and cancer. Two SNPs, rs41553913 at POLRMT and rs9905016 at POLG2, significantly increased risk of oral leukoplakia and cancer, respectively, at both genotypic and allelic levels. Gene-environment interaction models also revealed that tobacco habits and SNPs at POLG2 and TFAM may modulate risk of both leukoplakia and cancer. In silico analysis of published data-set also revealed that variant heterozygote (TC) significantly increased transcription of POLG2 compared to wild genotype (p=0.03). Cancer tissues having variant allele genotypes (TC+CC) at POLG2 contained 1.6 times (p

Mammalian mitochondrial DNA (mtDNA) encodes 13 core proteins of oxidative phosphorylation, 12S and 16S ribosomal RNAs, and 22 transfer RNAs. Mutations and deletions of mtDNA and/or nuclear genes encoding mitochondrial proteins have been implicated in a wide range of diseases. Thus, cell survival and health of the organism require some steady-state level of the mitochondrial genome and its expression. In mammalian systems, the mitochondrial transcription factor B2 (mtTFB2 or TFB2M) is indispensable for transcription initiation. TFB2M along with two other proteins, mitochondrial RNA polymerase (mtRNAP or POLRMT) and mitochondrial transcription factor A (mtTFA or TFAM), are key components of the core mitochondrial transcription apparatus. Structural information for POLRMT and TFAM from humans is available; however, there is no available structure for TFB2M. In the present study, three-dimensional structure of TFB2M from humans was modeled using a combination of homology modeling and small-angle X-ray scattering (SAXS). The TFB2M structural model adds substantively to our understanding of TFB2M function. An explanation for the low or absent RNA methyltransferase activity is provided. A putative nucleic acid-binding site is revealed. The amino and carboxy termini, while likely lacking defined secondary structure, appear to adopt compact, globular conformations, thus "capping" the ends of the protein. Finally, sites of interaction of TFB2M with other factors, protein and/or nucleic acid, are suggested by the identification of species-specific clusters on the surface of the protein.

The present research approaches for finding new therapies of acute myeloid leukemia (AML) are much focused on targeting the different genetic alterations in AML but many of them fail, probably due to the heterogeneity of the leukemic cells to be targeted. Recently, mitochondrial dependency is shown to play an important role in the progression of AML. AML cells exhibit high expression of mitochondrial RNA polymerase (POLRMT) which indirectly leads to high oxidative phosphorylation. Based on its upregulation as well as unique structure, POLRMT would have a great potential as a therapeutic target in AML. Recently, a novel inhibitor of mitochondrial transcription (IMT) has been developed which exhibits an anti-cancer potential by specifically targeting POLRMT. Here, we investigated the effect of IMT on AML. First, we showed that AML cell lines are sensitive to IMT. We could see a significant decline in proliferation in all human AML cell lines in a concentration-dependent manner, acquired by measurement of ATP production. Next, we investigated the effect of IMT in vivo in immunodeficient mice which were transplanted with an AML cell line that harbors FLT3-ITD mutation. IMT was administrated for 21 days from two weeks post-transplantation and compared with mice either treated with AC220 (FLT inhibitor) or vehicle (n=10/group). Results show that IMT was well tolerated by the mice and there was no sign of toxicity in normal tissues despite a significant delay or prevention of the onset of leukemia. Median survival of IMT treated mice=114 days vs vehicle treated=45 days (p

The human genome comprises large chromosomes in the nucleus and mitochondrial DNA (mtDNA) housed in the dynamic mitochondrial network. Human cells contain up to thousands of copies of the double-stranded, circular mtDNA molecule that encodes essential subunits of the oxidative phosphorylation complexes and the rRNAs and tRNAs needed to translate these in the organelle matrix. Transcription of human mtDNA is directed by a single-subunit RNA polymerase, POLRMT, which requires two primary transcription factors, TFB2M (transcription factor B2, mitochondrial) and TFAM (transcription factor A, mitochondrial), to achieve basal regulation of the system. Here, we review recent advances in understanding the structure and function of the primary human transcription machinery and the other factors that facilitate steps in transcription beyond initiation and provide more intricate control over the system.

Transcription of the human mitochondrial genome is required for the expression of 13 subunits of the respiratory chain complexes involved in oxidative phosphorylation, which is responsible for meeting the cells' energy demands in the form of ATP. Also transcribed are the two rRNAs and 22 tRNAs required for mitochondrial translation. This process is accomplished, with the help of several accessory proteins, by the human mitochondrial RNA polymerase (POLRMT, also known as h-mtRNAP), a nuclear-encoded single-subunit DNA-dependent RNA polymerase (DdRp or RNAP) that is distantly related to the bacteriophage T7 class of single-subunit RNAPs. In addition to its role in transcription, POLRMT serves as the primase for mitochondrial DNA replication. Therefore, this enzyme is of fundamental importance for both expression and replication of the human mitochondrial genome. Over the past several years rapid progress has occurred in understanding POLRMT and elucidating the molecular mechanisms of mitochondrial transcription. Important accomplishments include development of recombinant systems that reconstitute human mitochondrial transcription in vitro, determination of the X-ray crystal structure of POLRMT, identification of distinct mechanisms for promoter recognition and transcription initiation, elucidation of the kinetic mechanism for POLRMT-catalyzed nucleotide incorporation and discovery of unique mechanisms of mitochondrial transcription inhibition including the realization that POLRMT is an off target for antiviral ribonucleoside analogs. This review summarizes the current understanding of POLRMT structure–function, mechanism and inhibition. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

Eukaryotes control nearly every cellular process in part by modulating the transcription of genes encoded by their nuclear genome. However, these cells are faced with the added complexity of possessing a second genome, within the mitochondria, which encodes critical components of several essential processes, including energy metabolism and macromolecule biosynthesis. As these cellular processes require gene products encoded by both genomes, cells have adopted strategies for linking mitochondrial gene expression to nuclear gene expression and other dynamic cellular events. Here we discuss examples of several mechanisms that have been identified, by which eukaryotic cells link extramitochondrial signals to dynamic alterations in mitochondrial transcription. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

Mitochondria are organelles whose main function is to generate power by oxidative phosphorylation. Some of the essential genes required for this energy production are encoded by the mitochondrial genome, a small circular double stranded DNA molecule. Human mtDNA is replicated by a specialized machinery distinct from the nuclear replisome. Defects in the mitochondrial replication machinery can lead to loss of genetic information by deletion and/or depletion of the mtDNA, which subsequently may cause disturbed oxidative phosphorylation and neuromuscular symptoms in patients. We discuss here the different components of the mitochondrial replication machinery and their role in disease. We also review the mode of mammalian mtDNA replication.

The core human mitochondrial transcription machinery comprises a single subunit bacteriophage-related RNA polymerase, POLRMT, the high mobility group box DNA-binding protein h-mtTFA/TFAM, and two transcriptional co-activator proteins, h-mtTFB1 and h-mtTFB2 that also have rRNA methyltransferase activity. Recapitulation of specific initiation of transcription in vitro can be achieved by a complex of POL-RMT, h-mtTFA, and either h-mtTFB1 or h-mtTFB2. However, the nature of mitochondrial transcription complexes in vivo and the potential involvement of additional proteins in the transcription process in human mitochondria have not been extensively investigated. In Saccharomyces cerevisiae, transcription and translation are physically coupled via the formation of a multiprotein complex nucleated by the binding of Nam1p to the amino-terminal domain of mtRNA polymerase (Rpo41p). This model system paradigm led us to search for proteins that interact with POLRMT to regulate mitochondrial gene expression in humans. Using an affinity capture strategy to identify POL-RMT-binding proteins, we identified mitochondrial ribosomal protein L7/L12 (MRPL12) as a protein in HeLa mitochondrial extracts that interacts specifically with POLRMT in vitro. Purified recombinant MRPL12 binds to POLRMT and stimulates mitochondrial transcription activity in vitro, demonstrating that this interaction is both direct and functional. Finally, from HeLa cells that overexpress FLAG epitope-tagged MRPL12, increased steady-state levels of mtDNA-encoded transcripts are observed and MRPL12-POLRMT complexes can be co-immunoprecipitated, providing strong evidence that this interaction enhances mitochondrial transcription or RNA stability in vivo. We speculate that the MRPL12 interaction with POLRMT is likely part of a novel regulatory mechanism that coordinates mitochondrial transcription with translation and/or ribosome biogenesis during human mitochondrial gene expression.

Initiation of transcription at mitochondrial promoters in mammalian cells requires the simultaneous presence of a monomeric mitochondrial RNA polymerase, mitochondrial transcription factor A, and either transcription factor B1 or B2. We here review recent progress in our understanding of how these basal factors cooperate in the initiation and regulation of mitochondrial transcription. We describe the evolutionary origin of individual transcription factors and discuss how these phylogenetic relationships may facilitate a molecular understanding of the mitochondrial transcription machinery.