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

2016 ◽  
Vol 2 (8) ◽  
pp. e1600963 ◽  
Author(s):  
Inge Kühl ◽  
Maria Miranda ◽  
Viktor Posse ◽  
Dusanka Milenkovic ◽  
Arnaud Mourier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Knockout Mice ◽  
Transcription Initiation ◽  
Mitochondrial Gene ◽  
Conditional Knockout ◽  
Regulatory Role ◽  
Mitochondrial Rna ◽  
Mitochondrial Transcription ◽  
Mtdna Replication

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 is debated whether POLRMT also serves as the primase for the initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in the 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 the absence of POLRMT. Last, we report that mitochondrial transcription elongation factor may compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role of this factor in transcription. In conclusion, we present in vivo evidence that POLRMT has a key regulatory role in the replication of mammalian mtDNA and is part of a transcriptional mechanism that provides a switch between primer formation for mtDNA replication and mitochondrial gene expression.

scholarly journals Quantitative proteomics revealed C6orf203/MTRES1 as a factor preventing stress-induced transcription deficiency in human mitochondria

2019 ◽  
Vol 47 (14) ◽  
pp. 7502-7517 ◽  
Author(s):  
Anna V Kotrys ◽  
Dominik Cysewski ◽  
Sylwia D Czarnomska ◽  
Zbigniew Pietras ◽  
Lukasz S Borowski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Binding ◽  
Mitochondrial Gene ◽  
Binding Activity ◽  
Stress Conditions ◽  
Mitochondrial Rna ◽  
Mitochondrial Gene Expression ◽  
Compensatory Mechanisms ◽  
Temporary Reduction ◽  
Mitochondrial Transcription

AbstractMaintenance of mitochondrial gene expression is crucial for cellular homeostasis. Stress conditions may lead to a temporary reduction of mitochondrial genome copy number, raising the risk of insufficient expression of mitochondrial encoded genes. Little is known how compensatory mechanisms operate to maintain proper mitochondrial transcripts levels upon disturbed transcription and which proteins are involved in them. Here we performed a quantitative proteomic screen to search for proteins that sustain expression of mtDNA under stress conditions. Analysis of stress-induced changes of the human mitochondrial proteome led to the identification of several proteins with poorly defined functions among which we focused on C6orf203, which we named MTRES1 (Mitochondrial Transcription Rescue Factor 1). We found that the level of MTRES1 is elevated in cells under stress and we show that this upregulation of MTRES1 prevents mitochondrial transcript loss under perturbed mitochondrial gene expression. This protective effect depends on the RNA binding activity of MTRES1. Functional analysis revealed that MTRES1 associates with mitochondrial RNA polymerase POLRMT and acts by increasing mitochondrial transcription, without changing the stability of mitochondrial RNAs. We propose that MTRES1 is an example of a protein that protects the cell from mitochondrial RNA loss during stress.

scholarly journals Mitochondrial RNA capping: highly efficient 5’-RNA capping with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

2018 ◽  
Author(s):  
Jeremy G. Bird ◽  
Urmimala Basu ◽  
David Kuster ◽  
Aparna Ramachandran ◽  
Ewa Grudzien-Nogalska ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Mitochondrial Gene ◽  
Promoter Sequence ◽  
Mitochondrial Rna ◽  
Sensors And Actuators ◽  
Mitochondrial Rna Polymerase ◽  
Mitochondrial Transcripts ◽  
Rna Capping ◽  
Reduced Forms

AbstractBacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~10% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial gene expression, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

scholarly journals Human Mitochondrial Transcription Factor B1 Interacts with the C-Terminal Activation Region of h-mtTFA and Stimulates Transcription Independently of Its RNA Methyltransferase Activity

2003 ◽  
Vol 23 (16) ◽  
pp. 5816-5824 ◽  
Author(s):  
Vicki McCulloch ◽  
Gerald S. Shadel
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Mitochondrial Gene ◽  
Factor H ◽  
Dual Function ◽  
Mitochondrial Rna ◽  
Methyltransferase Activity ◽  
Mitochondrial Transcription

ABSTRACT A significant advancement in understanding mitochondrial gene expression is the recent identification of two new human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2. Both proteins stimulate transcription in collaboration with the high-mobility group box transcription factor, h-mtTFA, and are homologous to rRNA methyltransferases. In fact, the dual-function nature of h-mtTFB1 was recently demonstrated by its ability to methylate a conserved rRNA substrate. Here, we demonstrate that h-mtTFB1 binds h-mtTFA both in HeLa cell mitochondrial extracts and in direct-binding assays via an interaction that requires the C-terminal tail of h-mtTFA, a region necessary for transcriptional activation. In addition, point mutations in conserved methyltransferase motifs of h-mtTFB1 revealed that it stimulates transcription in vitro independently of S-adenosylmethionine binding and rRNA methyltransferase activity. Furthermore, one mutation (G65A) eliminated the ability of h-mtTFB1 to bind DNA yet did not affect transcriptional activation. These results, coupled with the observation that h-mtTFB1 and human mitochondrial RNA (h-mtRNA) polymerase can also be coimmunoprecipitated, lead us to propose a model in which h-mtTFA demarcates mitochondrial promoter locations and where h-mtTFB proteins bridge an interaction between the C-terminal tail of h-mtTFA and mtRNA polymerase to facilitate specific initiation of transcription. Altogether, these data provide important new insight into the mechanism of transcription initiation in human mitochondria and indicate that the dual functions of h-mtTFB1 can be separated.

scholarly journals Direct Regulation of Mitochondrial RNA Synthesis by Thyroid Hormone

1999 ◽  
Vol 19 (1) ◽  
pp. 657-670 ◽  
Author(s):  
José A. Enríquez ◽  
Patricio Fernández-Silva ◽  
Nuria Garrido-Pérez ◽  
Manuel J. López-Pérez ◽  
Acisclo Pérez-Martos ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Transcription Initiation ◽  
Dimethyl Sulfate ◽  
Nuclear Genes ◽  
Regulation Of Transcription ◽  
Mitochondrial Rna ◽  
Mitochondrial Transcription ◽  
In Organello

ABSTRACT We have analyzed the influence of in vivo treatment and in vitro addition of thyroid hormone on in organello mitochondrial DNA (mtDNA) transcription and, in parallel, on the in organello footprinting patterns at the mtDNA regions involved in the regulation of transcription. We found that thyroid hormone modulates mitochondrial RNA levels and the mRNA/rRNA ratio by influencing the transcriptional rate. In addition, we found conspicuous differences between the mtDNA dimethyl sulfate footprinting patterns of mitochondria derived from euthyroid and hypothyroid rats at the transcription initiation sites but not at the mitochondrial transcription termination factor (mTERF) binding region. Furthermore, direct addition of thyroid hormone to the incubation medium of mitochondria isolated from hypothyroid rats restored the mRNA/rRNA ratio found in euthyroid rats as well as the mtDNA footprinting patterns at the transcription initiation area. Therefore, we conclude that the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription machinery. Particularly, the influence on the mRNA/rRNA ratio is achieved by selective modulation of the alternative H-strand transcription initiation sites and does not require the previous activation of nuclear genes. These results provide the first functional demonstration that regulatory signals, such as thyroid hormone, that modify the expression of nuclear genes can also act as primary signals for the transcriptional apparatus of mitochondria.

scholarly journals Mitochondrial Mistranslation in Brain Provokes a Metabolic Response Which Mitigates the Age-Associated Decline in Mitochondrial Gene Expression

2021 ◽  
Vol 22 (5) ◽  
pp. 2746
Author(s):  
Dimitri Shcherbakov ◽  
Reda Juskeviciene ◽  
Adrián Cortés Sanchón ◽  
Margarita Brilkova ◽  
Hubert Rehrauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Response ◽  
Mitochondrial Gene ◽  
Tca Cycle ◽  
Brain Mitochondria ◽  
Mitochondrial Gene Expression ◽  
Rna Seq ◽  
The Tca Cycle ◽  
Neurological Phenotype

Mitochondrial misreading, conferred by mutation V338Y in mitoribosomal protein Mrps5, in-vivo is associated with a subtle neurological phenotype. Brain mitochondria of homozygous knock-in mutant Mrps5V338Y/V338Y mice show decreased oxygen consumption and reduced ATP levels. Using a combination of unbiased RNA-Seq with untargeted metabolomics, we here demonstrate a concerted response, which alleviates the impaired functionality of OXPHOS complexes in Mrps5 mutant mice. This concerted response mitigates the age-associated decline in mitochondrial gene expression and compensates for impaired respiration by transcriptional upregulation of OXPHOS components together with anaplerotic replenishment of the TCA cycle (pyruvate, 2-ketoglutarate).

scholarly journals Sirt1 deacetylates and stabilizes p62 to promote hepato-carcinogenesis

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Lifeng Feng ◽  
Miaoqin Chen ◽  
Yiling Li ◽  
Muchun Li ◽  
Shiman Hu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Overall Survival ◽  
Cell Growth ◽  
Knockout Mice ◽  
Liver Tumors ◽  
Underlying Mechanism ◽  
Carcinoma Cells ◽  
Conditional Knockout

Abstractp62/SQSTM1 is frequently up-regulated in many cancers including hepatocellular carcinoma. Highly expressed p62 promotes hepato-carcinogenesis by activating many signaling pathways including Nrf2, mTORC1, and NFκB signaling. However, the underlying mechanism for p62 up-regulation in hepatocellular carcinoma remains largely unclear. Herein, we confirmed that p62 was up-regulated in hepatocellular carcinoma and its higher expression was associated with shorter overall survival in patients. The knockdown of p62 in hepatocellular carcinoma cells decreased cell growth in vitro and in vivo. Intriguingly, p62 protein stability could be reduced by its acetylation at lysine 295, which was regulated by deacetylase Sirt1 and acetyltransferase GCN5. Acetylated p62 increased its association with the E3 ligase Keap1, which facilitated its poly-ubiquitination-dependent proteasomal degradation. Moreover, Sirt1 was up-regulated to deacetylate and stabilize p62 in hepatocellular carcinoma. Additionally, Hepatocyte Sirt1 conditional knockout mice developed much fewer liver tumors after Diethynitrosamine treatment, which could be reversed by the re-introduction of exogenous p62. Taken together, Sirt1 deacetylates p62 at lysine 295 to disturb Keap1-mediated p62 poly-ubiquitination, thus up-regulating p62 expression to promote hepato-carcinogenesis. Therefore, targeting Sirt1 or p62 is a reasonable strategy for the treatment of hepatocellular carcinoma.

scholarly journals Enhancers Predominantly Regulate Gene Expression in vivo Via Transcription Initiation

2019 ◽  
Author(s):  
Martin Stephen Charles Larke ◽  
Takayuki Nojima ◽  
Jelena Telenius ◽  
Jacqueline A. Sharpe ◽  
Jacqueline A. Sloane-Stanley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Regulate Gene Expression ◽  
Regulate Gene

scholarly journals Epithelial (E)-Cadherin is a Novel Mediator of Platelet Aggregation and Clot Stability

2019 ◽  
Vol 119 (05) ◽  
pp. 744-757 ◽  
Author(s):  
Vanessa Scanlon ◽  
Alexandra Teixeira ◽  
Tarun Tyagi ◽  
Siying Zou ◽  
Ping-Xia Zhang ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Knockout Mice ◽  
Glycogen Synthase ◽  
Conditional Knockout ◽  
Platelet Production ◽  
Conditional Knockout Mice ◽  
Clot Stability ◽  
E Cadherin

AbstractCadherins play a major role in mediating cell–cell adhesion, which shares many parallels with platelet–platelet interactions during aggregate formation and clot stabilization. Platelets express epithelial (E)-cadherin, but its contribution to platelet function and/or platelet production is currently unknown. To assess the role of E-cadherin in platelet production and function in vitro and in vivo, we utilized a megakaryocyte-specific E-cadherin knockout mouse model. Loss of E-cadherin in megakaryocytes does not affect megakaryocyte maturation, platelet number or size. However, platelet dysfunction in the absence of E-cadherin is revealed when conditional knockout mice are challenged with acute antibody-mediated platelet depletion. Unlike wild-type mice that recover fully, knockout mice die within 72 hours post-antibody administration, likely from haemorrhage. Furthermore, conditional knockout mice have prolonged tail bleeding times, unstable clot formation, reduced clot retraction and reduced fibrin deposition in in vivo injury models. Murine platelet aggregation in vitro in response to thrombin and thrombin receptor activating peptide is compromised in E-cadherin null platelets, while aggregation in response to adenosine diphosphate (ADP) is not significantly different. Consistent with this, in vitro aggregation of primary human platelets in response to thrombin is decreased by an inhibitory E-cadherin antibody. Integrin activation and granule secretion in response to ADP and thrombin are not affected in E-cadherin null platelets, but Akt and glycogen synthase kinase 3β (GSK3β) activation are attenuated, suggesting a that E-cadherin contributes to aggregation, clot stabilization and retraction that is mediated by phosphoinositide 3-kinase/Akt/GSK3β signalling. In summary, E-cadherin plays a salient role in platelet aggregation and clot stability.

scholarly journals Murine FSH Production Depends on the Activin Type II Receptors ACVR2A and ACVR2B

Endocrinology ◽  
2020 ◽  
Vol 161 (7) ◽  
Author(s):  
Gauthier Schang ◽  
Luisina Ongaro ◽  
Hailey Schultz ◽  
Ying Wang ◽  
Xiang Zhou ◽  
...  
Keyword(s):  
Cell Line ◽  
Bone Morphogenetic Protein ◽  
Litter Size ◽  
Knockout Mice ◽  
Conditional Knockout ◽  
Type Ii ◽  
Morphogenetic Protein ◽  
Testicular Weight ◽  
Critical Type

Abstract Activins are selective regulators of FSH production by pituitary gonadotrope cells. In a gonadotrope-like cell line, LβT2, activins stimulate FSH via the activin type IIA receptor (ACVR2A) and/or bone morphogenetic protein type II receptor (BMPR2). Consistent with these observations, FSH is greatly reduced, though still present, in global Acvr2a knockout mice. In contrast, FSH production is unaltered in gonadotrope-specific Bmpr2 knockout mice. In light of these results, we questioned whether an additional type II receptor might mediate the actions of activins or related TGF-β ligands in gonadotropes. We focused on the activin type IIB receptor (ACVR2B), even though it does not mediate activin actions in LβT2 cells. Using a Cre-lox strategy, we ablated Acvr2a and/or Acvr2b in murine gonadotropes. The resulting conditional knockout (cKO) animals were compared with littermate controls. Acvr2a cKO (cKO-A) females were subfertile (~70% reduced litter size), cKO-A males were hypogonadal, and both sexes showed marked decreases in serum FSH levels compared with controls. Acvr2b cKO (cKO-B) females were subfertile (~20% reduced litter size), cKO-B males had a moderate decrease in testicular weight, but only males showed a significant decrease in serum FSH levels relative to controls. Simultaneous deletion of both Acvr2a and Acvr2b in gonadotropes led to profound hypogonadism and FSH deficiency in both sexes; females were acyclic and sterile. Collectively, these data demonstrate that ACVR2A and ACVR2B are the critical type II receptors through which activins or related TGF-β ligands induce FSH production in mice in vivo.

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