scholarly journals The PPR domain of mitochondrial RNA polymerase is a ribonuclease required for mtDNA replication

2021 ◽  
Author(s):  
Yi Liu ◽  
Zhe Chen ◽  
Zong-Heng Wang ◽  
Katherine Delaney ◽  
Juanjie Tang ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Mitochondrial Rna ◽  
Cellular Energy ◽  
Cellular Energy Metabolism ◽  
Dna And Rna ◽  
Mitochondrial Rna Polymerase ◽  
Evolutionarily Conserved ◽  
Mtdna Replication

AbstractMitochondrial DNA (mtDNA) replication and transcription are of paramount importance to cellular energy metabolism. Mitochondrial RNA polymerase (POLRMT) is thought to be the primase for mtDNA replication. However, it is unclear how POLRMT, which normally transcribes long polycistronic RNAs, can produce short RNA oligos to initiate mtDNA replication. Here we show that the PPR domain of Drosophila POLRMT is a 3’ to 5’ exoribonuclease. The exoribonuclease activity is indispensable for POLRMT to synthesize short RNA oligos and to prime DNA replication in vitro. An exoribonuclease deficient POLRMT, POLRMTE423P partially restores mitochondrial transcription but fails to support mtDNA replication when expressed in POLRMT mutant background, indicating that the exoribonuclease activity is necessary for mtDNA replication. Overexpression of POLRMTE423P in adult flies leads to severe neuromuscular defects and a marked increase of mtDNA transcripts errors, suggesting that exoribonuclease activity may contribute to the proofreading of mtDNA transcription. PPR domain of human POLRMT also has exoribonuclease activity, indicating evolutionarily conserved roles of PPR domain in mitochondrial DNA and RNA metabolism.

scholarly journals Distinct roles for two purified factors in transcription of Xenopus mitochondrial DNA.

1995 ◽  
Vol 15 (12) ◽  
pp. 7032-7042 ◽  
Author(s):  
I Antoshechkin ◽  
D F Bogenhagen
Keyword(s):  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Sequence Similarity ◽  
Protein A ◽  
Mitochondrial Rna ◽  
Basal Transcription ◽  
Hmg Box ◽  
Mitochondrial Rna Polymerase ◽  
Dnase I Footprinting

Transcription of Xenopus laevis mitochondrial DNA (xl-mtDNA) by the mitochondrial RNA polymerase requires a dissociable factor. This factor was purified to near homogeneity and identified as a 40-kDa protein. A second protein implicated in the transcription of mtDNA, the Xenopus homolog of the HMG box protein mtTFA, was also purified to homogeneity and partially sequenced. The sequence of a cDNA clone encoding xl-mtTFA revealed a high degree of sequence similarity to human and Saccharomyces cerevisiae mtTFA. xl-mtTFA was not required for basal transcription from a minimal mtDNA promoter, and this HMG box factor could not substitute for the basal factor, which is therefore designated xl-mtTFB. An antibody directed against the N terminus of xl-mtTFA did not cross-react with xl-mtTFB. xl-mtTFA is an abundant protein that appears to have at least two functions in mitochondria. First, it plays a major role in packaging mtDNA within the organelle. Second, DNase I footprinting experiments identified preferred binding sites for xl-mtTFA within the control region of mtDNA next to major mitochondrial promoters. We show that binding of xl-mtTFA to a site separating the two clusters of bidirectional promoters selectively stimulates specific transcription in vitro by the basal transcription machinery, comprising mitochondrial RNA polymerase and xl-mtTFB.

scholarly journals Non-DNA-Templated Addition of Nucleotides to the 3′ End of RNAs by the Mitochondrial RNA Polymerase of Physarum polycephalum

2008 ◽  
Vol 28 (18) ◽  
pp. 5795-5802 ◽  
Author(s):  
Mara L. Miller ◽  
Dennis L. Miller
Keyword(s):  
Mitochondrial Dna ◽  
Rna Polymerase ◽  
Rna Editing ◽  
Physarum Polycephalum ◽  
Mitochondrial Gene ◽  
In Vitro Transcription ◽  
Open Reading Frames ◽  
Mitochondrial Rna ◽  
Mitochondrial Rna Polymerase

ABSTRACT Mitochondrial gene expression is necessary for proper mitochondrial biogenesis. Genes on the mitochondrial DNA are transcribed by a dedicated mitochondrial RNA polymerase (mtRNAP) that is encoded in the nucleus and imported into mitochondria. In the myxomycete Physarum polycephalum, nucleotides that are not specified by the mitochondrial DNA templates are inserted into some RNAs, a process called RNA editing. This is an essential step in the expression of these RNAs, as the insertion of the nontemplated nucleotides creates open reading frames for the production of proteins from mRNAs or produces required secondary structure in rRNAs and tRNAs. The nontemplated nucleotide is added to the 3′ end of the RNA as the RNA is being synthesized during mitochondrial transcription. Because RNA editing is cotranscriptional, the mtRNAP is implicated in RNA editing as well as transcription. We have cloned the cDNA for the mtRNAP of Physarum and have expressed the mtRNAP in Escherichia coli. We have used in vitro transcription assays based on the Physarum mtRNAP to identify a novel activity associated with the mtRNAP in which non-DNA-templated nucleotides are added to the 3′ end of RNAs. Any of the four ribonucleoside triphosphates (rNTPs) can act as precursors for this process, and this novel activity is observed when only one rNTP is supplied, a condition under which transcription does not occur. The implications of this activity for the mechanism of RNA editing are discussed.

Direct in vitro action of thyroid hormones on mitochondrial RNA-polymerase

1986 ◽  
Vol 11 (4) ◽  
pp. 205-211 ◽  
Author(s):  
G. Martino ◽  
C. Covello ◽  
R. De Giovanni ◽  
R. Filippelli ◽  
G. Pitrelli
Keyword(s):  
Thyroid Hormones ◽  
Rna Polymerase ◽  
Mitochondrial Rna ◽  
Mitochondrial Rna Polymerase

Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro

1989 ◽  
Vol 9 (8) ◽  
pp. 3193-3202
Author(s):  
G T Marczynski ◽  
P W Schultz ◽  
J A Jaehning
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
In Vitro Transcription ◽  
Catalytic Rna ◽  
Mitochondrial Rna ◽  
Promoter Sequences ◽  
Sequence Recognition ◽  
Mitochondrial Rna Polymerase

We have extended an earlier observation that the TATA box for the nuclear GAL10 gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL1 genes, which are regulated in concert with the GAL10 gene, the sigma repetitive element, and the 2 microns plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This extended sequence, -ACTATAAACGatcATAG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo41-) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested.

Accurate in vitro transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters

1986 ◽  
Vol 6 (7) ◽  
pp. 2543-2550
Author(s):  
D F Bogenhagen ◽  
B K Yoza
Keyword(s):  
Mitochondrial Dna ◽  
Xenopus Laevis ◽  
Rna Polymerase ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Xenopus Laevis Oocytes ◽  
Mitochondrial Rna ◽  
Bidirectional Promoters

The mitochondrial RNA polymerase from Xenopus laevis oocytes was partially purified by heparin-Sepharose chromatography and phosphocellulose chromatography. This RNA polymerase preparation specifically initiated the transcription of X. laevis mitochondrial DNA (mtDNA) from two bidirectional promoters contained within a 123-base-pair segment of the mtDNA between the heavy-strand replication origin and the rRNA cistrons. Transcription in vitro initiated from precisely the same start sites previously mapped as initiation sites for transcription in vivo. At each of the four sites, initiation occurred within a conserved nucleotide sequence, ACPuTTATA. This consensus sequence is not related to promoters for transcription of human mtDNA.

scholarly journals Nucleotide Substrate Specificity of Anti-Hepatitis C Virus Nucleoside Analogs for Human Mitochondrial RNA Polymerase

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Maryam Ehteshami ◽  
Longhu Zhou ◽  
Sheida Amiralaei ◽  
Jadd R. Shelton ◽  
Jong Hyun Cho ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Substrate Specificity ◽  
Rna Polymerase ◽  
Rna Virus ◽  
Antiviral Agents ◽  
Nucleoside Analogs ◽  
Mitochondrial Rna ◽  
Mitochondrial Rna Polymerase

ABSTRACT Nucleoside analog inhibitors (NAIs) are an important class of antiviral agents. Although highly effective, some NAIs with activity against hepatitis C virus (HCV) can cause toxicity, presumably due to off-target inhibition of host mitochondrial RNA polymerase (POLRMT). The in vitro nucleotide substrate specificity of POLRMT was studied in order to explore structure-activity relationships that can facilitate the identification of nontoxic NAIs. These findings have important implications for the development of all anti-RNA virus NAIs.

scholarly journals Heteroplasmy concordance between mitochondrial DNA and RNA

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ruoyu Zhang ◽  
Kiichi Nakahira ◽  
Augustine M. K. Choi ◽  
Zhenglong Gu
Keyword(s):  
Mitochondrial Dna ◽  
Cell Line ◽  
Mitochondrial Rna ◽  
Sequencing Data ◽  
Cell Functions ◽  
Dna And Rna ◽  
Public Data ◽  
Rapid Changes ◽  
Sequencing Studies

Abstract Mitochondrial DNA (mtDNA) heteroplasmies are associated with various diseases but the transmission of heteroplasmy from mtDNA to mitochondrial RNA (mtRNA) remains unclear. We compared heteroplasmies in mtRNA from 446 human B-lymphoblastoid cell lines to their corresponding mtDNA using deep sequencing data from two independent studies. We observed 2786 heteroplasmies presenting in both DNA and RNA at 1% frequency cutoff. Among them, the frequencies of 2427 (87.1%) heteroplasmies were highly consistent (less than 5% frequency difference) between DNA and RNA. To validate these frequency consistencies, we isolated DNA and RNA simultaneously from GM12282 cell line used in those two sequencing studies, and resequenced its heteroplasmy sites. Interestingly, we also observed the rapid changes of heteroplasmy frequencies during 4 weeks of the cell culture: the frequencies at Day 14 increased by >25% than those at Day 0. However, the heteroplasmy frequencies from the same time point were highly consistent. In summary, our analysis on public data together with in vitro study indicates that the heteroplasmies in DNA can be transcribed into RNA with high fidelity. Meanwhile, the observed rapid-changing heteroplasmy frequency can potentially disturb cell functions, which could be an overlooked confounding factor in cell line related studies.

scholarly journals A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence.

1995 ◽  
Vol 15 (1) ◽  
pp. 580-589 ◽  
Author(s):  
B Xu ◽  
D A Clayton
Keyword(s):  
Mitochondrial Dna ◽  
Dna Replication ◽  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Mitochondrial Rna ◽  
Hybrid Formation ◽  
Dna Replication Origin ◽  
Mitochondrial Replication ◽  
Mitochondrial Dna Polymerase

Critical features of the mitochondrial leading-strand DNA replication origin are conserved from Saccharomyces cerevisiae to humans. These include a promoter and a downstream GC-rich sequence block (CSBII) that encodes rGs within the primer RNA. During in vitro transcription at yeast mitochondrial replication origins, there is stable and persistent RNA-DNA hybrid formation that begins at the 5' end of the rG region. The short rG-dC sequence is the necessary and sufficient nucleic acid element for establishing stable hybrids, and the presence of rGs within the RNA strand of the RNA-DNA hybrid is required. The efficiency of hybrid formation depends on the length of RNA synthesized 5' to CSBII and the type of RNA polymerase employed. Once made, the RNA strand of an RNA-DNA hybrid can serve as an effective primer for mitochondrial DNA polymerase. These results reveal a new mechanism for persistent RNA-DNA hybrid formation and suggest a step in priming mitochondrial DNA replication that requires both mitochondrial RNA polymerase and an rG-dC sequence-specific event to form an extensive RNA-DNA hybrid.

scholarly journals Anti-viral nucleotide incorporation by recombinant human mitochondrial RNA polymerase is predictive for increased in vivo mitochondrial toxicity risk

2016 ◽  
pp. AAC.01253-16 ◽  
Author(s):  
Martijn Fenaux ◽  
Xiaodong Lin ◽  
Fumiaki Yokokawa ◽  
Zachary Sweeney ◽  
Oliver Saunders ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mitochondrial Dysfunction ◽  
Elevated Serum ◽  
Nucleoside Analogs ◽  
Hepatic Function ◽  
Gene Signature ◽  
Mitochondrial Rna ◽  
Mitochondrial Rna Polymerase

Nucleoside or nucleotide inhibitors are a highly successful class of antivirals due to selectivity, potency, broad coverage, and high barrier to resistance. Nucleosides are the backbone of combination treatments for HIV, hepatitis B and - since the FDA approval of sofosbuvir in 2013 - also for hepatitis C (HCV). However, many promising nucleotides have advanced to clinical trials only to be terminated due to unexpected toxicity. Here we describe the in vitro pharmacology of1, a monophosphate prodrug of a 2’ -ethynyluridine developed for the treatment of HCV.1inhibits multiple HCV genotypes in vitro (EC50= 0.05-0.1 μM) with a selectivity index of >300 (CC50= 30 μM in MT-4 cells). The active triphosphate metabolite of1,2, does not inhibit human α, β or γ DNA polymerases, but was a substrate for incorporation by the human mitochondrial RNA polymerase (POLRMT). In dog, the oral administration of1resulted in elevated serum liver enzymes and microscopic changes in the liver. Transmission electron microscopy showed significant mitochondrial swelling and lipid accumulation in hepatocytes. Gene expression analysis revealed dose-proportional gene signature changes linked to loss of hepatic function and increased mitochondrial dysfunction. The potential of in vivo toxicity through mitochondrial polymerase incorporation by nucleoside analogs has been previously shown. This study shows that even moderate levels of nucleotide analog incorporation by POLRMT increase the risk of in vivo mitochondrial dysfunction. Based on these results, further development of1as an anti-HCV compound was terminated.

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