Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase

ABSTRACT There is a growing body of evidence suggesting that some ribonucleoside/ribonucleotide analogs may be incorporated into mitochondrial RNA by human mitochondrial DNA-dependent RNA polymerase (POLRMT) and disrupt mitochondrial RNA synthesis. An assessment of the incorporation efficiency of a ribonucleotide analog 5′-triphosphate by POLRMT may be used to evaluate the potential mitochondrial toxicity of the analog early in the development process. In this report, we provide a simple method to prepare active recombinant POLRMT. A robust in vitro nonradioactive primer extension assay was developed to assay the incorporation efficiency of ribonucleotide analog 5′-triphosphates. Our results show that many ribonucleotide analogs, including some antiviral compounds currently in various preclinical or clinical development stages, can be incorporated into newly synthesized RNA by POLRMT and that the incorporation of some of them can lead to chain termination. The discrimination (D) values of ribonucleotide analog 5′-triphosphates over those of natural ribonucleotide triphosphates (rNTPs) were measured to evaluate the incorporation efficiency of the ribonucleotide analog 5′-triphosphates by POLRMT. The discrimination values of natural rNTPs under the condition of misincorporation by POLRMT were used as a reference to evaluate the potential mitochondrial toxicity of ribonucleotide analogs. We propose the following criteria for the potential mitochondrial toxicity of ribonucleotide analogs based on D values: a safe compound has a D value of >105; a potentially toxic compound has a D value of >104 but <105; and a toxic compound has a D value of <104. This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.

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

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.7032 ◽

1995 ◽

Vol 15 (12) ◽

pp. 7032-7042 ◽

Cited By ~ 59

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00356-08 ◽

2008 ◽

Vol 28 (18) ◽

pp. 5795-5802 ◽

Cited By ~ 10

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.

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Development of a Simple In Vitro Assay To Identify and Evaluate Nucleotide Analogs against SARS-CoV-2 RNA-Dependent RNA Polymerase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01508-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01508-20

Author(s):

Gaofei Lu ◽

Xi Zhang ◽

Weinan Zheng ◽

Jialei Sun ◽

Lan Hua ◽

...

Keyword(s):

Rna Polymerase ◽

Antiviral Treatment ◽

Screening Method ◽

Chain Termination ◽

Rna Dependent Rna Polymerase ◽

Nucleotide Analogs ◽

Vitro Assay ◽

Assay Conditions ◽

Incorporation Efficiency

ABSTRACTNucleotide analogs targeting viral RNA polymerase have been proved to be an effective strategy for antiviral treatment and are promising antiviral drugs to combat the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In this study, we developed a robust in vitro nonradioactive primer extension assay to quantitatively evaluate the efficiency of incorporation of nucleotide analogs by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analogs over those of natural nucleotides were measured to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RdRp. In agreement with the data published in the literature, we found that the incorporation efficiency of remdesivir-TP is higher than that of ATP and incorporation of remdesivir-TP caused delayed chain termination, which can be overcome by higher concentrations of the next nucleotide to be incorporated. Our data also showed that the delayed chain termination pattern caused by remdesivir-TP incorporation is different for different template sequences. Multiple incorporations of remdesivir-TP caused chain termination under our assay conditions. Incorporation of sofosbuvir-TP is very low, suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2′-C-methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2′-C-methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful for evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp and for studying the mechanism of action of selected nucleotide analogs.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2543-2550.1986 ◽

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.

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Development of a simple in vitro assay to identify and evaluate nucleotide analogs against SARS-CoV-2 RNA-dependent RNA polymerase

10.1101/2020.07.16.205799 ◽

2020 ◽

Cited By ~ 1

Author(s):

Gaofei Lu ◽

Xi Zhang ◽

Weinan Zheng ◽

Jialei Sun ◽

Lan Hua ◽

...

Keyword(s):

Rna Polymerase ◽

Mechanism Of Action ◽

Antiviral Treatment ◽

Chain Termination ◽

Rna Dependent Rna Polymerase ◽

Nucleotide Analogs ◽

Vitro Assay ◽

Nucleotide Analog ◽

Incorporation Efficiency

AbstractNucleotide analogs targeting viral RNA polymerase have been approved to be an effective strategy for antiviral treatment and are attracting antiviral drugs to combat the current SARS-CoV-2 pandemic. In this report, we develop a robust in vitro nonradioactive primer extension assay to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) quantitively. Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp, and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analog over those of natural nucleotide were measured to evaluate the incorporation efficiency of nucleotide analog by RdRp. We found that the incorporation efficiency of Remdesivir-TP is higher than ATP, and we did not observe chain termination or delayed chain termination caused by single Remdesivir-TP incorporation, while multiple incorporations of Remdesivir-TP caused chain termination in our assay condition. The incorporation efficiency of Ribavirin-TP and Favipiravir-TP is very low either as ATP or GTP analogs, which suggested that mutagenesis may not be the mechanism of action of those two drugs against SARS-CoV-2. Incorporation of Sofosbuvir-TP is also very low suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2’-C-Methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2’-C-Methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful in evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp, and for studying the mechanism of action of selected nucleotide analog.

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

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.580 ◽

1995 ◽

Vol 15 (1) ◽

pp. 580-589 ◽

Cited By ~ 88

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2543 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2543-2550 ◽

Cited By ~ 24

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

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

10.1101/2021.03.12.435139 ◽

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.

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