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The PPR domain of mitochondrial RNA polymerase is an exoribonuclease required for mtDNA replication in Drosophila melanogaster

Nature Cell Biology volume 24, pages 757–765 (2022)Cite this article

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Abstract

Mitochondrial DNA (mtDNA) replication and transcription are of paramount importance to cellular energy metabolism. Mitochondrial RNA polymerase is thought to be the primase for mtDNA replication. However, it is unclear how this enzyme, which normally transcribes long polycistronic RNAs, can produce short RNA oligonucleotides to initiate mtDNA replication. We show that the PPR domain of Drosophila mitochondrial RNA polymerase (PolrMT) has 3′-to-5′ exoribonuclease activity, which is indispensable for PolrMT to synthesize short RNA oligonucleotides and prime DNA replication in vitro. An exoribonuclease-deficient mutant, PolrMTE423P, partially restores mitochondrial transcription but fails to support mtDNA replication when expressed in PolrMT-mutant flies, indicating that the exoribonuclease activity is necessary for mtDNA replication. In addition, overexpression of PolrMTE423P in adult flies leads to severe neuromuscular defects and a marked increase in mtDNA transcript errors, suggesting that exoribonuclease activity may contribute to the proofreading of mtDNA transcription.

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Fig. 1: PolrMT is required for de novo mtDNA synthesis in Drosophila.
Fig. 2: The PPR domain of PolrMT is a 3′-to-5′ exoribonuclease.
Fig. 3: The exoribonuclease activity of PolrMT is essential for priming DNA replication in vitro.
Fig. 4: PolrMT or its PPR domain preferentially degrades a mismatched RNA–DNA hybrid.
Fig. 5: The exoribonuclease activity of PolrMT is required for mtDNA replication in vivo.
Fig. 6: Expression of the PolrMTE423P transgene in adult flies impairs mitochondrial activity and animal fitness.

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Data availability

The RNA-sequencing data generated in this study have been deposited at the Gene Expression Omnibus of NCBI with the following accession numbers: GSE154310, GSE192549 and GSE164324. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank A. Freitas for comments and edits on the manuscript, R. Levine for his advice on protein purification and structural modelling, M. Falkenberg for the pBac‐POLγA and pBac‐POLγB plasmids, J. -W. Zhang for advice on riboproteins analyses, the BDSC for various fly stocks, Bestgene Inc. for the transgenic service and Arraystar Inc. for the tRNA-sequencing service. This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute.

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  1. National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Yi Liu, Zhe Chen, Zong-Heng Wang, Katherine M. Delaney, Juanjie Tang, Mehdi Pirooznia, Duck-Yeon Lee, Ilker Tunc, Yuesheng Li & Hong Xu

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Y.L. and H.X. conceived the project and designed the experiments. Y.L., Z.C., Z.-H.W., K.M.D., J.T., D.-Y.L. and Y.L. performed the experiments. Y.L., Z.-H.W., M.P., I.T. and H.X. analysed the data. Y.L. and H.X. wrote the manuscript.

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Correspondence to Hong Xu.

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Extended data

Extended Data Fig. 1 Expression, purification, and biochemical characterization of the PPR domain of PolrMT.

a, A silver-stained gel showing the purified PPR domain (residues 314-464). b, HPLC-UV (210 nm) chromatogram of the purified PPR domain. c, Mass spectrometry analysis of the purified PPR domain indicating a single peak at 18166.9 dalton, the exact molecular weight of the recombinant PPR domain. d, 50 nM 5’-6-FAM-U29 (left panel), or 50 nM 5’-6-FAM-U29/dA29 hybrid (right panel) was incubated in the ribonuclease assay buffer without any protein and analysed at indicated time points (min), showing no degradation of the substrate over 30 min. e, Exoribonuclease activity of the PPR domain requires divalent cations. A 5’-6-FAM- ssRNA substrate (100 nM) was incubated with the PPR domain or PPRE423P (50 nM) in the presence of 10 mM EDTA, 5 mM Ca2+, 5 mM Mg2+, or 5 mM Mn2+ as indicated. The reaction was incubated for 15 min at 32 °C. f, The PPR domain is not sensitive to broad-spectrum RNase inhibitors. A 5’-6-FAM-ssRNA (50 nM) was incubated with the PPR domain (100 nM) in the absence or the presence of RNase inhibitors and analysed at indicated time points (min). RNase A was used as a control. Data shown represent three independent experiments in a-f.

Source data

Extended Data Fig. 2 The exoribonuclease activity is a conserved feature of metazoan mtRNAPs.

a, Phylogenetic analysis of PPR domains in different PPR proteins using the neighbour-joining algorithm. Distance scale, 0.2 (20%) divergence. b, Sequence alignment of metazoan mtRNAPs’ PPR domains. Conserved residues are highlighted. Abbreviations in (a) and (b): Hs, Homo sapiens; Rn, Rattus norvegicus; Mm, Mus musculus; Xl, Xenopus laevis; Dm, Drosophila melanogaster; Aa, Aedes aegypti. c, Other Drosophila PPR proteins do not have ribonuclease activity. A 5’-Biotin-labelled target ssRNA (50 nM) was incubated with recombinant CG4611, CG4679, CG10302, or CG14786 (100 nM) proteins and analysed at indicated time points (min). Arrowhead indicates the full-length substrate. d, The PPR domain (residues 218-368) of human POLRMT has exoribonuclease activity. A 5’-6-FAM-labelled ssRNA (50 nM) was incubated with recombinant PPR protein of human POLRMT (100 nM) and analysed at indicated time points (min). e, Pseudo-first-order cleavage kinetics of a 5’-6-FAM-labelled target ssRNA by the PPR domain of human POLRMT. Data are presented as mean ± SD of n = 3 independent experiments. f, A Coomassie-stained gel showing the purified POLRMT and POLRMTΔPPR proteins. g, RNA synthesis by human POLRMT or POLRMTΔPPR on a 359 bp PCR fragment of human mtDNA (202-560) spanning the light strand promoter. The arrow indicates the 207 nt run-off product; the arrowhead indicates the 120 nt prematurely terminated transcripts at the CSBII region; the long transcript (open arrowhead) is presumably resulted from the non-specific transcription of the full-length template. Note that POLRMT generates both the run-off product and the prematurely terminated transcript, while POLRMTΔPPR generates the run-off transcript only. Data shown represent three independent experiments in c, d, f and g. h, Schematic illustration showing a 359-bp segment of a human mtDNA noncoding region spanning the light-strand promoter (LSP). CSB, conserved sequence block.

Source data

Extended Data Fig. 3 The E423P substitution abolishes both nuclease and RNA–DNA hybrid binding activities of the PPR domain.

a, A 5’-6-FAM-U29/dA29 hybrid (50 nM; left panel), or a mismatched hybrid of 5’-6-FAM-U29/dC-dA28 (50 nM; right panel) was incubated with 100 nM PPRE423P and analysed at indicated time points (min), showing no degradation of the substrate over 30 min. b, A 5’-6-FAM-U29/dA29 hybrid (50 nM; left panel), or a mismatched hybrid of 5’-6-FAM-U29/dC-dA28 (50 nM; right panel) was incubated with 100 nM PolrMTE423P and analysed at indicated time points (min), showing no degradation of the substrate over 30 min. c, EMSA of 3’-Biotin-labelled matched and mismatched RNA/DNA hybrids (50 nM) in the presence of the PPR domain or PPRE423P at indicated concentrations (μM). The open arrowhead indicates the RNA/DNA hybrid-protein complex; the arrowhead indicates the free probe. d, EMSA of 3’-Biotin-labelled matched and mismatched RNA/DNA hybrids (50 nM) in the presence of PolrMT or PolrMTE423P at indicated concentrations (μM). The open arrowhead indicates the RNA/DNA hybrid-protein complex; the arrowhead indicates the free probe. Data shown represent three independent experiments in a-d.

Source data

Extended Data Fig. 4 Overexpression of PolrMTE423P does not affect mitochondrial nucleoids and mitochondrial transcript level.

a, EdU incorporation (Green) in Drosophila midgut (left panel), and indirect flight muscle (right panel) that were co-stained for ATP synthase (ATPs; Red) to mark mitochondria. EdU puncta localized in mitochondria indicate mtDNA replication. Arrows indicate nuclear genome replication. Note the lack of mtDNA replication in adult muscle. Scale bars, 10 μm. b, Western blot showing the overexpression of PolrMT or PolrMTE423P in adult flies activated by an Act-Gal4:PR. A Bac clone transgene carrying gfp ORF inserted in-frame at the 3’ end of PolrMT gene was included as a reference for the endogenous level of PolrMT protein. Actin was used as the loading control. c, Relative levels of mtDNA and mtRNA in adult thoraxes of PolrMT and PolrMTE423P overexpressing flies. Value of each sample was normalized to the average of PolrMT, and data are presented as mean ± SD of n = 3 biologically independent experiments. d, Indirect flight muscle of PolrMT or PolrMTE423P overexpressing flies stained for TFAM (Red) and ATP synthase (ATPs, Green) that mark mtDNA and mitochondria, respectively. Scale bars, 10 μm. Images in a, b, and d represent three independent experiments. e, Quantification of mitochondrial contents by normalizing mitochondrial volume (voxels) to total voxels of the tissue. ns: p = 0.8168. f, Number of TFAM puncta per μm3 in indirect flight muscles. ns: p = 0.8901. g, Mean arbitrary TFAM intensity in indirect flight muscles. ns: p = 0.2397. In (e-g), n = 20 animals were examined over 3 independent experiments for each genotype; the middle lines denote the median. P values were determined using the Two‐tailed Student’s t-test. Genotypes of all lines used in (c-g) are provided in Supplementary Table 1.

Source data

Extended Data Fig. 5 Poly(A) tail length and transcription errors of mitochondrial transcripts in PolrMT- and PolrMTE423P-overexpressing flies.

a, Quantification of poly(A) tail length of all 13 mitochondrial transcripts in control (ctrl), PolrMT and PolrMTE423P overexpressing flies. Data are presented as mean ± SD of n = 10 clones for each transcript. b, The mitochondrial transcription errors in PolrMT and PolrMTE423P overexpression flies were distributed randomly across the mitochondrial genome. Most errors were in highly transcribed genes. “Error reads” indicates the total number of errors detected in a 100-bp bin. “Error frequency” was calculated by normalizing the number of error reads to the total number of reads within that interval. c, The spectrum and frequency of transcripts errors in PolrMT and PolrMTE423P overexpressing flies. Genotypes of all lines in (a-c) are provided in Supplementary Table 1.

Source data

Supplementary information

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Supplementary Tables 1–5

All five supplementary tables in one MS Excel file. Supplementary Table 1. All D. melanogaster lines used in this study. The genotypes, sources and identification information are listed. Supplementary Table 2. List of all unprocessed RNA junctions and their frequencies in POLRMT- and POLRMTE423P-overexpressing flies. Supplementary Table 3. List of all tRNA counts in POLRMT- and POLRMTE423P-overexpressing flies. Supplementary Table 4. List of all plasmids generated in this study. Supplementary Table 5. List of all oligonucleotides used in this study.

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Liu, Y., Chen, Z., Wang, ZH. et al. The PPR domain of mitochondrial RNA polymerase is an exoribonuclease required for mtDNA replication in Drosophila melanogaster. Nat Cell Biol 24, 757–765 (2022). https://doi.org/10.1038/s41556-022-00887-y

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