Research ArticleMOLECULAR GENETICS

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

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Science Advances  05 Aug 2016:
Vol. 2, no. 8, e1600963
DOI: 10.1126/sciadv.1600963
  • Fig. 1 Knockout of Polrmt in germline and heart.

    (A) RT-PCR analysis of Polrmt transcripts from control (L/L) and tissue-specific knockout mice (L/L, cre). Different primer sets were used as indicated; exon 3, 551 bp. UTR, untranslated region. (B) Survival curve of control (n = 60) and tissue-specific knockout (n = 37) mice. (C) Cardiac phenotype: Vertical (upper panels) and transverse (lower panels) sections through the midportion of hearts of control and tissue-specific knockout mouse hearts at 4 weeks of age. Scale bars, 2 mm. (D) Heart–to–body weight ratio of control (n = 62) and tissue-specific knockout mice (n = 57) at different time points. Error bars indicate ±SEM (***P < 0.001; two-tailed Student’s t test).

  • Fig. 2 Reduced OXPHOS capacity in Polrmt knockout mouse heart.

    (A) Transmission electron micrographs of myocardium of 5-week-old control (n = 2) and tissue-specific knockout mice (n = 2). Scale bars, 2 μm (upper panel) and 0.5 μm (lower panel). (B) Relative enzyme activities of respiratory chain enzymes measured in mitochondria isolated from hearts of control and tissue-specific knockout mice at different ages. Citrate synthase (CS) was used as an internal control for normalization of the samples. The enzymes measured are citrate synthase; complex II, succinate dehydrogenase; complex I, NADH ubiquinone oxidoreductase; complex IV, cytochrome c oxidase; complex V, adenosine triphosphatase (ATPase) oligomycin-sensitive. All error bars indicate ±SEM (**P < 0.01 and ***P < 0.001; n = 4; two-tailed Student’s t test). (C) BN-PAGE analyses of mitochondria isolated from 5-week-old control and tissue-specific knockout hearts. OXPHOS complexes were detected with subunit-specific antibodies or Coomassie Brilliant Blue staining. NDUFA9, complex I; SDHA, 70 kD subunit of complex II; COX2, complex IV; sub α F1, complex V; respiratory supercomplexes, InIIInIVn and InIIIn.

  • Fig. 3 Decreased mtDNA replication in Polrmt knockout mice.

    (A) 7S RNA levels in control and tissue-specific knockout hearts at different ages by Northern blot on total RNA; loading, 18S rRNA. RNAs from hearts of Mterf4 conditional knockout mice (72) with increased 7S RNA levels were loaded as controls. (B) Southern blot analyses on mtDNA to assess 7S DNA levels of 4-week-old control and tissue-specific knockout mice. To allow relative comparison, the loaded amount of mtDNA from knockouts was higher than the amount loaded from control samples. (C) Southern blot analyses on total DNA to assess mtDNA levels of control and tissue-specific knockout mice at different ages; loading, 18S rDNA. (D) Quantification of Southern blots: mtDNA levels were normalized to 18S rDNA and presented as the percentage of controls. Error bars indicate ±SEM (*P < 0.05; two-tailed Student’s t test). (E) Levels of de novo–synthesized DNA of isolated heart mitochondria of 4-week-old tissue-specific knockout and control mice. Equal input was ensured by Western blot analysis [voltage-dependent anion channel (VDAC)] on isolated mitochondria after labeling before mtDNA extraction. (F) Quantification of the results from (E).

  • Fig. 4 Loss of POLRMT results in an increased mtDNA-free pool of TFAM.

    (A) Steady-state protein levels of nuclear-encoded factors of mtDNA expression analyzed by Western blotting on mitochondrial extracts from hearts of control and tissue-specific knockout mice; loading, VDAC; asterisk, cross-reacting band (28); for quantification, see fig. S4A. (B) Quantitative RT-PCR (qRT-PCR) of transcript levels of nuclear-encoded mitochondrial proteins. Normalization, β2M (β2-microglobulin). Error bars indicate ±SEM (*P < 0.05 and ***P < 0.001; two-tailed Student’s t test; see table S1). (C and D) Linear glycerol density gradient fractionations of mitochondrial lysates from tissue-specific knockout and control mice followed by Western blot analysis; for quantification, see figs. S5 (A to C) and S6. Samples taken from fractions 1 to 16 are of increasing density (that is, from top to bottom of the tube after separation by ultracentrifugation; as indicated by the schematic representation of the centrifuge tube to the left). Fractions were loaded from left to right on the gels as indicated by the lane numbering; input, aliquots of unfractionated lysates. The mtDNA content of the fractions was determined by Southern blotting. (E) Relative TFAM and POLRMT protein distribution across the gradient from control and knockout heart mitochondria.

  • Fig. 5 LSP and HSP show different sensitivities at low POLRMT concentrations.

    (A and B) Northern blot analyses of mitochondrial mRNAs, rRNAs, and tRNAs from hearts of 4-week-old control and tissue-specific knockout mice; loading, 18S rRNA. (C) Relative mitochondrial RNA abundance of mRNA and rRNA levels in hearts of 4-week-old tissue-specific knockout and control mice normalized to the upper quartile of the gene count distribution. The data analyzed are from three independent RNA-seq experiments; all RNAs have ***P ≤ 0.0001. Error bars indicate ±SEM. (D) In vitro transcription assay at different POLRMT levels. All reactions contained a cut plasmid template (containing the human LSP and HSP promoters giving a run-off product of 101 and 180 nt, respectively). POLRMT was added at 128, 32, 8, 2, and 0.5 nM in lanes 1 to 5, respectively; lane 6, control without POLRMT; lane 7, molecular weight marker (New England Biolabs). (E) Quantification of the results from (D). The experiment was performed in triplicates, and HSP transcription levels were normalized to LSP for each POLRMT concentration; bars, mean value. Error bars indicate ±SD (n = 3).

  • Fig. 6 Characterization of heterozygous Polrmt knockout mice.

    (A) POLRMT steady-state protein levels in heart from wild-type (+/+) and heterozygous Polrmt knockout (+/−) mice; loading, VDAC; for quantification, see fig. S7C. (B) Steady-state levels of mitochondrial mRNAs, rRNAs, and tRNAs; loading, 18S rRNA; for quantification, see fig. S8A. (C) Steady-state protein levels of nuclear-encoded factors of mtDNA expression analyzed by Western blotting on mitochondrial heart extracts; loading, VDAC; for quantification, see fig. S9A; asterisk, cross-reacting band (28). (D) De novo–synthesized mitochondrial transcripts from hearts of 52-week-old mice. Steady-state levels of individual mitochondrial transcripts were verified with a radiolabeled probe (mt-Co1); input, Western blot analysis (POLRMT and VDAC) after labeling. (E) 7S RNA levels in mouse hearts by Northern blotting on total RNA; loading, 18S rRNA. (F) Quantification of mtDNA by quantitative PCR (qPCR) with mt-Co1, mt-Nd1, and mt-Nd5 probes on mouse heart. Signals were normalized to the 18S signal; n = 3. Error bars indicate ±SEM. (G) De novo–synthesized DNA of isolated mitochondria from hearts of 12-week-old mice. The mtDNA was radioactively labeled in organello, isolated and boiled to release newly synthesized 7S DNA before Southern blotting; input, Western blotting (POLRMT and VDAC) after labeling; for quantification, see fig. S10B.

  • Fig. 7 Model of POLRMT regulating replication primer formation and expression of mtDNA.

    At high POLRMT levels, mitochondrial transcription initiation is activated from both the HSP and LSP resulting in mtDNA gene expression. At low POLRMT levels, only LSP is active and an RNA primer for replication of mtDNA is synthesized.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/8/e1600963/DC1

    fig. S1. Targeting strategy for disrupting the Polrmt gene in mouse.

    fig. S2. ECG analysis of conditional Polrmt knockout mice.

    fig. S3. Total protein levels of nuclear-encoded factors involved in mtDNA expression.

    fig. S4. Steady-state mitochondrial protein levels of conditional Polrmt knockout mice.

    fig. S5. Relative mitochondrial protein distribution across a gradient.

    fig. S6. Relative mitochondrial protein distribution in Polrmt knockout mice.

    fig. S7. Respiratory chain complex protein levels in heterozygous Polrmt knockout mice.

    fig. S8. Relative mitochondrial RNA abundance in heterozygous Polrmt knockout mice.

    fig. S9. Steady-state mitochondrial protein levels in heterozygous Polrmt knockout hearts.

    fig. S10. mtDNA levels in heterozygous Polrmt knockout hearts.

    table S1. Summary statistics and P values of RT-qPCR presented in Fig. 4B.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Targeting strategy for disrupting the Polrmt gene in mouse.
    • fig. S2. ECG analysis of conditional Polrmt knockout mice.
    • fig. S3. Total protein levels of nuclear-encoded factors involved in mtDNA expression.
    • fig. S4. Steady-state mitochondrial protein levels of conditional Polrmt knockout mice.
    • fig. S5. Relative mitochondrial protein distribution across a gradient.
    • fig. S6. Relative mitochondrial protein distribution in Polrmt knockout mice.
    • fig. S7. Respiratory chain complex protein levels in heterozygous Polrmt knockout mice.
    • fig. S8. Relative mitochondrial RNA abundance in heterozygous Polrmt knockout mice.
    • fig. S9. Steady-state mitochondrial protein levels in heterozygous Polrmt knockout hearts.
    • fig. S10. mtDNA levels in heterozygous Polrmt knockout hearts.
    • table S1. Summary statistics and P values of RT-qPCR presented in Fig. 4B.

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