Relative abundance of the human mitochondrial transcription system and distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression

Abstract

Human mitochondrial transcription requires the bacteriophage-related RNA polymerase, POLRMT, the mtDNA-binding protein, h-mtTFA/TFAM, and two transcription factors/rRNA methyltransferases, h-mtTFB1 and h-mtTFB2. Here, we determined the steady-state levels of these core transcription components and examined the consequences of purposeful elevation of h-mtTFB1 or h-mtTFB2 in HeLa cells. On a per molecule basis, we find an approximately 6-fold excess of POLRMT to mtDNA and approximately 3-fold more h-mtTFB2 than h-mtTFB1. We also estimate h-mtTFA at approximately 50 molecules/mtDNA, a ratio predicted to support robust transcription, but not to coat mtDNA. Consistent with a role for h-mtTFB2 in transcription and transcription-primed replication, increased mitochondrial DNA and transcripts result from its over-expression. This is accompanied by increased translation rates of most, but not all mtDNA-encoded proteins. Over-expression of h-mtTFB1 did not significantly influence these parameters, but did result in increased mitochondrial biogenesis. Furthermore, h-mtTFB1 mRNA and protein are elevated in response to h-mtTFB2 over-expression, suggesting the existence of a retrograde signal to the nucleus to coordinately regulate expression of these related factors. Altogether, our results provide a framework for understanding the regulation of human mitochondrial transcription in vivo and define distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression that together likely fine-tune mitochondrial function.

Figure 1.

h-mtTFB2 is processed in vivo and its over-expression in HeLa cells results in a coordinated increase of h-mtTFB1, but not POLRMT or h-mtTFA. (A) Shown are western blots on 200 ng of recombinant h-mtTFB1 and h-mtTFB2 proteins probed with four peptide antibodies: TFB1-1, TFB1-2, TFB2-1 and TFB2-2. Antibodies were found to specifically recognize their full-length recombinant peptide and not cross react with the paralogous protein. Coomassie staining of full-length recombinant h-mtTFB1 and h-mtTFB2 (top panel) demonstrates loading and their difference in molecular weight. (B) Western blot of mitochondrial extracts (100 μg protein) from HeLa cell lines over-expressing h-mtTFB1 or h-mtTFB2 used in this study in parallel with recombinant h-mtTFB1 and h-mtTFB2 run as controls. The blot was probed as indicated using peptide antibodies that distinguish h-mtTFB1 and h-mtTFB2 (α-h-mtTFB1 and α-h-mtTFB2) and an antibody that recognizes HSP60 (α-HSP60) that was used as a mitochondrial loading control. The lanes are loaded as follows: lane 1, molecular weight markers; lane 2, recombinant h-mtTFB1; lanes 3, recombinant h-mtTFB2; lanes 4–8, mitochondrial extracts from an empty pcDNA 3.1 zeo (+) vector-control, h-mtTFB1 over-expression, and three different h-mtTFB2 stable over-expression HeLa cell lines, respectively. (C) Shown are the results of real-time RT-PCR measurement of h-mtTFB1 mRNA (see Supplementary Figure S3) in h-mtTFB1 and h-mtTFB2 over-expression cell lines relative to empty-vector control cells. Reverse transcriptase real-time PCR was used to measure C_t values for cDNA samples from listed cell lines. The C_t values for h-mtTFB1 mRNA were normalized to those of β-actin and the values shown were normalized to the ratio obtained in the empty-vector control, which was given a value of 1. Values shown are the mean ± SD for three separate measurements. (D) Western blot of mitochondrial lysates from the same cell lines described in B probed using antibodies that recognize h-mtTFA (α-h-mtTFA), POLRMT (α-POLRMT), and the outer mitochondrial membrane protein VDAC (α-VDAC/porin) as a mitochondrial loading control.

Figure 2.

Over-expression of h-mtTFB2 increases the steady-state levels of mtDNA-encoded transcripts and proteins, and doubles mtDNA copy number. (A) Northern analysis of the mtDNA-encoded 12S, 16S, ND2 and ND6 from the same cell lines described in Figure 1B. Total RNA (2 μg) from the indicated cell line was loaded in each lane and the analysis was performed in triplicate on samples from three independent cultures. Ethidium bromide staining of the cytoplasmic 28S rRNA is shown as a loading control. Results of a quantification of the blots are graphed below. The relative transcript level (ratio of the mitochondrial signal to that of the 28S control) is plotted with the ratios obtained in the h-mtTFB1 and h-mtTFB2 cell lines normalized to that obtained from the empty-vector control cells, which was given a value of 1. The values are the mean ± SD. (B) Western blot of mitochondrial extracts from the indicated cell lines as in Figure 1B, probed using antibodies that recognize the mtDNA-encoded COX1 and COX2 protein or porin (VDAC) as a loading control. This demonstrates that there is an increase (on a per mitochondria basis) of these components unlike h-mtTFA and POLRMT. (C) Plotted is the relative mtDNA copy number (mtDNA relative to the nuclear 18S rDNA) normalized to that of the empty-vector control cells, whose ratio was given a value if 1. The analysis was done in triplicate and values shown are the mean ± SD.

Figure 3.

Analysis of mitochondrial translation rates and kasugamycin sensitivity in h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines. (A) Mitochondrial translation products were labeled in vivo with ³⁵S-methionine in the presence of the cytoplasmic translation inhibitor emetine. Mitochondria were purified after labeling and 100 μg of mitochondrial protein were loaded on a linear gradient polyacrylamide gel. Shown on the left is a coomassie stain of the resulting gel demonstrating equal protein loading and on the right is an autoradiogram of the radiolabeled mitochondrial proteins (specific proteins are indicated on the right). Changes in the profile of labeled products only occur during over-expression of h-mtTFB2. (B) Plotted is the percent viable cells of the indicated cell lines after growth for 72 h in the indicated amount of the aminoglycoside kasugamycin. The analysis was done in triplicate and values shown are the mean ± SD.

Figure 4.

Analysis of mitochondrial biogenesis and membrane potential in h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines. (A) Shown are representative results from FACS analysis of the indicated cell lines (see key in figure) stained with Mitotracker Green FM, as a measure of mitochondrial mass, or Mitotracker Red, as a measure of mitochondrial membrane potential. Plotted below is a quantification of a triplicate analysis of these parameters, with the mean fluorescence of the vector control cell line given a value of 1. Error bars represent SD of three experiments and asterisks indicate statistically significant differences in mean fluorescence as determined by t-tests, **P < 0.005 and ***P < 0.0005. (B) Western blot of whole-cell lysate (100 μg protein) from empty-vector control, h-mtTFB1 over-expression, and h-mtTFB2 over-expression cell lines with antibodies that recognize tubulin (α-tubulin) and α-porin (VDAC). The ratio of the VDAC signal to the tubulin signal is interpreted as a measure of the amount of mitochondria per cell.