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

Cotney J, Wang Z, Shadel GS - Nucleic Acids Res. (2007)

Bottom Line: Consistent with a role for h-mtTFB2 in transcription and transcription-primed replication, increased mitochondrial DNA and transcripts result from its over-expression.Over-expression of h-mtTFB1 did not significantly influence these parameters, but did result in increased mitochondrial biogenesis.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, 440 Clifton Road N.E., Atlanta, Georgia 30322, USA.

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.

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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.
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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.

Mentions: To address how altered levels of h-mtTFB1 and h-mtTFB2 affect mitochondrial gene expression, we next examined the h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines for changes in the steady-state levels of mtDNA-encoded transcripts and proteins, as well as for alterations in mtDNA copy number. Northern analysis of the mitochondrial 16S and 12S rRNAs and of ND2 and ND6 transcripts (representing mRNAs transcribed from each strand of mtDNA) revealed a ∼2-fold increase in their steady-state levels in the h-mtTFB2 over-expression cell line, but no change in the h-mtTFB1 over-expression line (Figure 2A). Similar results were obtained when immunoblots of the mtDNA-encoded COX1 and COX2 proteins was performed and mtDNA copy number was measured. That is, over-expression of h-mtTFB2, but not h-mtTFB1, led to a significant increase in the steady-state levels of COX1 and COX2 proteins (Figure 2B) and a doubling of the mtDNA copy number (Figure 2C). Altogether, these results are consistent with a role for h-mtTFB2 in transcription and in transcription-primed mtDNA replication. However, the fact that h-mtTFB1 is up-regulated in the h-mtTFB2 over-expression lines makes it difficult to assign these functions to h-mtTFB2 acting independently of h-mtTFB1.Figure 2.


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

Cotney J, Wang Z, Shadel GS - Nucleic Acids Res. (2007)

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.
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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.
Mentions: To address how altered levels of h-mtTFB1 and h-mtTFB2 affect mitochondrial gene expression, we next examined the h-mtTFB1 and h-mtTFB2 over-expression HeLa cell lines for changes in the steady-state levels of mtDNA-encoded transcripts and proteins, as well as for alterations in mtDNA copy number. Northern analysis of the mitochondrial 16S and 12S rRNAs and of ND2 and ND6 transcripts (representing mRNAs transcribed from each strand of mtDNA) revealed a ∼2-fold increase in their steady-state levels in the h-mtTFB2 over-expression cell line, but no change in the h-mtTFB1 over-expression line (Figure 2A). Similar results were obtained when immunoblots of the mtDNA-encoded COX1 and COX2 proteins was performed and mtDNA copy number was measured. That is, over-expression of h-mtTFB2, but not h-mtTFB1, led to a significant increase in the steady-state levels of COX1 and COX2 proteins (Figure 2B) and a doubling of the mtDNA copy number (Figure 2C). Altogether, these results are consistent with a role for h-mtTFB2 in transcription and in transcription-primed mtDNA replication. However, the fact that h-mtTFB1 is up-regulated in the h-mtTFB2 over-expression lines makes it difficult to assign these functions to h-mtTFB2 acting independently of h-mtTFB1.Figure 2.

Bottom Line: Consistent with a role for h-mtTFB2 in transcription and transcription-primed replication, increased mitochondrial DNA and transcripts result from its over-expression.Over-expression of h-mtTFB1 did not significantly influence these parameters, but did result in increased mitochondrial biogenesis.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, 440 Clifton Road N.E., Atlanta, Georgia 30322, USA.

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.

Show MeSH
Related in: MedlinePlus