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TEFM (c17orf42) is necessary for transcription of human mtDNA.

Minczuk M, He J, Duch AM, Ettema TJ, Chlebowski A, Dzionek K, Nijtmans LG, Huynen MA, Holt IJ - Nucleic Acids Res. (2011)

Bottom Line: After RNase treatment only POLRMT remained associated with TEFM, and in human cultured cells TEFM formed foci coincident with newly synthesized mitochondrial RNA.TEFM contains two HhH motifs and a Ribonuclease H fold, similar to the nuclear transcription elongation regulator Spt6.These findings lead us to propose that TEFM is a mitochondrial transcription elongation factor.

View Article: PubMed Central - PubMed

Affiliation: MRC Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK. michal.minczuk@mrc-mbu.cam.ac.uk

ABSTRACT
Here we show that c17orf42, hereafter TEFM (transcription elongation factor of mitochondria), makes a critical contribution to mitochondrial transcription. Inactivation of TEFM in cells by RNA interference results in respiratory incompetence owing to decreased levels of H- and L-strand promoter-distal mitochondrial transcripts. Affinity purification of TEFM from human mitochondria yielded a complex comprising mitochondrial transcripts, mitochondrial RNA polymerase (POLRMT), pentatricopeptide repeat domain 3 protein (PTCD3), and a putative DEAD-box RNA helicase, DHX30. After RNase treatment only POLRMT remained associated with TEFM, and in human cultured cells TEFM formed foci coincident with newly synthesized mitochondrial RNA. Based on deletion mutants, TEFM interacts with the catalytic region of POLRMT, and in vitro TEFM enhanced POLRMT processivity on ss- and dsDNA templates. TEFM contains two HhH motifs and a Ribonuclease H fold, similar to the nuclear transcription elongation regulator Spt6. These findings lead us to propose that TEFM is a mitochondrial transcription elongation factor.

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Steady-state levels of mtDNA and mitochondrial transcripts in cells with inactivated TEFM. (A) mtDNA copy number as measured by comparative qPCR of the mitochondrial Cox2 gene and single copy nuclear gene (APP) in controls (Untransfected and siRNA GFP) and cells treated with TEFM siRNA (siRNA TEFM 1 and 2). *P < 0.05, n = 3, error bars indicate 1 SD. (B) Northern blot analyses of mitochondrial transcripts transcribed from the HSP1 or LSP promoter in control cells (untransfected and treated with GFP siRNA) and cells treated with TEFM siRNA for 3 or 6 days. Nuclear 28S rRNA was used as a loading control. (C and D) Quantification of steady-state levels of the H-strand mitochondrial transcripts in cells treated with TEFM siRNA for 3 days (C) and 6 days (D) analysed by northern blots. The values of the relative RNA level (mtRNA/28S rRNA) were obtained by quantifying PhosphoImager scans of blots in the ImageQuant software and normalized for the values obtained for control cells transfected with siRNA GFP. The relative RNA level of each transcript for siRNA TEFM 1 (square) and 2 (triangle) was plotted in the function of the distance of its 3′ end from HSP. Dotted line, trend for siRNA GFP control; solid line, trend for siRNA TEFM 1; dashed line, trend for siRNA TEFM 2. Red symbols indicate the RNA19 transcript. n = 3, error bars = 1 SD. The P-values (two-tailed Student’s t-test) for each transcript calculated for combined values for both TEFM siRNAs for 3 days: 12S = 0.103, 16S = 0.719, RNA19 = 0.124, ND1 = 0.492, ND2 = 0.234, COI = 0.009, COII = 0.031, ATP6/8 = 0.064, COIII = 0.023, ND3 = 0.890, ND4/4L = 0.006, ND5 = 0.007, CytB < 0.001, ND6 = 0.502; and for 6 days: 12S = 0.813, 16S = 0.092, RNA19 = 0.187, ND1 = 0.026, ND2 = 0.285, COI = 0.003, COII = 0.129, ATP6/8 = 0.025, COIII = 0.027, ND3 = 0.169, ND4/4L < 0.001, ND5 = 0.002, CytB < 0.001, ND6 = 0.137. The quantification of the steady-state level of the ND6 transcript that is transcribed from LSP is shown in Supplementary Figure S2.
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Figure 3: Steady-state levels of mtDNA and mitochondrial transcripts in cells with inactivated TEFM. (A) mtDNA copy number as measured by comparative qPCR of the mitochondrial Cox2 gene and single copy nuclear gene (APP) in controls (Untransfected and siRNA GFP) and cells treated with TEFM siRNA (siRNA TEFM 1 and 2). *P < 0.05, n = 3, error bars indicate 1 SD. (B) Northern blot analyses of mitochondrial transcripts transcribed from the HSP1 or LSP promoter in control cells (untransfected and treated with GFP siRNA) and cells treated with TEFM siRNA for 3 or 6 days. Nuclear 28S rRNA was used as a loading control. (C and D) Quantification of steady-state levels of the H-strand mitochondrial transcripts in cells treated with TEFM siRNA for 3 days (C) and 6 days (D) analysed by northern blots. The values of the relative RNA level (mtRNA/28S rRNA) were obtained by quantifying PhosphoImager scans of blots in the ImageQuant software and normalized for the values obtained for control cells transfected with siRNA GFP. The relative RNA level of each transcript for siRNA TEFM 1 (square) and 2 (triangle) was plotted in the function of the distance of its 3′ end from HSP. Dotted line, trend for siRNA GFP control; solid line, trend for siRNA TEFM 1; dashed line, trend for siRNA TEFM 2. Red symbols indicate the RNA19 transcript. n = 3, error bars = 1 SD. The P-values (two-tailed Student’s t-test) for each transcript calculated for combined values for both TEFM siRNAs for 3 days: 12S = 0.103, 16S = 0.719, RNA19 = 0.124, ND1 = 0.492, ND2 = 0.234, COI = 0.009, COII = 0.031, ATP6/8 = 0.064, COIII = 0.023, ND3 = 0.890, ND4/4L = 0.006, ND5 = 0.007, CytB < 0.001, ND6 = 0.502; and for 6 days: 12S = 0.813, 16S = 0.092, RNA19 = 0.187, ND1 = 0.026, ND2 = 0.285, COI = 0.003, COII = 0.129, ATP6/8 = 0.025, COIII = 0.027, ND3 = 0.169, ND4/4L < 0.001, ND5 = 0.002, CytB < 0.001, ND6 = 0.137. The quantification of the steady-state level of the ND6 transcript that is transcribed from LSP is shown in Supplementary Figure S2.

Mentions: Inactivation of TEFM had little effect on mtDNA copy number (Figure 3A) or mitochondrial replication intermediates (data not shown) in HOS cells. Next, mitochondrial transcripts were extracted from HOS cells after TEFM RNAi. Quantification of the steady-state levels of mRNAs and rRNAs generated by transcription from HSP and LSP of mtDNA revealed marked reductions in many mitochondrial transcripts of cells subjected to TEFM RNAi (Figure 3B–E). There was a clear pattern to the changes in transcript levels: transcripts at promoter-distal locations on the H-strand polycistronic transcript were much less abundant than transcripts mapping closer to the promoter, with the exception of ND1 and ND2 mRNAs (Figure 3C–D). The exceptions could be explained by changes in transcript stability, as the half-life of ND1 was markedly increased in the TEFM silenced cells (Supplementary Figure S2B). Up-regulation of the steady-state level of ND1 mRNA has been observed previously in cells with inactivated genes that play a role in mitochondrial transcription and/or processing (24). RNA19, which contains 16S rRNA, tRNA-LeuUUR and ND1, followed the general trend of the other transcripts (Figure 3C and D, red). There was no detectable increase in any precursor RNA in northern blots (Supplementary Figure S3) and so the changes in mitochondrial mRNAs levels were not attributable to perturbed RNA processing.Figure 3.


TEFM (c17orf42) is necessary for transcription of human mtDNA.

Minczuk M, He J, Duch AM, Ettema TJ, Chlebowski A, Dzionek K, Nijtmans LG, Huynen MA, Holt IJ - Nucleic Acids Res. (2011)

Steady-state levels of mtDNA and mitochondrial transcripts in cells with inactivated TEFM. (A) mtDNA copy number as measured by comparative qPCR of the mitochondrial Cox2 gene and single copy nuclear gene (APP) in controls (Untransfected and siRNA GFP) and cells treated with TEFM siRNA (siRNA TEFM 1 and 2). *P < 0.05, n = 3, error bars indicate 1 SD. (B) Northern blot analyses of mitochondrial transcripts transcribed from the HSP1 or LSP promoter in control cells (untransfected and treated with GFP siRNA) and cells treated with TEFM siRNA for 3 or 6 days. Nuclear 28S rRNA was used as a loading control. (C and D) Quantification of steady-state levels of the H-strand mitochondrial transcripts in cells treated with TEFM siRNA for 3 days (C) and 6 days (D) analysed by northern blots. The values of the relative RNA level (mtRNA/28S rRNA) were obtained by quantifying PhosphoImager scans of blots in the ImageQuant software and normalized for the values obtained for control cells transfected with siRNA GFP. The relative RNA level of each transcript for siRNA TEFM 1 (square) and 2 (triangle) was plotted in the function of the distance of its 3′ end from HSP. Dotted line, trend for siRNA GFP control; solid line, trend for siRNA TEFM 1; dashed line, trend for siRNA TEFM 2. Red symbols indicate the RNA19 transcript. n = 3, error bars = 1 SD. The P-values (two-tailed Student’s t-test) for each transcript calculated for combined values for both TEFM siRNAs for 3 days: 12S = 0.103, 16S = 0.719, RNA19 = 0.124, ND1 = 0.492, ND2 = 0.234, COI = 0.009, COII = 0.031, ATP6/8 = 0.064, COIII = 0.023, ND3 = 0.890, ND4/4L = 0.006, ND5 = 0.007, CytB < 0.001, ND6 = 0.502; and for 6 days: 12S = 0.813, 16S = 0.092, RNA19 = 0.187, ND1 = 0.026, ND2 = 0.285, COI = 0.003, COII = 0.129, ATP6/8 = 0.025, COIII = 0.027, ND3 = 0.169, ND4/4L < 0.001, ND5 = 0.002, CytB < 0.001, ND6 = 0.137. The quantification of the steady-state level of the ND6 transcript that is transcribed from LSP is shown in Supplementary Figure S2.
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Figure 3: Steady-state levels of mtDNA and mitochondrial transcripts in cells with inactivated TEFM. (A) mtDNA copy number as measured by comparative qPCR of the mitochondrial Cox2 gene and single copy nuclear gene (APP) in controls (Untransfected and siRNA GFP) and cells treated with TEFM siRNA (siRNA TEFM 1 and 2). *P < 0.05, n = 3, error bars indicate 1 SD. (B) Northern blot analyses of mitochondrial transcripts transcribed from the HSP1 or LSP promoter in control cells (untransfected and treated with GFP siRNA) and cells treated with TEFM siRNA for 3 or 6 days. Nuclear 28S rRNA was used as a loading control. (C and D) Quantification of steady-state levels of the H-strand mitochondrial transcripts in cells treated with TEFM siRNA for 3 days (C) and 6 days (D) analysed by northern blots. The values of the relative RNA level (mtRNA/28S rRNA) were obtained by quantifying PhosphoImager scans of blots in the ImageQuant software and normalized for the values obtained for control cells transfected with siRNA GFP. The relative RNA level of each transcript for siRNA TEFM 1 (square) and 2 (triangle) was plotted in the function of the distance of its 3′ end from HSP. Dotted line, trend for siRNA GFP control; solid line, trend for siRNA TEFM 1; dashed line, trend for siRNA TEFM 2. Red symbols indicate the RNA19 transcript. n = 3, error bars = 1 SD. The P-values (two-tailed Student’s t-test) for each transcript calculated for combined values for both TEFM siRNAs for 3 days: 12S = 0.103, 16S = 0.719, RNA19 = 0.124, ND1 = 0.492, ND2 = 0.234, COI = 0.009, COII = 0.031, ATP6/8 = 0.064, COIII = 0.023, ND3 = 0.890, ND4/4L = 0.006, ND5 = 0.007, CytB < 0.001, ND6 = 0.502; and for 6 days: 12S = 0.813, 16S = 0.092, RNA19 = 0.187, ND1 = 0.026, ND2 = 0.285, COI = 0.003, COII = 0.129, ATP6/8 = 0.025, COIII = 0.027, ND3 = 0.169, ND4/4L < 0.001, ND5 = 0.002, CytB < 0.001, ND6 = 0.137. The quantification of the steady-state level of the ND6 transcript that is transcribed from LSP is shown in Supplementary Figure S2.
Mentions: Inactivation of TEFM had little effect on mtDNA copy number (Figure 3A) or mitochondrial replication intermediates (data not shown) in HOS cells. Next, mitochondrial transcripts were extracted from HOS cells after TEFM RNAi. Quantification of the steady-state levels of mRNAs and rRNAs generated by transcription from HSP and LSP of mtDNA revealed marked reductions in many mitochondrial transcripts of cells subjected to TEFM RNAi (Figure 3B–E). There was a clear pattern to the changes in transcript levels: transcripts at promoter-distal locations on the H-strand polycistronic transcript were much less abundant than transcripts mapping closer to the promoter, with the exception of ND1 and ND2 mRNAs (Figure 3C–D). The exceptions could be explained by changes in transcript stability, as the half-life of ND1 was markedly increased in the TEFM silenced cells (Supplementary Figure S2B). Up-regulation of the steady-state level of ND1 mRNA has been observed previously in cells with inactivated genes that play a role in mitochondrial transcription and/or processing (24). RNA19, which contains 16S rRNA, tRNA-LeuUUR and ND1, followed the general trend of the other transcripts (Figure 3C and D, red). There was no detectable increase in any precursor RNA in northern blots (Supplementary Figure S3) and so the changes in mitochondrial mRNAs levels were not attributable to perturbed RNA processing.Figure 3.

Bottom Line: After RNase treatment only POLRMT remained associated with TEFM, and in human cultured cells TEFM formed foci coincident with newly synthesized mitochondrial RNA.TEFM contains two HhH motifs and a Ribonuclease H fold, similar to the nuclear transcription elongation regulator Spt6.These findings lead us to propose that TEFM is a mitochondrial transcription elongation factor.

View Article: PubMed Central - PubMed

Affiliation: MRC Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK. michal.minczuk@mrc-mbu.cam.ac.uk

ABSTRACT
Here we show that c17orf42, hereafter TEFM (transcription elongation factor of mitochondria), makes a critical contribution to mitochondrial transcription. Inactivation of TEFM in cells by RNA interference results in respiratory incompetence owing to decreased levels of H- and L-strand promoter-distal mitochondrial transcripts. Affinity purification of TEFM from human mitochondria yielded a complex comprising mitochondrial transcripts, mitochondrial RNA polymerase (POLRMT), pentatricopeptide repeat domain 3 protein (PTCD3), and a putative DEAD-box RNA helicase, DHX30. After RNase treatment only POLRMT remained associated with TEFM, and in human cultured cells TEFM formed foci coincident with newly synthesized mitochondrial RNA. Based on deletion mutants, TEFM interacts with the catalytic region of POLRMT, and in vitro TEFM enhanced POLRMT processivity on ss- and dsDNA templates. TEFM contains two HhH motifs and a Ribonuclease H fold, similar to the nuclear transcription elongation regulator Spt6. These findings lead us to propose that TEFM is a mitochondrial transcription elongation factor.

Show MeSH
Related in: MedlinePlus