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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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Related in: MedlinePlus

Defects in respiratory chain function upon TEFM gene silencing. (A) Western blot analyses of steady-state protein level of TEFM and subunits of respiratory chain complexes in control cells (untransfected cells and siRNA GFP) and cells treated with siRNA TEFM for 3 and 6 days. (B) Oxygen consumption rate (OCR) measured in quadruplicate population of control cells transfected siRNA GFP (green), cells treated with siRNA TEFM for 3 days (siRNA 1 in dark blue and siRNA 2 in light blue) and cells lacking mtDNA (Rho0, yellow). (C) Respiratory control ratio (RCR) in cells treated with siRNA GFP or siRNA TEFM for 3 or 6 days.
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Figure 2: Defects in respiratory chain function upon TEFM gene silencing. (A) Western blot analyses of steady-state protein level of TEFM and subunits of respiratory chain complexes in control cells (untransfected cells and siRNA GFP) and cells treated with siRNA TEFM for 3 and 6 days. (B) Oxygen consumption rate (OCR) measured in quadruplicate population of control cells transfected siRNA GFP (green), cells treated with siRNA TEFM for 3 days (siRNA 1 in dark blue and siRNA 2 in light blue) and cells lacking mtDNA (Rho0, yellow). (C) Respiratory control ratio (RCR) in cells treated with siRNA GFP or siRNA TEFM for 3 or 6 days.

Mentions: In order to test experimentally the role of TEFM in OXPHOS function, the expression of the gene was inactivated by RNA interference. We identified two siRNAs that efficiently depleted the target mRNA (Supplementary Figure S2A) and protein (Figure 2A). We inactivated TEFM in HOS cells and analysed steady-state levels of respiratory chain subunits, as OXPHOS dysfunction is often associated with aberrant assembly or instability of mitochondrial respiratory complexes. TEFM RNAi markedly reduced the abundance of the complex IV subunit, COX2, and to a lesser extent, NDUFB8, a component of complex I (Figure 2A). The decreases in respiratory chain components were accompanied by reduced cellular OCR by ∼50% (Figure 2B) and uncoupling of the respiratory chain and ATP synthase based on the respiratory control ratio (Figure 2C). These results indicated that TEFM plays a key role in mitochondrial energy production and given its homology to nucleic acids modifying proteins (Figure 1A) this was most likely to occur via a contribution to mtDNA maintenance or expression.


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)

Defects in respiratory chain function upon TEFM gene silencing. (A) Western blot analyses of steady-state protein level of TEFM and subunits of respiratory chain complexes in control cells (untransfected cells and siRNA GFP) and cells treated with siRNA TEFM for 3 and 6 days. (B) Oxygen consumption rate (OCR) measured in quadruplicate population of control cells transfected siRNA GFP (green), cells treated with siRNA TEFM for 3 days (siRNA 1 in dark blue and siRNA 2 in light blue) and cells lacking mtDNA (Rho0, yellow). (C) Respiratory control ratio (RCR) in cells treated with siRNA GFP or siRNA TEFM for 3 or 6 days.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105396&req=5

Figure 2: Defects in respiratory chain function upon TEFM gene silencing. (A) Western blot analyses of steady-state protein level of TEFM and subunits of respiratory chain complexes in control cells (untransfected cells and siRNA GFP) and cells treated with siRNA TEFM for 3 and 6 days. (B) Oxygen consumption rate (OCR) measured in quadruplicate population of control cells transfected siRNA GFP (green), cells treated with siRNA TEFM for 3 days (siRNA 1 in dark blue and siRNA 2 in light blue) and cells lacking mtDNA (Rho0, yellow). (C) Respiratory control ratio (RCR) in cells treated with siRNA GFP or siRNA TEFM for 3 or 6 days.
Mentions: In order to test experimentally the role of TEFM in OXPHOS function, the expression of the gene was inactivated by RNA interference. We identified two siRNAs that efficiently depleted the target mRNA (Supplementary Figure S2A) and protein (Figure 2A). We inactivated TEFM in HOS cells and analysed steady-state levels of respiratory chain subunits, as OXPHOS dysfunction is often associated with aberrant assembly or instability of mitochondrial respiratory complexes. TEFM RNAi markedly reduced the abundance of the complex IV subunit, COX2, and to a lesser extent, NDUFB8, a component of complex I (Figure 2A). The decreases in respiratory chain components were accompanied by reduced cellular OCR by ∼50% (Figure 2B) and uncoupling of the respiratory chain and ATP synthase based on the respiratory control ratio (Figure 2C). These results indicated that TEFM plays a key role in mitochondrial energy production and given its homology to nucleic acids modifying proteins (Figure 1A) this was most likely to occur via a contribution to mtDNA maintenance or expression.

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