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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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In silico identification and mitochondrial localization of TEFM. (A) Domain architecture of proteins homologous to TEFM. Green blocks indicate the tandem helix-hairpin-helix domains (HhH, Pfam PF00633) found in transcription related and DNA binding proteins. The RuvC and YqgF (Pfam PF03652) motifs (blue) exhibit the typical topology and structural elements of the RNase H fold. S1 domain (red) typically binds RNA whereas the SH2 domain (grey) is found in many proteins involved in signal transduction and is responsible for protein–protein interaction. SAP motif (Pfam PF02037, yellow) represents a putative DNA binding domain found in a diverse number of proteins involved in chromosomal organization (40). The MTS present in TEFM is indicated in orange. See Supplementary Data for further details. (B) Sub-cellular location of TEFM. The HOS cells were fractionated into fraction containing unbroken cells and cell debris (‘D’, lane 2), cytosol (‘C’, lane 3) and mitochondria (‘M’, lane 4) as described ‘Materials and Methods’ section. The protein fractions were analysed by western blotting using antibodies to endogenous TEFM. The location of TEFM was compared with that of the following marker proteins: TFAM (mitochondrial matrix), TOM22 (mitochondrial outer membrane), GAPDH (cytosol). (C) The intra-cellular localization of the HA-tagged variant of TEFM (green) by immunofluorescence, in human A549 cells, as described in ‘Materials and Methods’ section. Mitochondria were stained with MitoTracker Red CMXRos (red); the nucleus was stained with DAPI (blue). Yellow signal on digitally overlaid pictures indicated that TEFM has the same cellular distribution as mitochondria.
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Figure 1: In silico identification and mitochondrial localization of TEFM. (A) Domain architecture of proteins homologous to TEFM. Green blocks indicate the tandem helix-hairpin-helix domains (HhH, Pfam PF00633) found in transcription related and DNA binding proteins. The RuvC and YqgF (Pfam PF03652) motifs (blue) exhibit the typical topology and structural elements of the RNase H fold. S1 domain (red) typically binds RNA whereas the SH2 domain (grey) is found in many proteins involved in signal transduction and is responsible for protein–protein interaction. SAP motif (Pfam PF02037, yellow) represents a putative DNA binding domain found in a diverse number of proteins involved in chromosomal organization (40). The MTS present in TEFM is indicated in orange. See Supplementary Data for further details. (B) Sub-cellular location of TEFM. The HOS cells were fractionated into fraction containing unbroken cells and cell debris (‘D’, lane 2), cytosol (‘C’, lane 3) and mitochondria (‘M’, lane 4) as described ‘Materials and Methods’ section. The protein fractions were analysed by western blotting using antibodies to endogenous TEFM. The location of TEFM was compared with that of the following marker proteins: TFAM (mitochondrial matrix), TOM22 (mitochondrial outer membrane), GAPDH (cytosol). (C) The intra-cellular localization of the HA-tagged variant of TEFM (green) by immunofluorescence, in human A549 cells, as described in ‘Materials and Methods’ section. Mitochondria were stained with MitoTracker Red CMXRos (red); the nucleus was stained with DAPI (blue). Yellow signal on digitally overlaid pictures indicated that TEFM has the same cellular distribution as mitochondria.

Mentions: In order to construct pcDNA5-TEFM.HA used for the immunofluorescence localization of the TEFM protein (Figures 1C and 6C), the cDNA encoding TEFM (c17orf42) was modified by PCR to introduce the HA epitope tag (YPYDVPDYA) to the C-terminus of the ZFP and flanked with unique KpnI (5′) and XbaI (3′) restriction sites. The resulting fragment was cloned into pcDNA5/FRT/TO (Invitrogen) using the above restriction sites.Figure 1.


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)

In silico identification and mitochondrial localization of TEFM. (A) Domain architecture of proteins homologous to TEFM. Green blocks indicate the tandem helix-hairpin-helix domains (HhH, Pfam PF00633) found in transcription related and DNA binding proteins. The RuvC and YqgF (Pfam PF03652) motifs (blue) exhibit the typical topology and structural elements of the RNase H fold. S1 domain (red) typically binds RNA whereas the SH2 domain (grey) is found in many proteins involved in signal transduction and is responsible for protein–protein interaction. SAP motif (Pfam PF02037, yellow) represents a putative DNA binding domain found in a diverse number of proteins involved in chromosomal organization (40). The MTS present in TEFM is indicated in orange. See Supplementary Data for further details. (B) Sub-cellular location of TEFM. The HOS cells were fractionated into fraction containing unbroken cells and cell debris (‘D’, lane 2), cytosol (‘C’, lane 3) and mitochondria (‘M’, lane 4) as described ‘Materials and Methods’ section. The protein fractions were analysed by western blotting using antibodies to endogenous TEFM. The location of TEFM was compared with that of the following marker proteins: TFAM (mitochondrial matrix), TOM22 (mitochondrial outer membrane), GAPDH (cytosol). (C) The intra-cellular localization of the HA-tagged variant of TEFM (green) by immunofluorescence, in human A549 cells, as described in ‘Materials and Methods’ section. Mitochondria were stained with MitoTracker Red CMXRos (red); the nucleus was stained with DAPI (blue). Yellow signal on digitally overlaid pictures indicated that TEFM has the same cellular distribution as mitochondria.
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Related In: Results  -  Collection

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Figure 1: In silico identification and mitochondrial localization of TEFM. (A) Domain architecture of proteins homologous to TEFM. Green blocks indicate the tandem helix-hairpin-helix domains (HhH, Pfam PF00633) found in transcription related and DNA binding proteins. The RuvC and YqgF (Pfam PF03652) motifs (blue) exhibit the typical topology and structural elements of the RNase H fold. S1 domain (red) typically binds RNA whereas the SH2 domain (grey) is found in many proteins involved in signal transduction and is responsible for protein–protein interaction. SAP motif (Pfam PF02037, yellow) represents a putative DNA binding domain found in a diverse number of proteins involved in chromosomal organization (40). The MTS present in TEFM is indicated in orange. See Supplementary Data for further details. (B) Sub-cellular location of TEFM. The HOS cells were fractionated into fraction containing unbroken cells and cell debris (‘D’, lane 2), cytosol (‘C’, lane 3) and mitochondria (‘M’, lane 4) as described ‘Materials and Methods’ section. The protein fractions were analysed by western blotting using antibodies to endogenous TEFM. The location of TEFM was compared with that of the following marker proteins: TFAM (mitochondrial matrix), TOM22 (mitochondrial outer membrane), GAPDH (cytosol). (C) The intra-cellular localization of the HA-tagged variant of TEFM (green) by immunofluorescence, in human A549 cells, as described in ‘Materials and Methods’ section. Mitochondria were stained with MitoTracker Red CMXRos (red); the nucleus was stained with DAPI (blue). Yellow signal on digitally overlaid pictures indicated that TEFM has the same cellular distribution as mitochondria.
Mentions: In order to construct pcDNA5-TEFM.HA used for the immunofluorescence localization of the TEFM protein (Figures 1C and 6C), the cDNA encoding TEFM (c17orf42) was modified by PCR to introduce the HA epitope tag (YPYDVPDYA) to the C-terminus of the ZFP and flanked with unique KpnI (5′) and XbaI (3′) restriction sites. The resulting fragment was cloned into pcDNA5/FRT/TO (Invitrogen) using the above restriction sites.Figure 1.

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