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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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TEFM interacts with C-terminal, catalytic part of POLRMT. (A) Schematic representation of the myc-tagged POLRMT constructs used to map the interaction with TEFM in comparison with the T7 phage RNA polymerase (T7RNAP). The N-terminal extension present in the POLRMT (dark gray) is missing in the T7RNAP. f.l., full-length. (B) Western blot of the control pull-down experiment with mock (left) and the full-length POLRMT (right) transfected HEK cells that inducibly express TEFM.STREP2. The blot was incubated with the antibodies indicated to the right. T, total cell lysate; P, pulled-down material. The asterisk indicates a non-specific band. (C) Western blot of the pull-down experiment with the POLRMT lacking the N-terminal extension (POLRMT MTS-768-1230, left) or the C-terminal catalytic part (POLRMT 1-801, right) with TEFM.STREP2. The asterisk indicates a non-specific band.
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Figure 7: TEFM interacts with C-terminal, catalytic part of POLRMT. (A) Schematic representation of the myc-tagged POLRMT constructs used to map the interaction with TEFM in comparison with the T7 phage RNA polymerase (T7RNAP). The N-terminal extension present in the POLRMT (dark gray) is missing in the T7RNAP. f.l., full-length. (B) Western blot of the control pull-down experiment with mock (left) and the full-length POLRMT (right) transfected HEK cells that inducibly express TEFM.STREP2. The blot was incubated with the antibodies indicated to the right. T, total cell lysate; P, pulled-down material. The asterisk indicates a non-specific band. (C) Western blot of the pull-down experiment with the POLRMT lacking the N-terminal extension (POLRMT MTS-768-1230, left) or the C-terminal catalytic part (POLRMT 1-801, right) with TEFM.STREP2. The asterisk indicates a non-specific band.

Mentions: Human POLRMT displays significant homology to the RNA polymerase of T-odd bacteriophages, such as T7 (25,30). However, mitochondrial RNA polymerases contain N-terminal extensions not present in the T7 polymerase (T7RNAP) (Figure 7A) and so this was considered to be a potential binding region for TEFM. In order to map the region of human mitochondrial RNA polymerase that binds to TEFM, pull-down experiments with truncated variants of POLRMT were performed. Myc-tagged truncated versions of POLRMT (Figure 7A) were transiently expressed in HEK cells that simultaneously expressed TEFM, under the control of a doxycycline inducible promoter. Control experiments showed that full-length, Myc-tagged POLRMT could be pulled down only if TEFM was induced (Figure 7B). The POLRMT variant lacking residues 61–767 interacted with TEFM (Figure 7C, left), whereas the C-terminally truncated variant of POLRMT (lacking residues 802–1230) was no longer able to form a complex with TEFM (Figure 7C, right). Therefore, we concluded that TEFM interacts with the catalytic region of POLRMT, suggesting that it might be involved directly in the regulation of polymerization.Figure 7.


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)

TEFM interacts with C-terminal, catalytic part of POLRMT. (A) Schematic representation of the myc-tagged POLRMT constructs used to map the interaction with TEFM in comparison with the T7 phage RNA polymerase (T7RNAP). The N-terminal extension present in the POLRMT (dark gray) is missing in the T7RNAP. f.l., full-length. (B) Western blot of the control pull-down experiment with mock (left) and the full-length POLRMT (right) transfected HEK cells that inducibly express TEFM.STREP2. The blot was incubated with the antibodies indicated to the right. T, total cell lysate; P, pulled-down material. The asterisk indicates a non-specific band. (C) Western blot of the pull-down experiment with the POLRMT lacking the N-terminal extension (POLRMT MTS-768-1230, left) or the C-terminal catalytic part (POLRMT 1-801, right) with TEFM.STREP2. The asterisk indicates a non-specific band.
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Figure 7: TEFM interacts with C-terminal, catalytic part of POLRMT. (A) Schematic representation of the myc-tagged POLRMT constructs used to map the interaction with TEFM in comparison with the T7 phage RNA polymerase (T7RNAP). The N-terminal extension present in the POLRMT (dark gray) is missing in the T7RNAP. f.l., full-length. (B) Western blot of the control pull-down experiment with mock (left) and the full-length POLRMT (right) transfected HEK cells that inducibly express TEFM.STREP2. The blot was incubated with the antibodies indicated to the right. T, total cell lysate; P, pulled-down material. The asterisk indicates a non-specific band. (C) Western blot of the pull-down experiment with the POLRMT lacking the N-terminal extension (POLRMT MTS-768-1230, left) or the C-terminal catalytic part (POLRMT 1-801, right) with TEFM.STREP2. The asterisk indicates a non-specific band.
Mentions: Human POLRMT displays significant homology to the RNA polymerase of T-odd bacteriophages, such as T7 (25,30). However, mitochondrial RNA polymerases contain N-terminal extensions not present in the T7 polymerase (T7RNAP) (Figure 7A) and so this was considered to be a potential binding region for TEFM. In order to map the region of human mitochondrial RNA polymerase that binds to TEFM, pull-down experiments with truncated variants of POLRMT were performed. Myc-tagged truncated versions of POLRMT (Figure 7A) were transiently expressed in HEK cells that simultaneously expressed TEFM, under the control of a doxycycline inducible promoter. Control experiments showed that full-length, Myc-tagged POLRMT could be pulled down only if TEFM was induced (Figure 7B). The POLRMT variant lacking residues 61–767 interacted with TEFM (Figure 7C, left), whereas the C-terminally truncated variant of POLRMT (lacking residues 802–1230) was no longer able to form a complex with TEFM (Figure 7C, right). Therefore, we concluded that TEFM interacts with the catalytic region of POLRMT, suggesting that it might be involved directly in the regulation of polymerization.Figure 7.

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