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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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Stimulation of the POLRMT activity by TEFM. (A) Coomassie Brilliant Blue stained SDS–PAGE gel showing E. coli purified the GST.TEFM protein fusion. MW, molecular weight marker. (B) The synthesis of 32P-labelled RNA by POLRMT (0.35 pmol) on M13mp18(+) ssDNA in the absence (lane2) and the presence of 1.0 pmol (lane 4), 0.33 pmol (lane 5) and 0.11 pmol (lane 6) of GST.TEFM was performed as described in ‘Materials and Methods’ section. The products were separated on a 5% UREA polyacrylamide gel and subjected to autoradiography. (C) Relative abundance of the RNA products of indicated lengths synthesized by POLRMT on the ssDNA template in the presence of different concentrations of GST.TEFM. The values were obtained by quantifying PhosphoImager scans of dried UREA gels in the ImageQuant software and normalized for the values obtained from the reaction with POLRMT only. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student’s t-test; n = 4, Error bars = 1 SD. (D) Schematic representation of the construction of long 3′-tailed dsDNA templates. (E) The synthesis of 32P-labelled RNA by POLRMT (0.35 pmol) on 3′-tailed dsDNA of different length (20, 100 and 400 bp) in the absence (lanes 1, 5 and 9) and the presence of 1.0 pmol (lanes 2, 6 and 10), 0.33 pmol (lanes 3, 7 and 11) and 0.11 pmol (lanes 4, 8 and 12) of GST.TEFM for the indicated time. (F) The ratio between the 400 and 20 nt RNA products synthesized by POLRMT on the 3′-tailed dsDNA template in the presence of different concentrations of TEFM. The values were normalized with respect to the reaction with POLRMT alone. *P < 0.05; two-tailed Student’s t- test; n = 3, Error bars = 1 SD.
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Figure 8: Stimulation of the POLRMT activity by TEFM. (A) Coomassie Brilliant Blue stained SDS–PAGE gel showing E. coli purified the GST.TEFM protein fusion. MW, molecular weight marker. (B) The synthesis of 32P-labelled RNA by POLRMT (0.35 pmol) on M13mp18(+) ssDNA in the absence (lane2) and the presence of 1.0 pmol (lane 4), 0.33 pmol (lane 5) and 0.11 pmol (lane 6) of GST.TEFM was performed as described in ‘Materials and Methods’ section. The products were separated on a 5% UREA polyacrylamide gel and subjected to autoradiography. (C) Relative abundance of the RNA products of indicated lengths synthesized by POLRMT on the ssDNA template in the presence of different concentrations of GST.TEFM. The values were obtained by quantifying PhosphoImager scans of dried UREA gels in the ImageQuant software and normalized for the values obtained from the reaction with POLRMT only. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student’s t-test; n = 4, Error bars = 1 SD. (D) Schematic representation of the construction of long 3′-tailed dsDNA templates. (E) The synthesis of 32P-labelled RNA by POLRMT (0.35 pmol) on 3′-tailed dsDNA of different length (20, 100 and 400 bp) in the absence (lanes 1, 5 and 9) and the presence of 1.0 pmol (lanes 2, 6 and 10), 0.33 pmol (lanes 3, 7 and 11) and 0.11 pmol (lanes 4, 8 and 12) of GST.TEFM for the indicated time. (F) The ratio between the 400 and 20 nt RNA products synthesized by POLRMT on the 3′-tailed dsDNA template in the presence of different concentrations of TEFM. The values were normalized with respect to the reaction with POLRMT alone. *P < 0.05; two-tailed Student’s t- test; n = 3, Error bars = 1 SD.

Mentions: To test the above hypothesis, recombinant GST.TEFM protein was purified to homogeneity (Figure 8A, Supplementary Figure S5 and ‘Materials and Methods’ section) and the polymerase activity of recombinant POLRMT on ssDNA assayed in vitro, with or without recombinant GST.TEFM. POLRMT incubated with ssDNA in the presence of radiolabelled UTP yielded short RNA fragments (∼25–75 nt), and longer RNAs when recombinant GST.TEFM was included in the reaction mixture (Figure 8B and C). The highest concentration of GST.TEFM tested (50 pM), revealed a >2-fold increase in RNA products of 200 and 400 nt in length, compared to POLRMT transcripts synthesized without TEFM (Figure 8C). In a further test, the promoter independent activity of POLRMT was assayed for 5–20 min on short or long 3′-tailed dsDNA of 20, 100 or 400 bp, with or without recombinant GST.TEFM (Figure 8D–F). In 5 min reactions containing the highest concentration of GST.TEFM the ratio of 400:20 nt product was 75% higher than that of reactions lacking TEFM (Figure 8F). Therefore, POLRMT in concert with TEFM needs less time to make transcripts 400 nt in length than POLRMT alone; i.e. POLRMT processivity is enhanced by TEFM in vitro.Figure 8.


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)

Stimulation of the POLRMT activity by TEFM. (A) Coomassie Brilliant Blue stained SDS–PAGE gel showing E. coli purified the GST.TEFM protein fusion. MW, molecular weight marker. (B) The synthesis of 32P-labelled RNA by POLRMT (0.35 pmol) on M13mp18(+) ssDNA in the absence (lane2) and the presence of 1.0 pmol (lane 4), 0.33 pmol (lane 5) and 0.11 pmol (lane 6) of GST.TEFM was performed as described in ‘Materials and Methods’ section. The products were separated on a 5% UREA polyacrylamide gel and subjected to autoradiography. (C) Relative abundance of the RNA products of indicated lengths synthesized by POLRMT on the ssDNA template in the presence of different concentrations of GST.TEFM. The values were obtained by quantifying PhosphoImager scans of dried UREA gels in the ImageQuant software and normalized for the values obtained from the reaction with POLRMT only. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student’s t-test; n = 4, Error bars = 1 SD. (D) Schematic representation of the construction of long 3′-tailed dsDNA templates. (E) The synthesis of 32P-labelled RNA by POLRMT (0.35 pmol) on 3′-tailed dsDNA of different length (20, 100 and 400 bp) in the absence (lanes 1, 5 and 9) and the presence of 1.0 pmol (lanes 2, 6 and 10), 0.33 pmol (lanes 3, 7 and 11) and 0.11 pmol (lanes 4, 8 and 12) of GST.TEFM for the indicated time. (F) The ratio between the 400 and 20 nt RNA products synthesized by POLRMT on the 3′-tailed dsDNA template in the presence of different concentrations of TEFM. The values were normalized with respect to the reaction with POLRMT alone. *P < 0.05; two-tailed Student’s t- test; n = 3, Error bars = 1 SD.
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Figure 8: Stimulation of the POLRMT activity by TEFM. (A) Coomassie Brilliant Blue stained SDS–PAGE gel showing E. coli purified the GST.TEFM protein fusion. MW, molecular weight marker. (B) The synthesis of 32P-labelled RNA by POLRMT (0.35 pmol) on M13mp18(+) ssDNA in the absence (lane2) and the presence of 1.0 pmol (lane 4), 0.33 pmol (lane 5) and 0.11 pmol (lane 6) of GST.TEFM was performed as described in ‘Materials and Methods’ section. The products were separated on a 5% UREA polyacrylamide gel and subjected to autoradiography. (C) Relative abundance of the RNA products of indicated lengths synthesized by POLRMT on the ssDNA template in the presence of different concentrations of GST.TEFM. The values were obtained by quantifying PhosphoImager scans of dried UREA gels in the ImageQuant software and normalized for the values obtained from the reaction with POLRMT only. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student’s t-test; n = 4, Error bars = 1 SD. (D) Schematic representation of the construction of long 3′-tailed dsDNA templates. (E) The synthesis of 32P-labelled RNA by POLRMT (0.35 pmol) on 3′-tailed dsDNA of different length (20, 100 and 400 bp) in the absence (lanes 1, 5 and 9) and the presence of 1.0 pmol (lanes 2, 6 and 10), 0.33 pmol (lanes 3, 7 and 11) and 0.11 pmol (lanes 4, 8 and 12) of GST.TEFM for the indicated time. (F) The ratio between the 400 and 20 nt RNA products synthesized by POLRMT on the 3′-tailed dsDNA template in the presence of different concentrations of TEFM. The values were normalized with respect to the reaction with POLRMT alone. *P < 0.05; two-tailed Student’s t- test; n = 3, Error bars = 1 SD.
Mentions: To test the above hypothesis, recombinant GST.TEFM protein was purified to homogeneity (Figure 8A, Supplementary Figure S5 and ‘Materials and Methods’ section) and the polymerase activity of recombinant POLRMT on ssDNA assayed in vitro, with or without recombinant GST.TEFM. POLRMT incubated with ssDNA in the presence of radiolabelled UTP yielded short RNA fragments (∼25–75 nt), and longer RNAs when recombinant GST.TEFM was included in the reaction mixture (Figure 8B and C). The highest concentration of GST.TEFM tested (50 pM), revealed a >2-fold increase in RNA products of 200 and 400 nt in length, compared to POLRMT transcripts synthesized without TEFM (Figure 8C). In a further test, the promoter independent activity of POLRMT was assayed for 5–20 min on short or long 3′-tailed dsDNA of 20, 100 or 400 bp, with or without recombinant GST.TEFM (Figure 8D–F). In 5 min reactions containing the highest concentration of GST.TEFM the ratio of 400:20 nt product was 75% higher than that of reactions lacking TEFM (Figure 8F). Therefore, POLRMT in concert with TEFM needs less time to make transcripts 400 nt in length than POLRMT alone; i.e. POLRMT processivity is enhanced by TEFM in vitro.Figure 8.

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