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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 mitochondrial tRNAs in TEFM-depleted cells. (A)–(B) Northern blot analyses of mitochondrial tRNAs transcribed from the HSP (A) or LSP (B) promoter in control cells (untransfected and treated with GFP siRNA) and cells treated with TEFM siRNA for 3 or 6 days. Nuclear 18S rRNA was used as a loading control. (C)–(F) Quantification of steady-state levels of the H-strand (C and D) or L-strand (E and F) mitochondrial tRNAs in cells treated with TEFM siRNA for 3 days (C and E) and 6 days (D and F) analysed by Northern blots. The values of the relative RNA level (tRNA/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 tRNA for siRNA TEFM 1 (square) and 2 (triangle) was plotted in the function of the distance of its 3′-end from the promoters. Dotted line, trend for siRNA GFP control; solid line, trend for siRNA TEFM 1; dashed line, trend for siRNA TEFM 2. n = 3, error bars = 1 SD. The P-values (two-tailed Student’s t-test) for each tRNA calculated for combined values for both TEFM siRNAs for 3 days: F = 0.553, L(UUA/G) = 0.303, K = 0.002, S(AGY) = 0.001, T = 0.002, P = 0.103, S(UCN) = 0.004, Q < 0.001; and for 6 days: F = 0.656, L(UUA/G) = 0.154, K < 0.001, S(AGY) < 0.001, T = 0.002, P = 0.297, S(UCN) = 0.002, Q = 0.004.
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Figure 4: Steady-state levels of mitochondrial tRNAs in TEFM-depleted cells. (A)–(B) Northern blot analyses of mitochondrial tRNAs transcribed from the HSP (A) or LSP (B) promoter in control cells (untransfected and treated with GFP siRNA) and cells treated with TEFM siRNA for 3 or 6 days. Nuclear 18S rRNA was used as a loading control. (C)–(F) Quantification of steady-state levels of the H-strand (C and D) or L-strand (E and F) mitochondrial tRNAs in cells treated with TEFM siRNA for 3 days (C and E) and 6 days (D and F) analysed by Northern blots. The values of the relative RNA level (tRNA/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 tRNA for siRNA TEFM 1 (square) and 2 (triangle) was plotted in the function of the distance of its 3′-end from the promoters. Dotted line, trend for siRNA GFP control; solid line, trend for siRNA TEFM 1; dashed line, trend for siRNA TEFM 2. n = 3, error bars = 1 SD. The P-values (two-tailed Student’s t-test) for each tRNA calculated for combined values for both TEFM siRNAs for 3 days: F = 0.553, L(UUA/G) = 0.303, K = 0.002, S(AGY) = 0.001, T = 0.002, P = 0.103, S(UCN) = 0.004, Q < 0.001; and for 6 days: F = 0.656, L(UUA/G) = 0.154, K < 0.001, S(AGY) < 0.001, T = 0.002, P = 0.297, S(UCN) = 0.002, Q = 0.004.

Mentions: In addition, we measured the abundance of several mitochondrial tRNAs (mt-tRNA) encoded on the H- and L-strands, from cells treated with TEFM-targeted dsRNAs (Figure 4A–B). As with mitochondrial mRNAs and rRNAs, TEFM gene-silencing decreased the steady-state level of promoter-distal tRNAs encoded both on H- and L-strand to a greater extent than promoter-proximal tRNAs (Figure 4C–F). The steady-state levels of mt-tRNAs that map in the last third of the mitochondrial genome (with respect to the promoter) were decreased by ∼90% (e.g. tRNA-SerAGY or tRNA-Thr) in cells treated with TEFM siRNA for 6 days. The effective loss of 90% tRNAs due to pathological mutation has a substantial effect on complex I activity and mitochondrial translation (Dunbar et al., 1996), and so the decrease in mt-tRNAs caused by TEFM siRNA can explain the associated severe decreases in OCR and mitochondrially encoded respiratory chain components (Figure 2A). In vertebrates mitochondria, transcription from the HSP and LSP promoters produces polycistronic precursor RNAs that are processed to yield the individual mRNAs, tRNAs and rRNAs, and so reduced processivity of POLRMT is the most straightforward explanation for the larger decreases in the levels of promoter-distal RNAs than promoter-proximal RNAs. Thus, the data are consistent with the hypothesis of TEFM enhancing transcription processivity of both stands of mtDNA.Figure 4.


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 mitochondrial tRNAs in TEFM-depleted cells. (A)–(B) Northern blot analyses of mitochondrial tRNAs transcribed from the HSP (A) or LSP (B) promoter in control cells (untransfected and treated with GFP siRNA) and cells treated with TEFM siRNA for 3 or 6 days. Nuclear 18S rRNA was used as a loading control. (C)–(F) Quantification of steady-state levels of the H-strand (C and D) or L-strand (E and F) mitochondrial tRNAs in cells treated with TEFM siRNA for 3 days (C and E) and 6 days (D and F) analysed by Northern blots. The values of the relative RNA level (tRNA/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 tRNA for siRNA TEFM 1 (square) and 2 (triangle) was plotted in the function of the distance of its 3′-end from the promoters. Dotted line, trend for siRNA GFP control; solid line, trend for siRNA TEFM 1; dashed line, trend for siRNA TEFM 2. n = 3, error bars = 1 SD. The P-values (two-tailed Student’s t-test) for each tRNA calculated for combined values for both TEFM siRNAs for 3 days: F = 0.553, L(UUA/G) = 0.303, K = 0.002, S(AGY) = 0.001, T = 0.002, P = 0.103, S(UCN) = 0.004, Q < 0.001; and for 6 days: F = 0.656, L(UUA/G) = 0.154, K < 0.001, S(AGY) < 0.001, T = 0.002, P = 0.297, S(UCN) = 0.002, Q = 0.004.
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Figure 4: Steady-state levels of mitochondrial tRNAs in TEFM-depleted cells. (A)–(B) Northern blot analyses of mitochondrial tRNAs transcribed from the HSP (A) or LSP (B) promoter in control cells (untransfected and treated with GFP siRNA) and cells treated with TEFM siRNA for 3 or 6 days. Nuclear 18S rRNA was used as a loading control. (C)–(F) Quantification of steady-state levels of the H-strand (C and D) or L-strand (E and F) mitochondrial tRNAs in cells treated with TEFM siRNA for 3 days (C and E) and 6 days (D and F) analysed by Northern blots. The values of the relative RNA level (tRNA/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 tRNA for siRNA TEFM 1 (square) and 2 (triangle) was plotted in the function of the distance of its 3′-end from the promoters. Dotted line, trend for siRNA GFP control; solid line, trend for siRNA TEFM 1; dashed line, trend for siRNA TEFM 2. n = 3, error bars = 1 SD. The P-values (two-tailed Student’s t-test) for each tRNA calculated for combined values for both TEFM siRNAs for 3 days: F = 0.553, L(UUA/G) = 0.303, K = 0.002, S(AGY) = 0.001, T = 0.002, P = 0.103, S(UCN) = 0.004, Q < 0.001; and for 6 days: F = 0.656, L(UUA/G) = 0.154, K < 0.001, S(AGY) < 0.001, T = 0.002, P = 0.297, S(UCN) = 0.002, Q = 0.004.
Mentions: In addition, we measured the abundance of several mitochondrial tRNAs (mt-tRNA) encoded on the H- and L-strands, from cells treated with TEFM-targeted dsRNAs (Figure 4A–B). As with mitochondrial mRNAs and rRNAs, TEFM gene-silencing decreased the steady-state level of promoter-distal tRNAs encoded both on H- and L-strand to a greater extent than promoter-proximal tRNAs (Figure 4C–F). The steady-state levels of mt-tRNAs that map in the last third of the mitochondrial genome (with respect to the promoter) were decreased by ∼90% (e.g. tRNA-SerAGY or tRNA-Thr) in cells treated with TEFM siRNA for 6 days. The effective loss of 90% tRNAs due to pathological mutation has a substantial effect on complex I activity and mitochondrial translation (Dunbar et al., 1996), and so the decrease in mt-tRNAs caused by TEFM siRNA can explain the associated severe decreases in OCR and mitochondrially encoded respiratory chain components (Figure 2A). In vertebrates mitochondria, transcription from the HSP and LSP promoters produces polycistronic precursor RNAs that are processed to yield the individual mRNAs, tRNAs and rRNAs, and so reduced processivity of POLRMT is the most straightforward explanation for the larger decreases in the levels of promoter-distal RNAs than promoter-proximal RNAs. Thus, the data are consistent with the hypothesis of TEFM enhancing transcription processivity of both stands of mtDNA.Figure 4.

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