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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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Functional interaction of TEFM with RNA. (A) SDS–PAGE analysis of the affinity purified TEFM complex (lanes 1–3) treated with DNase I (lanes 4–6) lanes or RNase A (lanes 7–9) prior to the loading on the Strep-Tactin column. M, mitochondrial lysate; FT, flow through; E2, elution fraction 2; single asterisk, DNase I; two asterisks, RNase A. (B) Western blotting analysis of the SDS–PAGE gel shown in (C). The blot was incubated with the antibodies indicated to the right of the panel. (C) The co-localization of the HA-tagged version of TEFM with mtDNA and mtRNA analysed by immunofluorescence in A549 cells as described in ‘Materials and Methods’ section. Incorporation of BrU in RNA was visualized in cultured cells with BrU-specific mAbs. Black and white images shown in the top row were pseudo-coloured in red or green as indicated in the top right corner of each image and digitally overlaid; yellow staining is indicative of co-localization.
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Figure 6: Functional interaction of TEFM with RNA. (A) SDS–PAGE analysis of the affinity purified TEFM complex (lanes 1–3) treated with DNase I (lanes 4–6) lanes or RNase A (lanes 7–9) prior to the loading on the Strep-Tactin column. M, mitochondrial lysate; FT, flow through; E2, elution fraction 2; single asterisk, DNase I; two asterisks, RNase A. (B) Western blotting analysis of the SDS–PAGE gel shown in (C). The blot was incubated with the antibodies indicated to the right of the panel. (C) The co-localization of the HA-tagged version of TEFM with mtDNA and mtRNA analysed by immunofluorescence in A549 cells as described in ‘Materials and Methods’ section. Incorporation of BrU in RNA was visualized in cultured cells with BrU-specific mAbs. Black and white images shown in the top row were pseudo-coloured in red or green as indicated in the top right corner of each image and digitally overlaid; yellow staining is indicative of co-localization.

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)

Functional interaction of TEFM with RNA. (A) SDS–PAGE analysis of the affinity purified TEFM complex (lanes 1–3) treated with DNase I (lanes 4–6) lanes or RNase A (lanes 7–9) prior to the loading on the Strep-Tactin column. M, mitochondrial lysate; FT, flow through; E2, elution fraction 2; single asterisk, DNase I; two asterisks, RNase A. (B) Western blotting analysis of the SDS–PAGE gel shown in (C). The blot was incubated with the antibodies indicated to the right of the panel. (C) The co-localization of the HA-tagged version of TEFM with mtDNA and mtRNA analysed by immunofluorescence in A549 cells as described in ‘Materials and Methods’ section. Incorporation of BrU in RNA was visualized in cultured cells with BrU-specific mAbs. Black and white images shown in the top row were pseudo-coloured in red or green as indicated in the top right corner of each image and digitally overlaid; yellow staining is indicative of co-localization.
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Figure 6: Functional interaction of TEFM with RNA. (A) SDS–PAGE analysis of the affinity purified TEFM complex (lanes 1–3) treated with DNase I (lanes 4–6) lanes or RNase A (lanes 7–9) prior to the loading on the Strep-Tactin column. M, mitochondrial lysate; FT, flow through; E2, elution fraction 2; single asterisk, DNase I; two asterisks, RNase A. (B) Western blotting analysis of the SDS–PAGE gel shown in (C). The blot was incubated with the antibodies indicated to the right of the panel. (C) The co-localization of the HA-tagged version of TEFM with mtDNA and mtRNA analysed by immunofluorescence in A549 cells as described in ‘Materials and Methods’ section. Incorporation of BrU in RNA was visualized in cultured cells with BrU-specific mAbs. Black and white images shown in the top row were pseudo-coloured in red or green as indicated in the top right corner of each image and digitally overlaid; yellow staining is indicative of co-localization.
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