Limits...
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.

Show MeSH

Related in: MedlinePlus

Mitochondrial RNA polymerase co-purifies with TEFM. (A) A SDS–PAGE gel stained with Coomassie Brilliant Blue showing the protein profile of the affinity purification of the TEFM.STREP2 from the mitochondria of HEK cells. The most intense protein band of ∼40 kDa seen in the elution fractions 2–4 corresponds to the purified TEFM.STREP2 protein. M, total mitochondrial lysate; FT, flow-through; MW, Molecular weight marker. (B) A SDS–PAGE gel stained with Coomassie Brilliant Blue with concentrated fractions from 2 to 5 (E2–5). Protein bands were cut from the gel and analysed by mass spectrometry. The identities of the protein are shown on the left-hand side. Some endogenous mitochondrial biotinylated proteins (e.g. 3-hydroxyacyl-CoA dehydrogenase α-subunit or hydroxyacyl dehydrogenase, subunit B) were detected in the analysis as affinity purification system used here is based on the interaction between the STREP2 tag and engineered streptavidin (Strep-Tactin), which also binds biotin. Only biotinylated proteins were detected in mock affinity capture experiments performed on parental HEK cells (data not shown). The presence of the mitochondrial chaperone protein, MTHSP75, in our preparation could well result from a mitochondrial stress response caused by overexpression of TEFM (41). (C)–(E) Western blots confirming the identity of the proteins that co-purify with TEFM that were detected by mass spectrometry (C) or documenting that there was no enrichment of highly abundant mitochondrial proteins (D) or mitochondrial proteins involved in the initiation of mtDNA transcription (E). (F) A SDS–PAGE gel stained with Coomassie Brilliant Blue showing the protein profile of the affinity purification of the POLRMT.STREP2 from the mitochondria of HEK cells. The most intense protein band of ∼140 kDa seen in the elution fractions 3–4 corresponds to the purified POLRMT.STREP2 protein. M, total mitochondrial lysate; FT, flow-through; MW, Molecular weight marker. (G) Western blots illustrating the protein profile of the affinity purification of the POLRMT.STREP2 from the mitochondria of HEK cells. (H) Relative abundance of proteins that co-purify with POLRMT.STREP2. The values were obtained by quantifying PhosphoImager scans of western blots from (G) in the ImageQuant software and normalized for the values obtained for the total mitochondrial lysate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105396&req=5

Figure 5: Mitochondrial RNA polymerase co-purifies with TEFM. (A) A SDS–PAGE gel stained with Coomassie Brilliant Blue showing the protein profile of the affinity purification of the TEFM.STREP2 from the mitochondria of HEK cells. The most intense protein band of ∼40 kDa seen in the elution fractions 2–4 corresponds to the purified TEFM.STREP2 protein. M, total mitochondrial lysate; FT, flow-through; MW, Molecular weight marker. (B) A SDS–PAGE gel stained with Coomassie Brilliant Blue with concentrated fractions from 2 to 5 (E2–5). Protein bands were cut from the gel and analysed by mass spectrometry. The identities of the protein are shown on the left-hand side. Some endogenous mitochondrial biotinylated proteins (e.g. 3-hydroxyacyl-CoA dehydrogenase α-subunit or hydroxyacyl dehydrogenase, subunit B) were detected in the analysis as affinity purification system used here is based on the interaction between the STREP2 tag and engineered streptavidin (Strep-Tactin), which also binds biotin. Only biotinylated proteins were detected in mock affinity capture experiments performed on parental HEK cells (data not shown). The presence of the mitochondrial chaperone protein, MTHSP75, in our preparation could well result from a mitochondrial stress response caused by overexpression of TEFM (41). (C)–(E) Western blots confirming the identity of the proteins that co-purify with TEFM that were detected by mass spectrometry (C) or documenting that there was no enrichment of highly abundant mitochondrial proteins (D) or mitochondrial proteins involved in the initiation of mtDNA transcription (E). (F) A SDS–PAGE gel stained with Coomassie Brilliant Blue showing the protein profile of the affinity purification of the POLRMT.STREP2 from the mitochondria of HEK cells. The most intense protein band of ∼140 kDa seen in the elution fractions 3–4 corresponds to the purified POLRMT.STREP2 protein. M, total mitochondrial lysate; FT, flow-through; MW, Molecular weight marker. (G) Western blots illustrating the protein profile of the affinity purification of the POLRMT.STREP2 from the mitochondria of HEK cells. (H) Relative abundance of proteins that co-purify with POLRMT.STREP2. The values were obtained by quantifying PhosphoImager scans of western blots from (G) in the ImageQuant software and normalized for the values obtained for the total mitochondrial lysate.

Mentions: In order to identify mitochondrial proteins that interact with TEFM a FLAG- and STREP2-tagged version of TEFM (TEFM.STREP2) was introduced into Flp-In T-Rex™ HEK 293T cells. The STREP2 binding group was used in order to purify TEFM and potential interacting proteins via a streptavidin-coated matrix after induction of the transgene for 24 h with 20 ng/ml of doxycycline (Figure 5A). Streptavidin immobilized proteins from mitochondrial lysates of HEK cells expressing TEFM.STREP2 or control cells were analysed by mass spectrometry. Three mitochondrial proteins were identified consistently in TEFM.STREP2 expressing mitochondrial lysates: mitochondrial RNA polymerase—POLRMT (25), Pentatricopeptide repeat domain 3—PTCD3 (26) and a putative DEAH-box RNA helicase—DHX30 (27) (Figure 5B). The mass spectrometry analysis also revealed a variable set of mitochondrial ribosomal proteins co-purifying with TEFM (Supplementary Table S2).Figure 5.


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)

Mitochondrial RNA polymerase co-purifies with TEFM. (A) A SDS–PAGE gel stained with Coomassie Brilliant Blue showing the protein profile of the affinity purification of the TEFM.STREP2 from the mitochondria of HEK cells. The most intense protein band of ∼40 kDa seen in the elution fractions 2–4 corresponds to the purified TEFM.STREP2 protein. M, total mitochondrial lysate; FT, flow-through; MW, Molecular weight marker. (B) A SDS–PAGE gel stained with Coomassie Brilliant Blue with concentrated fractions from 2 to 5 (E2–5). Protein bands were cut from the gel and analysed by mass spectrometry. The identities of the protein are shown on the left-hand side. Some endogenous mitochondrial biotinylated proteins (e.g. 3-hydroxyacyl-CoA dehydrogenase α-subunit or hydroxyacyl dehydrogenase, subunit B) were detected in the analysis as affinity purification system used here is based on the interaction between the STREP2 tag and engineered streptavidin (Strep-Tactin), which also binds biotin. Only biotinylated proteins were detected in mock affinity capture experiments performed on parental HEK cells (data not shown). The presence of the mitochondrial chaperone protein, MTHSP75, in our preparation could well result from a mitochondrial stress response caused by overexpression of TEFM (41). (C)–(E) Western blots confirming the identity of the proteins that co-purify with TEFM that were detected by mass spectrometry (C) or documenting that there was no enrichment of highly abundant mitochondrial proteins (D) or mitochondrial proteins involved in the initiation of mtDNA transcription (E). (F) A SDS–PAGE gel stained with Coomassie Brilliant Blue showing the protein profile of the affinity purification of the POLRMT.STREP2 from the mitochondria of HEK cells. The most intense protein band of ∼140 kDa seen in the elution fractions 3–4 corresponds to the purified POLRMT.STREP2 protein. M, total mitochondrial lysate; FT, flow-through; MW, Molecular weight marker. (G) Western blots illustrating the protein profile of the affinity purification of the POLRMT.STREP2 from the mitochondria of HEK cells. (H) Relative abundance of proteins that co-purify with POLRMT.STREP2. The values were obtained by quantifying PhosphoImager scans of western blots from (G) in the ImageQuant software and normalized for the values obtained for the total mitochondrial lysate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105396&req=5

Figure 5: Mitochondrial RNA polymerase co-purifies with TEFM. (A) A SDS–PAGE gel stained with Coomassie Brilliant Blue showing the protein profile of the affinity purification of the TEFM.STREP2 from the mitochondria of HEK cells. The most intense protein band of ∼40 kDa seen in the elution fractions 2–4 corresponds to the purified TEFM.STREP2 protein. M, total mitochondrial lysate; FT, flow-through; MW, Molecular weight marker. (B) A SDS–PAGE gel stained with Coomassie Brilliant Blue with concentrated fractions from 2 to 5 (E2–5). Protein bands were cut from the gel and analysed by mass spectrometry. The identities of the protein are shown on the left-hand side. Some endogenous mitochondrial biotinylated proteins (e.g. 3-hydroxyacyl-CoA dehydrogenase α-subunit or hydroxyacyl dehydrogenase, subunit B) were detected in the analysis as affinity purification system used here is based on the interaction between the STREP2 tag and engineered streptavidin (Strep-Tactin), which also binds biotin. Only biotinylated proteins were detected in mock affinity capture experiments performed on parental HEK cells (data not shown). The presence of the mitochondrial chaperone protein, MTHSP75, in our preparation could well result from a mitochondrial stress response caused by overexpression of TEFM (41). (C)–(E) Western blots confirming the identity of the proteins that co-purify with TEFM that were detected by mass spectrometry (C) or documenting that there was no enrichment of highly abundant mitochondrial proteins (D) or mitochondrial proteins involved in the initiation of mtDNA transcription (E). (F) A SDS–PAGE gel stained with Coomassie Brilliant Blue showing the protein profile of the affinity purification of the POLRMT.STREP2 from the mitochondria of HEK cells. The most intense protein band of ∼140 kDa seen in the elution fractions 3–4 corresponds to the purified POLRMT.STREP2 protein. M, total mitochondrial lysate; FT, flow-through; MW, Molecular weight marker. (G) Western blots illustrating the protein profile of the affinity purification of the POLRMT.STREP2 from the mitochondria of HEK cells. (H) Relative abundance of proteins that co-purify with POLRMT.STREP2. The values were obtained by quantifying PhosphoImager scans of western blots from (G) in the ImageQuant software and normalized for the values obtained for the total mitochondrial lysate.
Mentions: In order to identify mitochondrial proteins that interact with TEFM a FLAG- and STREP2-tagged version of TEFM (TEFM.STREP2) was introduced into Flp-In T-Rex™ HEK 293T cells. The STREP2 binding group was used in order to purify TEFM and potential interacting proteins via a streptavidin-coated matrix after induction of the transgene for 24 h with 20 ng/ml of doxycycline (Figure 5A). Streptavidin immobilized proteins from mitochondrial lysates of HEK cells expressing TEFM.STREP2 or control cells were analysed by mass spectrometry. Three mitochondrial proteins were identified consistently in TEFM.STREP2 expressing mitochondrial lysates: mitochondrial RNA polymerase—POLRMT (25), Pentatricopeptide repeat domain 3—PTCD3 (26) and a putative DEAH-box RNA helicase—DHX30 (27) (Figure 5B). The mass spectrometry analysis also revealed a variable set of mitochondrial ribosomal proteins co-purifying with TEFM (Supplementary Table S2).Figure 5.

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