TEFM regulates both transcription elongation and RNA processing in mitochondria
2019; Springer Nature; Volume: 20; Issue: 6 Linguagem: Inglês
10.15252/embr.201948101
ISSN1469-3178
AutoresShan Jiang, Camilla Koolmeister, Jelena Misic, Stefan J. Siira, Inge Kühl, Eduardo Silva Ramos, María Miranda, Min Jiang, Viktor Posse, Oleksandr Lytovchenko, Ilian Atanassov, F. Schober, Rolf Wibom, Kjell Hultenby, Dusanka Milenkovic, Claes M. Gustafsson, Aleksandra Filipovska, Nils‐Göran Larsson,
Tópico(s)ATP Synthase and ATPases Research
ResumoArticle29 April 2019Open Access Transparent process TEFM regulates both transcription elongation and RNA processing in mitochondria Shan Jiang Shan Jiang orcid.org/0000-0003-2020-6447 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Camilla Koolmeister Camilla Koolmeister Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Jelena Misic Jelena Misic Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Stefan Siira Stefan Siira Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Inge Kühl Inge Kühl orcid.org/0000-0003-4797-0859 Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Institute of Integrative Biology of the Cell, UMR9198, CEA, CNRS, University Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France Search for more papers by this author Eduardo Silva Ramos Eduardo Silva Ramos Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Maria Miranda Maria Miranda orcid.org/0000-0002-0817-553X Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Min Jiang Min Jiang Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Viktor Posse Viktor Posse Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Oleksandr Lytovchenko Oleksandr Lytovchenko Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Ilian Atanassov Ilian Atanassov orcid.org/0000-0001-8259-2545 Proteomics Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Florian A Schober Florian A Schober Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Rolf Wibom Rolf Wibom Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Centre for Inherited Metabolic Diseases, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Kjell Hultenby Kjell Hultenby Division of Clinical Research Centre, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Dusanka Milenkovic Dusanka Milenkovic Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Claes M Gustafsson Claes M Gustafsson Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Aleksandra Filipovska Aleksandra Filipovska orcid.org/0000-0002-6998-8403 Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Nils-Göran Larsson Corresponding Author Nils-Göran Larsson [email protected] orcid.org/0000-0001-5100-996X Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Centre for Inherited Metabolic Diseases, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Shan Jiang Shan Jiang orcid.org/0000-0003-2020-6447 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Camilla Koolmeister Camilla Koolmeister Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Jelena Misic Jelena Misic Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Stefan Siira Stefan Siira Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Inge Kühl Inge Kühl orcid.org/0000-0003-4797-0859 Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Institute of Integrative Biology of the Cell, UMR9198, CEA, CNRS, University Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France Search for more papers by this author Eduardo Silva Ramos Eduardo Silva Ramos Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Maria Miranda Maria Miranda orcid.org/0000-0002-0817-553X Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Min Jiang Min Jiang Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Viktor Posse Viktor Posse Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Oleksandr Lytovchenko Oleksandr Lytovchenko Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Ilian Atanassov Ilian Atanassov orcid.org/0000-0001-8259-2545 Proteomics Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Florian A Schober Florian A Schober Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Rolf Wibom Rolf Wibom Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Centre for Inherited Metabolic Diseases, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Kjell Hultenby Kjell Hultenby Division of Clinical Research Centre, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Dusanka Milenkovic Dusanka Milenkovic Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Claes M Gustafsson Claes M Gustafsson Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Aleksandra Filipovska Aleksandra Filipovska orcid.org/0000-0002-6998-8403 Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, Perth, WA, Australia Search for more papers by this author Nils-Göran Larsson Corresponding Author Nils-Göran Larsson [email protected] orcid.org/0000-0001-5100-996X Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany Centre for Inherited Metabolic Diseases, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Author Information Shan Jiang1,2, Camilla Koolmeister1,2, Jelena Misic1,2, Stefan Siira3, Inge Kühl4,5, Eduardo Silva Ramos4, Maria Miranda4, Min Jiang4, Viktor Posse6, Oleksandr Lytovchenko1,2, Ilian Atanassov7, Florian A Schober2,8, Rolf Wibom1,9, Kjell Hultenby10, Dusanka Milenkovic4, Claes M Gustafsson6, Aleksandra Filipovska3 and Nils-Göran Larsson *,1,2,4,9 1Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden 2Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory, Karolinska Institutet, Stockholm, Sweden 3Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, Perth, WA, Australia 4Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany 5Institute of Integrative Biology of the Cell, UMR9198, CEA, CNRS, University Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France 6Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden 7Proteomics Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany 8Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden 9Centre for Inherited Metabolic Diseases, Karolinska University Hospital, Stockholm, Sweden 10Division of Clinical Research Centre, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden *Corresponding author. Tel: +46 8 524 83036; E-mail: [email protected] EMBO Reports (2019)20:e48101https://doi.org/10.15252/embr.201948101 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Regulation of replication and expression of mitochondrial DNA (mtDNA) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor (TEFM) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome-length transcription for mtDNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter-distal mitochondrial transcripts are drastically reduced in conditional Tefm-knockout hearts. In contrast, the promoter-proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mtDNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity-labeling (BioID) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria. Synopsis The mitochondrial transcription elongation factor TEFM provides primers for mtDNA replication and is required for mtDNA transcription and also for RNA processing. TEFM is essential for mouse embryonic development. TEFM is necessary for gene expression and replication of mtDNA. Loss of TEFM impairs RNA processing. Introduction Mitochondria harbor the oxidative phosphorylation (OXPHOS) system, which performs cellular energy conversion resulting in the production of adenosine triphosphate (ATP) 1, 2. Deficient OXPHOS is a well-established primary cause of mitochondrial disease and is also secondarily implicated in a variety of pathophysiological conditions, such as neurological disease, age-related diseases, and aging 3. The regulation of OXPHOS is complex because subunits of the respiratory chain and the ATP synthase are encoded by both the nuclear genome and mtDNA 4, 5. Mammalian mtDNA is a gene-dense, circular double-stranded DNA molecule, and the two strands are defined as the heavy (H) and the light (L) strand according to their base composition 6. The mtDNA only encodes 13 of the ∼90 proteins present in the OXPHOS system, but all of these are essential subunits 4, 5. In addition, mtDNA encodes 2 ribosomal RNAs (mt-rRNAs) and 22 transfer RNAs (mt-tRNAs), which are necessary for translation of mitochondrial mRNAs (mt-mRNAs). The compact mtDNA only contains one longer noncoding region named the displacement loop (D-loop), where the origin of replication of the H strand (OH) and the promoters for transcription of H and L strands (HSP and LSP) are located 7, 8. Transcription of mtDNA initiates from HSP and LSP and generates two, near genomic-length polycistronic precursor RNAs, which sequentially go through processing to yield individual mitochondrial RNAs (mtRNAs) 5. Transcription initiation has been extensively studied using in vitro and in vivo models, showing that the mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factors A (TFAM) and B2 (TFB2M) are core components in this process 9-11. In brief, TFAM unwinds the promoter region of double-stranded DNA (dsDNA) and introduces a transcription bubble covering the initiation site. This loose structure is essential for the recruitment of POLRMT and enables TFB2M binding, which completes the assembly of the initiation complex 12, 13. However, once POLRMT successfully transcribes the promoter region, TFB2M is released from the initiation complex 14, 15. Thus, TFB2M is necessary for transcription initiation, but not for elongation. Recent studies have also identified and characterized TEFM as another component of the mitochondrial transcription machinery that interacts with POLRMT and promotes transcription processivity to enable near genome-length transcription 16, 17. The structures of the transcription initiation 18 and elongation complexes 19 have recently been determined. Binding of TEFM to POLRMT allows the complex to form a "sliding clamp" around the DNA, which facilitates high processivity of transcription 19. TEFM also enhances POLRMT transcription by reducing the duration and frequencies of long-lived transcription pauses 20. Transcription initiated from LSP also supplies primers for the replication of mtDNA. Using a recombinant in vitro transcription system, previous studies have suggested that a large proportion of transcription events initiated at LSP are prematurely terminated at the conserved sequence block II (CSBII) within the D-loop region 21. This termination occurs because of the formation of a G-quadruplex structure between nascent RNA and the non-template strand of mtDNA and has been proposed to be linked to primer formation for initiation of H-strand DNA replication 22, 23. The newly transcribed RNA remains associated with the CSB region, where it forms an R-loop that is resistant to treatment by RNase A and RNase T1 24-26. Interestingly, TEFM has been reported to regulate the generation of replication primers in human mitochondria 27 by helping POLRMT to bypass the highly structured CSBII region 19, 20 and abolish R-loop formation 17. Results from an in vitro study have been interpreted to support a model where TEFM serves as a molecular switch that coordinates the balance between mtDNA transcription for replication primer formation and gene expression 27, whereas another in vitro study argues that TEFM is a general unspecific transcription elongator needed for mtDNA gene expression 17. Processing of the newly synthesized polycistronic precursor RNAs to release mature mtRNAs is thought to occur co-transcriptionally and is mainly performed in distinct foci, named mitochondrial RNA granules 28-30. The RNA granules provide an organizational platform for spatiotemporal regulation of mitochondrial RNA processing and maturation 31. The majority of mt-rRNAs and mt-mRNAs are flanked by mt-tRNAs and processing starts with the excision of these flanking mt-tRNAs, according to the widely accepted tRNA punctuation model 32. The endonucleolytic cleavage of the 5′- and 3′-ends of the tRNAs is performed by the mitochondrial RNase P complex, which consists of MRPP1, MRPP2, and MRPP3 33, 34 and the mitochondrial RNase Z (ELAC2) 35, 36, respectively, situated in close proximity to the RNA granules 29, 37. The G-rich RNA sequence binding factor 1 (GRSF1) can melt dsRNA 38 and is localized in RNA granules where it interacts with RNase P 29, 30. Other processing factors are also present in the RNA granules, such as the Fas-activated serine/threonine kinase (FASTK) protein family members FASTK, FASTK2 and FASTKD5 39, 40, the mitochondrial poly(A)-polymerase (mtPAP) 41, methyltransferases 42, RNA helicases, and the degradosome (SUPV3L1-PNPase) complex 43. In this study, we have established the in vivo function of TEFM by generating and characterizing conditional Tefm-knockout mice. We report that TEFM is essential for embryonic survival and that loss of TEFM in the mouse heart causes mitochondrial cardiomyopathy with severe OXPHOS deficiency. Depletion of TEFM drastically reduces the levels of promoter-distal transcripts encoded by both mtDNA strands. In contrast, there is a marked increase in the steady-state levels of promoter-proximal transcripts at both LSP and HSP in the absence of TEFM. At LSP, these short transcripts are prematurely terminated and shorter than the RNA primers needed for initiation of mtDNA replication. Consistently, de novo mtDNA replication is drastically decreased in isolated mitochondria lacking TEFM. Unexpectedly, RNA sequencing (RNA-Seq) and northern blot analyses show that there is an increase of unprocessed mitochondrial transcripts in the absence of TEFM. TEFM proximity-labeling (BioID) assays show that TEFM interacts with diverse RNA processing factors, including the RNase P complex (MRPP1-3), ELAC2, GRSF1, and SUPV3L1-PNPase, which may explain why transcription elongation affects RNA processing. Our results thus show that TEFM is necessary for transcription of mtDNA to generate both short replication primers and near genome-length transcripts for gene expression. Furthermore, we show that loss of TEFM affects both transcription elongation and RNA processing in vivo. Results TEFM is essential for embryonic survival To determine the in vivo function of TEFM, we generated a conditional knockout allele by flanking exon 2 of Tefm with loxP sites (Fig 1A). The targeted allele was transmitted through the germline, and heterozygous mice (Tefm+/loxP-puro) were mated to mice expressing flp-recombinase to excise the puromycin (puro) cassette to obtain mice that were heterozygous for the loxP-flanked Tefm allele (Tefm+/loxP). Heterozygous knockout mice (Tefm+/−) were obtained by breeding Tefm+/loxP mice to mice ubiquitously expressing cre-recombinase (+/β-actin-cre) (Fig 1A). Intercrossing of Tefm+/− mice produced no viable homozygous knockout (Tefm−/−) mice (analyzed offspring, n = 94; Tefm+/+, n = 34, Tefm+/−, n = 60 and Tefm−/−, n = 0), demonstrating that loss of Tefm results in embryonic lethality. Next, we performed an intercross of Tefm+/− mice and analyzed staged embryos at embryonic day 8.5 (E8.5; analyzed embryos, n = 43). All embryos with the Tefm−/− genotype (n = 11) were small and lacked heart structure, while embryos with the Tefm+/+ (n = 10) or Tefm+/− (n = 22) genotype were well developed with normal appearance typical of E8.5 embryos (Fig 1B). Thus, loss of TEFM causes severe developmental defects and embryonic lethality at E8.5, consistent with the phenotype of other knockout mice with homozygous disruption of genes critical for mtDNA expression and maintenance 11, 44-46. Figure 1. Disruption of Tefm in the germline and heart Targeting strategy for disruption of the Tefm gene. LoxP sites flanking exon 2 of the Tefm gene together with puromycin selection marker (PuroR) were inserted into the mouse genome by homologous recombination. The PuroR cassette was excised by mating with flp-recombinase mice to obtain heterozygous floxed Tefm mice (Tefm+/loxP). Whole-body and tissue-specific Tefm knockout mice were obtained by crossing Tefm+/loxP with different cre-recombinase mice. Morphology of wild-type (Tefm+/+) and Tefm homozygous knockout (Tefm−/−) embryos at embryonic day 8.5 (analyzed embryos, n = 43). Scale bar, 500 μm. Western blot analyses of the TEFM protein level in heart mitochondrial extracts from 8-week-old control (L/L) and tissue-specific Tefm knockout (L/L, cre) mice (n = 12 mice for each group). VDAC was used as loading control. Survival curve for control (L/L; n = 60) and Tefm knockout (L/L, cre; n = 22) mice. Heart weight to body weight ratio in 4- and 8-week-old control (L/L) and tissue-specific Tefm knockout mice (L/L, cre). At 4 weeks: L/L n = 22, L/L, cre n = 15; 8 weeks: L/L n = 41, L/L, cre n = 39. Data information: In (E), data are presented as mean ± SEM. ***P < 0.001; Student's t-test. 4 weeks P = 5.041476e-009, 8 weeks P = 1.096535e-033. Download figure Download PowerPoint Disruption of Tefm in heart leads to cardiomyopathy Next, we disrupted Tefm in heart and skeletal muscle by breeding Tefm+/loxP mice with transgenic mice expressing cre-recombinase from the muscle creatinine kinase promoter (Ckmm-cre). The tissue-specific Tefm knockout mice (TefmloxP/loxP, +/Ckmm-cre), hereafter denoted Tefm knockout mice (L/L, cre), were born at expected Mendelian ratios. The depletion of TEFM in heart was verified at the age of 8 weeks by western blot (Fig 1C) and reverse transcriptase quantitative PCR (RT–qPCR) analyses (Fig EV1A). The Tefm knockout mice had a drastically shortened life span with a maximal longevity of 9 weeks (Fig 1D). We also observed a progressively decreased body weight (Fig EV1B) and enlargement of the heart (Fig EV1C–E) in the tissue-specific Tefm knockout mice, resulting in a significant increase in the heart weight to body weight ratio (Fig 1E). TEFM is thus essential for normal heart function. Click here to expand this figure. Figure EV1. Disruption of Tefm in the germline and heart RT–qPCR analyses of the RNA level of Tefm in 8-week-old control (L/L) and Tefm knockout (L/L, cre) mice (n = 10 mice for each group). Body weight of 4- and 8-week-old control and Tefm knockout mice. At 4 weeks: L/L n = 22, L/L, cre n = 15; 8 weeks: L/L n = 41, L/L, cre n = 39. Heart weight of 4- and 8-week-old control and Tefm knockout mice. At 4 weeks: L/L n = 22, L/L, cre n = 15; 8 weeks: L/L n = 41, L/L, cre n = 39. Representative images of hearts of 8-week-old control and Tefm knockout mice (n = 5 mice for each group). Representative images of hematoxylin and eosin staining showing heart structure and morphology in 8-week-old control and Tefm knockout mice (n = 5 mice for each group). Scale bar, 100 μm. Data information: In (A–C), data are presented as mean ± SEM. ***P < 0.001; Student's t-test. (A) P = 3,666241e-010; (B) 4 weeks P = 0.0541737, 8 weeks P = 1.126271e-016; (C) 4 weeks P = 1.207998e-006, 8 weeks P = 1.305101e-026. Download figure Download PowerPoint Loss of TEFM causes severe mitochondrial dysfunction Transmission electron microscopy analysis of heart tissue from end-stage knockout mice showed disrupted mitochondrial alignment and disorganized cristae, consistent with severe respiratory chain dysfunction (Fig 2A). Quantification of mitochondrial density revealed a 1.5-fold increase of relative mitochondrial mass in Tefm knockout hearts in comparison with controls (Fig EV2A). We also observed an increased ratio of levels of mtDNA relative to nuclear DNA (18S rDNA) as determined by Southern blot (Fig 2B) and quantitative PCR (qPCR) (Fig EV2B) analyses. Further supporting an activation of mitochondrial biogenesis, we also found an increased ratio of VDAC to histone H3 protein levels as determined by western blots (Fig 2B). This increase of mitochondrial biogenesis typically occurs in severely respiratory chain deficient tissues, as exemplified by analysis of tissue-specific Lrpprc knockout and Mterf4 knockout hearts (Fig 2B) 45, 47. The increase of mitochondrial mass likely represents a compensatory biogenesis response typically seen in human patients and mice with severe mitochondrial dysfunction 48-50. Next, we measured the activities of the individual complexes in the respiratory chain 51. We found a drastic decrease in the activity for complexes I, III, and IV in the Tefm knockout mice (Fig 2C). A moderate decrease in complex II activity was also observed in the absence of TEFM (Fig 2C), similar to what has been observed in other models with severe reduction of mtDNA expression 52. Consistently, mitochondrial oxygen consumption assays showed decreased OXPHOS capacity after depletion of TEFM (Fig EV2C and D). Additionally, the expression and the assembly of the respiratory chain complexes were negatively affected as determined by western blot analyses (Fig EV2E) and blue native polyacrylamide gel electrophoresis (BN-PAGE), followed by western blotting (Fig EV2F). Thus, loss of TEFM profoundly impairs OXPHOS, demonstrating that mtDNA transcription elongation is essential for maintaining mitochondrial function. Figure 2. Knockout of Tefm in heart causes mitochondrial dysfunction Transmission electron micrographs of heart tissue in 8-week-old control and Tefm knockout mice (n = 5 mice for each group). Scale bar, 2 μm (upper panel) and 1 μm (lower panel). Southern blot analyses of mtDNA levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (upper 2 blots; n = 6 mice at each time-point for each group). 18S rDNA was used as loading control. DNA isolated from tissue-specific Lrpprc and Mterf4 knockout hearts used as controls. Western blot analyses of VDAC and histone H3 protein levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (bottom 2 blots; n = 6 mice at each time-point for each group). VDAC represents mitochondria loading, and histone H3 was used as a loading control for nucleus DNA. Respiratory chain complex activities were measured spectrophotometrically and normalized to citrate synthase activity in heart mitochondria from 8-week-old control and Tefm knockout mice (n = 5 mice for each group). The analyzed enzyme activities are NADH coenzyme Q reductase (complex I), NADH cytochrome c reductase (complex I/III), succinate dehydrogenase (complex II), and cytochrome c oxidase (complex IV). Barplot of the significantly changed proteins (Benjamini-Hochberg adjusted P < 0.05) in Tefm knockout mice compared to control mice (n = 5 mice for each group), organized by individual OXPHOS complexes. Proteins are organized in alphabetical order based on gene name. Boxplots of the protein intensity (LFQ) of TEFM (top) and POLRMT (bottom). Red dot: mean; blue dots: intensity value for each biological replicate (n = 5 mice for each group). Horizontal lines: median; box represents the interquartile range (the first quartile (bottom) and third quartile (top) of the intensity value of 5 replicates). The whiskers represent the maximum and minimum values excluding outliers that represent values extend beyond 1.5 times of the interquartile range. Western blot analyses of POLRMT protein levels in control and Tefm knockout heart mitochondria at the age of 8 weeks (n = 12 mice for each group). VDAC was used as loading control. Data information: In (C), data are presented as mean ± SEM. ***P < 0.001; Student's t-test. CI P = 4.27443E-06, CI/CIII P = 2.0553E-05, CII P = 2.76012E-05, CII/CIII P = 2.00732E-07, CIV P = 1.72156E-06. In (D), data are presented as mean (log2 [L/L, cre/L,L]) ± 95% confidence interval (CI). In (E), boxplot represents the individual intensity values, the adjusted P-values are calculated by the moderated t-test (limma). adj. P = 0.0001334 (top), adj. P = 2.69E-05 (bottom). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Depletion of TEFM in heart causes mitochondrial dysfunction A. Quantification of the relative mitochondrial mass by electron microscopy analysis of the heart of 8-week-old control and Tefm knockout mice (n = 5 mice for each group). B. Quantitative PCR analyses of the relative mtDNA levels in 4- and 8-week-old control and Tefm knockout mice (n = 8 mice at each time-point for each group). C, D. Oxygen consumption rates measured using an Oroboros oxygen electrode in heart mitochondria isolated from 8-week-old control and Tefm knockout mice (n = 5 mice for each group). Phosphorylating, non-phosphorylating, and uncoupled respiration under carbonyl cyanide 3-chlorophenylhydrazone states were measured using pyruvate, glutamate, and malate or succinate and rotenone as su
Referência(s)