LRPPRC is necessary for polyadenylation and coordination of translation of mitochondrial mRNAs
2011; Springer Nature; Volume: 31; Issue: 2 Linguagem: Inglês
10.1038/emboj.2011.392
ISSN1460-2075
AutoresBenedetta Ruzzenente, Metodi D. Metodiev, Anna Wredenberg, Ana Bratić, Chan Bae Park, Yolanda Cámara, Dusanka Milenkovic, Volker Zickermann, Rolf Wibom, Kjell Hultenby, Hediye Erdjument‐Bromage, Paul Tempst, Ulrich Brandt, James B. Stewart, Claes M. Gustafsson, Nils‐Göran Larsson,
Tópico(s)Metabolism and Genetic Disorders
ResumoArticle1 November 2011free access LRPPRC is necessary for polyadenylation and coordination of translation of mitochondrial mRNAs Benedetta Ruzzenente Benedetta Ruzzenente Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Department of Biology, University of Padova, Padova, Italy Search for more papers by this author Metodi D Metodiev Metodi D Metodiev Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Anna Wredenberg Anna Wredenberg Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Ana Bratic Ana Bratic Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Chan Bae Park Chan Bae Park Institute for Medical Sciences, Ajou University School of Medicine, Suwon, Korea Search for more papers by this author Yolanda Cámara Yolanda Cámara Unitat de Patologia Mitocondrial, Institut de Recerca, Hospital Universitari Vall d′Hebron, Barcelona, Spain Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain Search for more papers by this author Dusanka Milenkovic Dusanka Milenkovic Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Volker Zickermann Volker Zickermann Molecular Bioenergetics Group, Cluster of Excellence Frankfurt ‘Macromolecular Complexes’, Centre for Membrane Proteomics, Medical School, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Rolf Wibom Rolf Wibom Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Kjell Hultenby Kjell Hultenby Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Hediye Erdjument-Bromage Hediye Erdjument-Bromage Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Paul Tempst Paul Tempst Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Ulrich Brandt Ulrich Brandt Molecular Bioenergetics Group, Cluster of Excellence Frankfurt ‘Macromolecular Complexes’, Centre for Membrane Proteomics, Medical School, Goethe University, Frankfurt am Main, Germany Search for more papers by this author James B Stewart James B Stewart Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Claes M Gustafsson Claes M Gustafsson Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Nils-Göran Larsson Corresponding Author Nils-Göran Larsson Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Benedetta Ruzzenente Benedetta Ruzzenente Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Department of Biology, University of Padova, Padova, Italy Search for more papers by this author Metodi D Metodiev Metodi D Metodiev Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Anna Wredenberg Anna Wredenberg Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Ana Bratic Ana Bratic Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Chan Bae Park Chan Bae Park Institute for Medical Sciences, Ajou University School of Medicine, Suwon, Korea Search for more papers by this author Yolanda Cámara Yolanda Cámara Unitat de Patologia Mitocondrial, Institut de Recerca, Hospital Universitari Vall d′Hebron, Barcelona, Spain Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain Search for more papers by this author Dusanka Milenkovic Dusanka Milenkovic Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Volker Zickermann Volker Zickermann Molecular Bioenergetics Group, Cluster of Excellence Frankfurt ‘Macromolecular Complexes’, Centre for Membrane Proteomics, Medical School, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Rolf Wibom Rolf Wibom Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Kjell Hultenby Kjell Hultenby Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Hediye Erdjument-Bromage Hediye Erdjument-Bromage Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Paul Tempst Paul Tempst Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Ulrich Brandt Ulrich Brandt Molecular Bioenergetics Group, Cluster of Excellence Frankfurt ‘Macromolecular Complexes’, Centre for Membrane Proteomics, Medical School, Goethe University, Frankfurt am Main, Germany Search for more papers by this author James B Stewart James B Stewart Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Search for more papers by this author Claes M Gustafsson Claes M Gustafsson Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Nils-Göran Larsson Corresponding Author Nils-Göran Larsson Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Author Information Benedetta Ruzzenente1,2, Metodi D Metodiev1, Anna Wredenberg1, Ana Bratic1,3, Chan Bae Park4, Yolanda Cámara5,6, Dusanka Milenkovic1, Volker Zickermann7, Rolf Wibom3, Kjell Hultenby3, Hediye Erdjument-Bromage8, Paul Tempst8, Ulrich Brandt7, James B Stewart1, Claes M Gustafsson9 and Nils-Göran Larsson 1,3 1Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Cologne, Germany 2Department of Biology, University of Padova, Padova, Italy 3Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden 4Institute for Medical Sciences, Ajou University School of Medicine, Suwon, Korea 5Unitat de Patologia Mitocondrial, Institut de Recerca, Hospital Universitari Vall d′Hebron, Barcelona, Spain 6Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain 7Molecular Bioenergetics Group, Cluster of Excellence Frankfurt ‘Macromolecular Complexes’, Centre for Membrane Proteomics, Medical School, Goethe University, Frankfurt am Main, Germany 8Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 9Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden *Corresponding author. Department of Mitochondrial Genetics, Max Planck Institute for Biology of Ageing, Gleueler Strasse 50a, 50931 Cologne, Germany. Tel.: +49 221 478 89771; Fax: +49 221 478 97409; E-mail: [email protected] The EMBO Journal (2012)31:443-456https://doi.org/10.1038/emboj.2011.392 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 Regulation of mtDNA expression is critical for maintaining cellular energy homeostasis and may, in principle, occur at many different levels. The leucine-rich pentatricopeptide repeat containing (LRPPRC) protein regulates mitochondrial mRNA stability and an amino-acid substitution of this protein causes the French-Canadian type of Leigh syndrome (LSFC), a neurodegenerative disorder characterized by complex IV deficiency. We have generated conditional Lrpprc knockout mice and show here that the gene is essential for embryonic development. Tissue-specific disruption of Lrpprc in heart causes mitochondrial cardiomyopathy with drastic reduction in steady-state levels of most mitochondrial mRNAs. LRPPRC forms an RNA-dependent protein complex that is necessary for maintaining a pool of non-translated mRNAs in mammalian mitochondria. Loss of LRPPRC does not only decrease mRNA stability, but also leads to loss of mRNA polyadenylation and the appearance of aberrant mitochondrial translation. The translation pattern without the presence of LRPPRC is misregulated with excessive translation of some transcripts and no translation of others. Our findings point to the existence of an elaborate machinery that regulates mammalian mtDNA expression at the post-transcriptional level. Introduction The regulation of mammalian oxidative phosphorylation capacity in response to physiological demands and disease states is complex and requires the concerted action of both nuclear and mtDNA-encoded genes (Scarpulla, 2008). The mtDNA genome only encodes 13 proteins, but these are essential for the oxidative phosphorylation system (Larsson et al, 1998). Reduced mtDNA expression is a well-recognized cause of human mitochondrial disease (Tuppen et al, 2010) and is heavily implicated in age-associated diseases and ageing (Larsson, 2010; Wallace, 2010). Nuclear genes are necessary for maintenance and expression of mtDNA, for example, by controlling mtDNA copy number (Ekstrand et al, 2004), transcription initiation (Falkenberg et al, 2002) and translation (Metodiev et al, 2009; Camara et al, 2011). Control of mtDNA transcription initiation is thought to have a key role in regulation of oxidative phosphorylation capacity. The basal machinery for transcription of mtDNA consists of the nuclear-encoded mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM; Parisi and Clayton, 1991) and mitochondrial transcription factor B2 (TFB2M; Bogenhagen, 1996; Falkenberg et al, 2002), which together are sufficient and necessary for in vitro transcription initiation from mtDNA fragments containing the heavy and light strand promoter (HSP and LSP; Falkenberg et al, 2002). Mitochondrial transcription generates large polycistronic transcripts, which undergo RNA processing to release 13 mRNAs, 2 rRNAs and 22 tRNAs. In the polycistronic transcripts, mRNAs are often flanked by tRNAs and endonucleolytic processing to release tRNAs will therefore also release mRNAs, according to the so-called tRNA punctuation model (Ojala et al, 1981). The enzymatic excision of tRNAs involves two enzymatic activities, that is, RNase P at the 5′ end (Holzmann et al, 2008) and RNase Z suggested to process the 3′ end (Takaku et al, 2003; Dubrovsky et al, 2004). Most mRNAs are subsequently polyadenylated by the mitochondrial polyA polymerase (mtPAP; Tomecki et al, 2004) and polyadenylation is often necessary to generate the stop codon at the 3′ end of the open reading frame encoded by the mRNA. A number of enzymes are involved in rRNA (Metodiev et al, 2009; Camara et al, 2011) and tRNA modification (Nagaike et al, 2001; Suzuki et al, 2011). The function of polyadenylation, besides generating stop codons in some transcripts, is not fully understood. Polyadenylation is implicated in regulation of mitochondrial mRNA stability (Nagaike et al, 2005; Slomovic and Schuster, 2008; Wydro et al, 2010) and a mutation in the mtPAP gene has been reported to cause impaired mitochondrial function and ataxia in humans (Crosby et al, 2010). The mechanism whereby mature mRNAs are recognized by the ribosome for subsequent translation initiation is well characterized in prokaryotes. Most prokaryotic mRNAs have an untranslated region (UTR) upstream of the start codon containing a so-called Shine–Dalgarno (SD) sequence. This SD sequence is complementary to a sequence in the 16S rRNA of the 30S bacterial ribosomal subunit and allows the mRNA start codon to find the correct position at the P site of the ribosome (Shine and Dalgarno, 1974). In yeast mitochondria, mRNA recognition by the ribosome takes advantage of the affinity between the 5′ UTR of the mRNA and transcript-specific translational activators. One such example is PET309, a proposed homologue of leucine-rich pentatricopeptide repeat containing (LRPPRC), which acts as a specific translational activator for the COXI mRNA to promote translation initiation (Tavares-Carreon et al, 2008). Mammalian mitochondrial mRNAs do not have 5′ UTRs and an alternate mechanism must therefore be responsible for mRNA recognition by mammalian ribosomes. The pentatricopeptide repeat (PPR) protein family was first discovered in plants and is characterized by a canonical, often repeated, 35 amino acid motif involved in RNA binding. A surprisingly large number of PPR proteins have been reported in plants, where they are implicated in regulating processing, editing and stability of organelle genome transcripts in chloroplasts and mitochondria (Schmitz-Linneweber and Small, 2008; Zehrmann et al, 2011). Mammals have only seven PPR proteins and while the function of some has been at least partly elucidated (Holzmann et al, 2008; Xu et al, 2008; Davies et al, 2009; Rackham et al, 2009), the molecular mechanisms remain unclear. One of the mammalian PPR proteins, LRPPRC, was first discovered as being highly expressed in hepatoma cancer cell lines (Hou et al, 1994). Subsequent papers have associated LRPPRC with a ribonucleoprotein complex responsible for shuttling mature mRNAs from the nucleus to the cytosol (Mili and Pinol-Roma, 2003). LRPPRC has also been proposed to be a cofactor of the eukaryotic translation initiation factor 4E, which is involved in control of nuclear gene expression by regulating the export of specific mRNAs from the nucleus to the cytosol (Topisirovic et al, 2009). In addition, a nuclear role for LRPPRC has been reported as it has been shown to interact with the co-activator PGC-1α to regulate the expression of nuclear genes involved in mitochondrial biogenesis (Cooper et al, 2006). Recessive mutations of Lrpprc cause the French-Canadian type of Leigh syndrome (LSFC; Mootha et al, 2003), a mitochondrial disease which is characterized by infantile onset of severe neurodegeneration in the brain stem and a profound cytochrome c oxidase deficiency in liver and brain (Merante et al, 1993; Debray et al, 2011). Studies of the subcellular distribution of LRPPRC have demonstrated that it is mainly present in mitochondria (Tsuchiya et al, 2002; Mili and Pinol-Roma, 2003; Xu et al, 2004; Cooper et al, 2006; Sasarman et al, 2010; Sterky et al, 2010). We recently reported that transcription of the Lrpprc gene only seems to produce a single mRNA isoform, which is encoding an LRPPRC protein with a mitochondrial targeting sequence that is cleaved after import to the mitochondrial matrix (Sterky et al, 2010). Results from studies of cell lines indeed suggest that LRPPRC has an intramitochondrial role in regulation of mtDNA expression (Gohil et al, 2010; Sasarman et al, 2010), although the mechanism by which it acts is controversial (Sondheimer et al, 2010). On the one hand, it has been shown that knockdown of LRPPRC in tissue culture cells causes a general decrease in mitochondrial mRNA levels, impaired translation and a general decrease of the respiratory chain complexes (Sasarman et al, 2010). On the other hand, fibroblasts from LSFC patients have a respiratory chain deficiency mainly affecting complex IV. Immunoprecipitation experiments and blue native polyacrylamide gel electrophoresis (BN–PAGE) gel analyses have demonstrated that LRPPRC interacts with the stem-loop interacting RNA binding protein (SLIRP) in an RNA-independent way (Sasarman et al, 2010). SLIRP was initially described as a protein binding the nuclear RNA augmenting co-activation of nuclear receptors, but recent results suggest it is mainly present in mitochondria and has a role in maintaining mitochondrial mRNAs (Baughman et al, 2009). Recent studies have implicated LRPPRC in apoptosis (Michaud et al, 2011) and in autophagocytosis (Xie et al, 2011); however, these effects may be secondary because deficient oxidative phosphorylation is known to increase apoptosis (Wang et al, 2001; Kujoth et al, 2005) and has been reported to induce autophagy (Narendra et al, 2008). We have characterized the in vivo function of LRPPRC by generating and characterizing conditional knockout mice. We report here that LRPPRC is essential for embryonic survival and that loss of LRPPRC in the heart leads to a drastic reduction in steady-state levels of all mitochondrial mRNAs, except ND6. LRPPRC forms an RNA-dependent complex with SLIRP, which is necessary for maintaining a pool of non-translated mitochondrial mRNAs. Loss of LRPPRC does not only lead to decreased mRNA stability but also causes loss of mRNA polyadenylation and the appearance of a misregulated mitochondrial translation pattern. Thus, LRPPRC has important roles in post-transcriptional regulation of mtDNA expression and is an essential regulator of oxidative phosphorylation capacity in mammals. Results LRPPRC is essential for embryonic development in the mouse To determine the in vivo function of LRPPRC in mammals, we generated a conditional knockout allele of the mouse Lrpprc gene (Figure 1A and B). Lrpprc was targeted in embryonic stem (ES) cells and the mutated locus was transmitted through the germline to obtain heterozygous Lrpprc+/loxP−neo animals, which in turn, were crossed with transgenic mice expressing the Flp recombinase to excise the neomycin cassette (Figure 1A). The resulting Lrpprc+/loxP mice were mated to mice expressing cre recombinase under the control of the β-actin promoter to generate heterozygous Lrpprc knockout mice (Lrpprc+/−). Intercrossing of Lrpprc+/− mice produced no viable homozygous knockouts (Lrpprc−/−), whereas the other genotypes were recovered at expected Mendelian ratios (genotyped pups n=111; Lrpprc+/+ n=41, Lrpprc+/− n=70). We proceeded to dissect staged embryos derived from intercrossing of Lrpprc+/− mice. We analysed embryos at embryonic day (E) 8.5 and found ∼25% embryos with a mutant appearance (Figure 1C). Genotyping of these embryos (n=38) showed that all mutant embryos were homozygous knockouts (Lrpprc−/−, n=11) whereas the remaining normally appearing embryos had other genotypes (Lrpprc+/−, n=17 or Lrpprc +/+, n=10). These results show that loss of LRPPRC causes embryonic lethality at ∼E8.5, which is consistent with results from analyses of other mouse genes that are essential for mtDNA expression, for example, TFAM (Larsson et al, 1998), MTERF3 (Park et al, 2007), TFB1M (Metodiev et al, 2009) and MTERF4 (Camara et al, 2011). Figure 1.Disruption of Lrpprc in the germline and heart. (A) Targeting strategy for the conditional disruption of the Lrpprc gene. Probe and restriction sites used for the screening of the ES cells are shown in the picture. (B) Southern blot analysis of genomic DNA digested with KpnI in control (C) and targeted (T) ES cells. *Crossreacting band. (C) Morphology of wild-type (Lrpprc+/+) and homozygous knockout (Lrpprc−/−) embryos at day E8.5. Scale bar, 0.5 mm. (D) Heart weight to body weight ratio in control (L/L) and knockout mice (L/L, cre) at different ages. At 4 weeks L/L n=5, L/L, cre n=5; at 8 weeks L/L n=8, L/L, cre n=8; at 12 weeks L/L n=6, L/L, cre n=12. Error bars indicate s.e.m.; **P<0.01; ***P<0.001, Student's t-test. (E) Western blot analysis of LRPPRC and SLIRP levels in heart mitochondrial extracts from control (L/L) and knockout mice (L/L, cre) at different ages. VDAC was used as a loading control. *Crossreacting band. Download figure Download PowerPoint Next, we performed a genetic rescue experiment by introducing a bacterial artificial chromosome (BAC) clone encoding LRPPRC with a C-terminal Flag tag (Supplementary Figure S1A). This clone was modified by introduction of a silent mutation, which eliminated a BglII site in exon 3, thereby enabling the discrimination of LRPPRC mRNAs expressed from the transgene or the endogenous gene (Supplementary Figure S1B). We obtained viable and normally appearing Lrpprc germline knockout mice containing the BAC clone (genotype Lrpprc−/−, +/BAC-LRPPRC–Flag) (Supplementary Figure S1B). The homozygous Lrpprc knockout can, thus, be rescued by the BAC transgene, showing that the knockout of the Lrpprc gene is causing the observed embryonic lethality. Tissue-specific disruption of Lrpprc in heart causes mitochondrial cardiomyopathy We proceeded to cross Lrpprc+/loxP mice with transgenic mice expressing cre recombinase under the control of muscle creatinine kinase promoter (Ckmm-cre), in order to generate mice with tissue-specific knockout of Lrpprc in heart and skeletal muscle. These conditional knockout mice had a drastically shortened lifespan and all of them died before 16 weeks of age (Supplementary Figure S1C). We calculated the ratio of the heart weight to body weight at different ages and found a progressive enlargement of the heart in the knockouts (Figure 1D). Western blot analyses of heart mitochondria showed that the LRPPRC protein was present at very low levels in 4-week-old heart knockouts and it could thereafter not be detected (Figure 1E). We found an accompanying absence of the SLIRP protein in Lrpprc heart knockouts at all investigated ages (Figure 1E), despite normal SLIRP mRNA levels (Supplementary Figure S1D). These results are consistent with reports by others that LRPPRC and SLIRP form a complex (Sasarman et al, 2010), and additionally show that the stability of the SLIRP protein depends on the presence of the LRPPRC protein. Loss of LRPPRC in heart causes severe cytochrome c oxidase deficiency Electron micrographs of knockout heart tissue showed a progressive increase of mitochondrial mass and the presence of mitochondria with abnormally appearing cristae (Figure 2A and B). Consistently, we found increased enzyme activities of citrate synthase and glutamate dehydrogenase, two matrix proteins often used as markers for mitochondrial mass (Supplementary Figure S2A). The levels of mtDNA were normal in Lrpprc knockout hearts (Supplementary Figure S2B), demonstrating that LRPPRC is dispensable for mtDNA maintenance, despite being implicated as a component of the mitochondrial nucleoid (Bogenhagen et al, 2008). Measurement of respiratory chain function showed a profound reduction in complex IV activity in Lrpprc knockout hearts, whereas the activities of the other complexes were unaffected or showed a moderate decrease (Figure 2C). Also the nucleus-encoded complex II showed moderately decreased enzyme activity in end-stage knockout animals and this is likely a secondary phenomenon. We have previously seen a similar reduction of complex II activity in other mouse knockouts with disrupted mtDNA expression (Park et al, 2007; Metodiev et al, 2009). Complex II is dependent of FeS clusters for its function and superoxide-induced damage or impaired synthesis of FeS clusters caused by the deficient oxidative phosphorylation may provide an explanation for the observed enzyme deficiency. Figure 2.Loss of LRPPRC in heart causes mitochondrial dysfunction. (A) Electron micrographs of heart tissue from 12-week-old control (L/L) and knockout mice (L/L, cre). Scale bar, 1 μm. (B) Quantification of the relative mitochondrial mass obtained by electron microscopy analysis of control (L/L) and Lrpprc knockout (L/L, cre) hearts at 4 weeks (L/L n=2, L/L, cre n=2), 8 weeks (L/L n=3, L/L, cre n=3) and 12 weeks (L/L n=5, L/L, cre n=5). Error bars indicate s.e.m.; **P<0.01; ***P<0.001, Student's t-test. (C) Relative activities of respiratory chain enzyme complexes in heart mitochondria from control (L/L) and Lrpprc knockout mice (L/L, cre) at 4, 8 and 12 weeks of age. The analysed enzyme activities are NADH cytochrome c oxidoreductase (complex I/III), succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV). All activities are referred to milligrams of mitochondrial protein. The number of analysed animals at each time point were L/L (n=3) and L/L, cre (n=3). Error bars indicate s.e.m.; *P<0.05, **P<0.01, ***P<0.001, Student's t-test. (D) BN–PAGE analysis of the assembled respiratory chain complexes in heart mitochondria from control (L/L) and Lrpprc knockout (L/L, cre) mice at 4 and 12 weeks of age. Immunoblotting was performed to detect nuclear-encoded subunits of complex I (NDUFA9), complex II (SDHA), complex III (UQCRC2), complex IV (COXIV) and complex V (ATP5A1). The second panel from top shows a blot first hybridized with antibodies to detect complex III and then rehybridized with antibodies to detect complex V (without prior removal of complex III antibodies). (E) Heart mitochondria from 12-week-old control (L/L; left panel) and Lrpprc knockout mice (L/L, cre; right panel) were solubilized with DDM and separated by 2D BN/SDS–PAGE. Gels were silver stained. Assignment of oxidative phosphorylation complexes: complex I (I), complex V or ATP synthase (V), complex III (III), subcomplexes of ATP synthase (F1), complex IV (IV) and complex II (II). Download figure Download PowerPoint We proceeded to analyse levels of assembled respiratory chain complexes by using BN–PAGE and observed a profound reduction of complex IV and a moderate reduction of ATP synthase (complex V) in Lrpprc knockout hearts from age 4 weeks and onwards (Figure 2D). In 12-week-old knockouts, an antibody against the α-subunit of the F1 portion of complex V detected an abnormal complex which had a size corresponding to an F1 subcomplex (Figure 2D). We have previously reported a similar subcomplex in other mouse knockouts with impaired mtDNA expression (Park et al, 2007; Metodiev et al, 2009; Camara et al, 2011). We performed further characterization of respiratory chain complexes by two-dimensional electrophoresis (BN–PAGE followed by SDS–PAGE) and found profound reduction of complex IV, moderate reduction in complexes I and V, and increased levels of the F1 subcomplex (Figure 2E). The levels of complex I were normal or moderately decreased and the levels of complex III were normal or moderately increased in Lrpprc knockout hearts at age 12 weeks (Figure 2D and E). In conclusion, we report here that the activity of complex IV is much more impaired than the activities of the other complexes in Lrpprc knockout hearts, which is in good agreement with the observation that LFSC patients have a profound complex IV deficiency (Merante et al, 1993; Debray et al, 2011). Knockdown of LRPPRC in cell lines has been reported to cause a generalized respiratory chain deficiency (Sasarman et al, 2010), which suggests that the biochemical phenotype in cell lines is at least partly different from the effects we have observed in differentiated tissues in vivo, perhaps due to the continuous proliferation and glycolytic metabolism of tissue culture cells. LRPPRC regulates mitochondrial mRNA stability We performed northern blot analyses of mitochondrial transcripts in Lrpprc knockout hearts and found a profound decrease of most mRNAs already at 4 weeks of age (Figure 3A–C). The steady-state levels of the COXI, COXII, COXIII and Cytb mRNAs were only 2–20% of the levels in control hearts at age 12 weeks. These data are in agreement with the results obtained by knocking down LRPPRC in cell lines, which have shown decrease in steady-state levels of mRNAs (Cooper et al, 2006; Gohil et al, 2010; Sasarman et al, 2010). Interestingly, the levels of ND6, the only L strand-encoded mRNA, were not affected in the knockout hearts, demonstrating that LRPPRC is not important for stability of all mRNA species. In addition, we found that the levels of 12S rRNA and 16S rRNA were increased in the Lrpprc knockout hearts. We observed no significant change in the steady-state levels of a precursor transcript containing 16S and ND1 (RNA19; Supplementary Figure S3), suggesting that RNA processing is normal in the absence of LRPPRC. Figure 3.Steady-state levels of mitochondrial mRNAs, rRNAs and tRNAs in the absence of LRPPRC. (A) Linear map of mouse mtDNA indicating the relative position of each transcript analysed by northern blot. (B) Northern blot analysis of RNA isolated from heart tissues of control (L/L) and knockout (L/L, cre) mice at 12 weeks of age. A separate autoradiograph is shown for every analysed transcript. Nuclear ribosomal RNA (18S) was used as a loading control. (C) Quantification of steady-state levels of the transcripts from control (L/L) and knockout (L/L, cre) mice at different ages. At 4 weeks n=5, at 8 weeks n=5 and at 12 weeks n=4. Error bars indicate s.e.m.; *P<0.05, **P<0.01, ***P<0.001, Student's t-test. (D) Northern blot analysis of RNA isolated from heart tissue of control (L/L) and knockout (L/L, cre) mice at 12 weeks of age. Nuclear ribosomal RNA (18S) was used as a loading control. (E) Quantification of steady-state levels of mitochondrial tRNAs from control (L/L) and knockout (L/L, cre) mice at different ages. At 4 weeks n=5 and at 12 weeks n=5. Error bars indicate s.e.m.; *P<0.05, Student's t-test.
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