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Mitochondrial and Nuclear Genomic Responses to Loss of LRPPRC Expression*

  • Vishal M. Gohil
    Affiliations
    Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts 02114

    Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts 02142

    Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02446
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  • Roland Nilsson
    Affiliations
    Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts 02114

    Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts 02142

    Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02446
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  • Casey A. Belcher-Timme
    Affiliations
    Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts 02114

    Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts 02142

    Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02446
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  • Biao Luo
    Affiliations
    Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts 02142
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  • David E. Root
    Affiliations
    Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts 02142
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  • Vamsi K. Mootha
    Correspondence
    To whom correspondence should be addressed: Center for Human Genetic Research, Massachusetts General Hospital, 185 Cambridge St. CPZN 5-806, Boston, MA 02114. Tel.: 617-643-3096; Fax: 617-643-2335
    Affiliations
    Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts 02114

    Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts 02142

    Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02446
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R01DK081457 (to V. K. M.). This work was also supported by grants from the United Mitochondrial Disease Foundation (to V. M. G.), the Broad Institute Scientific Planning and Allocation of Resources Committee, and the American Diabetes Association.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1–3 and additional references and Figs. 1–4.
Open AccessPublished:March 10, 2010DOI:https://doi.org/10.1074/jbc.M109.098400
      Rapid advances in genotyping and sequencing technology have dramatically accelerated the discovery of genes underlying human disease. Elucidating the function of such genes and understanding their role in pathogenesis, however, remain challenging. Here, we introduce a genomic strategy to characterize such genes functionally, and we apply it to LRPPRC, a poorly studied gene that is mutated in Leigh syndrome, French-Canadian type (LSFC). We utilize RNA interference to engineer an allelic series of cellular models in which LRPPRC has been stably silenced to different levels of knockdown efficiency. We then combine genome-wide expression profiling with gene set enrichment analysis to identify cellular responses that correlate with the loss of LRPPRC. Using this strategy, we discovered a specific role for LRPPRC in the expression of all mitochondrial DNA-encoded mRNAs, but not the rRNAs, providing mechanistic insights into the enzymatic defects observed in the disease. Our analysis shows that nuclear genes encoding mitochondrial proteins are not collectively affected by the loss of LRPPRC. We do observe altered expression of genes related to hexose metabolism, prostaglandin synthesis, and glycosphingolipid biology that may either play an adaptive role in cell survival or contribute to pathogenesis. The combination of genetic perturbation, genomic profiling, and pathway analysis represents a generic strategy for understanding disease pathogenesis.

      Introduction

      In recent years, the discovery of genes underlying human disease has progressed rapidly due to the availability of the human genome sequence as well as methods for rapidly genotyping and sequencing. Genome-wide association studies have yielded a plethora of genes associated with common diseases such as cancer and diabetes (
      • McCarthy M.I.
      • Abecasis G.R.
      • Cardon L.R.
      • Goldstein D.B.
      • Little J.
      • Ioannidis J.P.
      • Hirschhorn J.N.
      ). Integrative genomic approaches made possible by the availability of large scale biological data sets have hastened the discovery of causative genes for rare Mendelian disorders (
      • Giallourakis C.
      • Henson C.
      • Reich M.
      • Xie X.
      • Mootha V.K.
      ). More recent advances in sequencing technologies further promise rapid identification of a multitude of mutant alleles responsible for Mendelian disorders as exemplified by the discovery of DHODH mutations in Miller syndrome (
      • Ng S.B.
      • Buckingham K.J.
      • Lee C.
      • Bigham A.W.
      • Tabor H.K.
      • Dent K.M.
      • Huff C.D.
      • Shannon P.T.
      • Jabs E.W.
      • Nickerson D.A.
      • Shendure J.
      • Bamshad M.J.
      ). Still, pinpointing the exact function of newly discovered genes and the global implications of mutations on cellular function remains extremely challenging. The identification of causative alleles is an important first step in understanding the molecular basis of a disorder; however, determining gene function and characterizing cellular responses to disease-causing mutations are necessary to understand pathogenesis. Although the discovery of disease genes is becoming simpler, the latter step remains a major bottleneck.
      One of the first successful applications of genome-wide association studies was Leigh syndrome, French-Canadian type (LSFC)
      The abbreviations used are: LSFC
      Leigh syndrome, French-Canadian type
      COX
      cytochrome c oxidase
      LRPPRC
      leucine-rich pentatricopeptide repeat motif-containing protein
      mtDNA
      mitochondrial DNA
      RNAi
      RNA interference
      shRNA
      short hairpin RNA
      PGC-1α
      peroxisome proliferator-activated receptor γ coactivator 1α
      GSEA
      gene set enrichment analysis
      PG
      prostaglandin
      qRT-PCR
      quantitative real-time PCR
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2- (hydroxymethyl)propane-1,3-diol
      OCR
      oxygen consumption rate
      ECAR
      extracellular acidification rate
      ES
      enrichment score.
      (
      • Lee N.
      • Daly M.J.
      • Delmonte T.
      • Lander E.S.
      • Xu F.
      • Hudson T.J.
      • Mitchell G.A.
      • Morin C.C.
      • Robinson B.H.
      • Rioux J.D.
      ). LSFC is a rare, monogenic, Mendelian mitochondrial disease that presents as a cytochrome c oxidase (COX) deficiency, common in the Saguenay-Lac-Saint-Jean region of Quebec. Lee et al. (
      • Lee N.
      • Daly M.J.
      • Delmonte T.
      • Lander E.S.
      • Xu F.
      • Hudson T.J.
      • Mitchell G.A.
      • Morin C.C.
      • Robinson B.H.
      • Rioux J.D.
      ) mapped the disease to a 2-cm region on chromosome 2 by performing a genome-wide screen for linkage disequilibrium. A follow-up integrative genomic analysis spotlighted LRPPRC, leucine-rich pentatricopeptide repeat motif-containing protein, a high scoring candidate gene whose product is co-expressed with known mitochondrial genes (
      • Mootha V.K.
      • Lepage P.
      • Miller K.
      • Bunkenborg J.
      • Reich M.
      • Hjerrild M.
      • Delmonte T.
      • Villeneuve A.
      • Sladek R.
      • Xu F.
      • Mitchell G.A.
      • Morin C.
      • Mann M.
      • Hudson T.J.
      • Robinson B.
      • Rioux J.D.
      • Lander E.S.
      ). Subsequent sequencing of LRPPRC identified two different causal mutations (
      • Mootha V.K.
      • Lepage P.
      • Miller K.
      • Bunkenborg J.
      • Reich M.
      • Hjerrild M.
      • Delmonte T.
      • Villeneuve A.
      • Sladek R.
      • Xu F.
      • Mitchell G.A.
      • Morin C.
      • Mann M.
      • Hudson T.J.
      • Robinson B.
      • Rioux J.D.
      • Lander E.S.
      ). LRPPRC encodes a 130-kDa RNA-binding protein (
      • Mili S.
      • Piñol-Roma S.
      ) that localizes primarily to the mitochondria (
      • Mootha V.K.
      • Lepage P.
      • Miller K.
      • Bunkenborg J.
      • Reich M.
      • Hjerrild M.
      • Delmonte T.
      • Villeneuve A.
      • Sladek R.
      • Xu F.
      • Mitchell G.A.
      • Morin C.
      • Mann M.
      • Hudson T.J.
      • Robinson B.
      • Rioux J.D.
      • Lander E.S.
      ,
      • Mili S.
      • Piñol-Roma S.
      ). It belongs to a family of pentatricopeptide repeat proteins common to mitochondria and chloroplast that are particularly abundant in plants (
      • Lurin C.
      • Andrés C.
      • Aubourg S.
      • Bellaoui M.
      • Bitton F.
      • Bruyère C.
      • Caboche M.
      • Debast C.
      • Gualberto J.
      • Hoffmann B.
      • Lecharny A.
      • Le Ret M.
      • Martin-Magniette M.L.
      • Mireau H.
      • Peeters N.
      • Renou J.P.
      • Szurek B.
      • Taconnat L.
      • Small I.
      ). As a class, pentatricopeptide repeat proteins tend to be sequence-specific RNA-binding proteins with direct roles in RNA editing, processing, splicing, and translation (
      • Schmitz-Linneweber C.
      • Small I.
      ).
      The precise molecular function of LRPPRC has remained controversial. Consistent with the predicted function of a pentatricopeptide repeat domain-containing protein, Xu et al. (
      • Xu F.
      • Morin C.
      • Mitchell G.
      • Ackerley C.
      • Robinson B.H.
      ) have shown that LRPPRC is required for the expression of mitochondrial DNA (mtDNA)-encoded COX subunits CO1 and CO3. However, Cooper et al. (
      • Cooper M.P.
      • Qu L.
      • Rohas L.M.
      • Lin J.
      • Yang W.
      • Erdjument-Bromage H.
      • Tempst P.
      • Spiegelman B.M.
      ,
      • Cooper M.P.
      • Uldry M.
      • Kajimura S.
      • Arany Z.
      • Spiegelman B.M.
      ) proposed a role for LRPPRC in transcriptional activation of nuclear genes via its interaction with PGC-1α. The global effect of loss of LRPPRC on cellular metabolic pathways has not been determined, although previous reports have implicated LRPPRC in hepatic glucose homeostasis and brown fat differentiation (
      • Cooper M.P.
      • Qu L.
      • Rohas L.M.
      • Lin J.
      • Yang W.
      • Erdjument-Bromage H.
      • Tempst P.
      • Spiegelman B.M.
      ,
      • Cooper M.P.
      • Uldry M.
      • Kajimura S.
      • Arany Z.
      • Spiegelman B.M.
      ). Like many rare diseases, unraveling the LSFC pathogenesis and determining the function of LRPPRC are hindered by the limited availability of patient samples. Because LSFC is characterized by a loss of function of LRPPRC, gene silencing by RNAi offers a tractable approach to create cellular models of LSFC.
      Here, we use short hairpin RNA (shRNA)-mediated graded knockdown of LRPPRC to construct cellular models of LSFC that recapitulate all of the reported disease phenotypes. To identify mitochondrial and nuclear responses to loss of LRPPRC systematically, we carry out genome-wide expression profiling of these cellular models, followed by gene set enrichment analysis (GSEA) (
      • Mootha V.K.
      • Lindgren C.M.
      • Eriksson K.F.
      • Subramanian A.
      • Sihag S.
      • Lehar J.
      • Puigserver P.
      • Carlsson E.
      • Ridderstråle M.
      • Laurila E.
      • Houstis N.
      • Daly M.J.
      • Patterson N.
      • Mesirov J.P.
      • Golub T.R.
      • Tamayo P.
      • Spiegelman B.
      • Lander E.S.
      • Hirschhorn J.N.
      • Altshuler D.
      • Groop L.C.
      ). We demonstrate that all mtDNA-encoded mRNA transcripts decrease in proportion to the loss of LRPPRC. In contrast, mtDNA-encoded rRNAs are unaffected, suggesting a specific role for LRPPRC in mt-mRNA homeostasis. In addition, we find up-regulation of key enzymes for fructose and mannose metabolism and of genes for prostaglandin (PG) biosynthesis, as well as down-regulation of genes for glycosphingolipid biosynthesis. These pathways could play a compensatory role in the face of mitochondrial dysfunction, or alternatively may contribute to pathogenesis.

      DISCUSSION

      It has been more than 6 years since LRPPRC was identified as a causative gene for LSFC, yet its precise function has remained unclear. Based on the presence of a pentatricopeptide repeat motif, sequence similarity to the yeast PET309 protein, and the mitochondrial localization of LRPPRC, Mootha et al. (
      • Mootha V.K.
      • Lepage P.
      • Miller K.
      • Bunkenborg J.
      • Reich M.
      • Hjerrild M.
      • Delmonte T.
      • Villeneuve A.
      • Sladek R.
      • Xu F.
      • Mitchell G.A.
      • Morin C.
      • Mann M.
      • Hudson T.J.
      • Robinson B.
      • Rioux J.D.
      • Lander E.S.
      ) postulated that this protein may play a role in mRNA processing. Indeed, a subsequent study on LSFC patient fibroblasts showed a selective decrease in mtDNA-encoded CO1 and CO3 transcripts (
      • Xu F.
      • Morin C.
      • Mitchell G.
      • Ackerley C.
      • Robinson B.H.
      ). This finding is consistent with the COX enzyme deficiency in LSFC patients. However, our study shows that LRPPRC is required for the expression of all of the mtDNA-encoded mRNAs and is not specific to COX subunits. This discordance may be due to differences in LRPPRC protein levels, that is, a more severe depletion in our knockdown cellular models compared with a milder decrease in LSFC patients with a point mutation in LRPPRC. Importantly, we have shown that LRPPRC depletion does not alter transcript levels of rRNA (Fig. 3D), suggesting a specific role for LRPPRC in the processing of mitochondrial mRNAs. At present, the exact molecular function of LRPPRC is not known. It is likely that other proteins may interact with LRPPRC to co-stabilize mRNA. In this regard, it is noteworthy that the loss of SLIRP, a RNA-binding protein, results in a remarkably similar pattern of mitochondrial mRNA depletion (
      • Baughman J.M.
      • Nilsson R.
      • Gohil V.M.
      • Arlow D.H.
      • Gauhar Z.
      • Mootha V.K.
      ).
      Mitochondrial disorders are frequently characterized by a variety of clinical features that are not readily linked to the biochemical defect within the respiratory chain. Our engineered LSFC cell lines offer a powerful tool to discover cellular responses that may help us understand pathogenesis. This approach is effective because LRPPRC protein appears to be limiting in cells: a decrease in its level strongly correlates with mitochondrial mRNA expression (Fig. 3A) and cellular energetics (Fig. 1D). The allelic series of LRPPRC knockdown cell lines offers multiple advantages: it cancels the off-target effects of individual shRNAs; it provides an isogenic background that eliminates genotype specific effects; stable silencing allows us to understand the long term adaptations of the cell to the loss of gene function; and it recapitulates the phenotypes observed in mitochondrial disease where mitochondrial respiration is defective. The transcriptional responses to a graded loss of LRPPRC and reduced mitochondrial function may be either dose-dependent or exhibit a threshold behavior. By analyzing gene expression data based on linear (Pearson) correlation with the LRPPRC expression level, the current analysis is focused on dose-dependent responses; however, our data could be reanalyzed with different similarity metrics to identify genes exhibiting nonlinear responses as well. Of the pathways showing a dose-dependent response, we were particularly interested in gene sets that were negatively correlated with LRPPRC expression because these may represent activation of an adaptive cellular response to the loss of mitochondrial energy-generating capacity. We found three such anticorrelated, statistically significant gene sets (Fig. 2B), representing hexose sugar metabolism and PG biosynthesis. The hexose sugar metabolism gene set included key glycolytic genes such as HK1, HK2, and HK3. Previous studies have shown increased expression of genes involved in glycolysis, including HK1, HK2, LDHB, and PFK in mtDNA mutant cells (
      • Heddi A.
      • Stepien G.
      • Benke P.J.
      • Wallace D.C.
      ,
      • Behan A.
      • Doyle S.
      • Farrell M.
      ). The up-regulation of these genes may in part facilitate increased ATP production via glycolysis, a necessary adaptation to the loss of mitochondrial respiratory capacity observed in mitochondrial disorders, including LSFC. Not surprisingly, LSFC cells displayed greater sensitivity to a glycolytic inhibitor (supplemental Fig. 2B). Notably, the gene set comprising hexose sugar metabolism includes genes required for the synthesis of GDP-mannose and GDP-fucose, which provide a carbohydrate backbone for glycoprotein synthesis. The increased expression of genes encoding this pathway may reflect alterations in glycoprotein biosynthesis secondary to mitochondrial dysfunction.
      The concerted up-regulation of genes involved in the PG biosynthetic pathway was surprising and has not been reported previously in mitochondrial respiratory chain disease. However, recent reports link PG biosynthesis to mitochondrial dysfunction. First, Cillero-Pastor et al. (
      • Cillero-Pastor B.
      • Caramés B.
      • Lires-Deán M.
      • Vaamonde-Garcia C.
      • Blanco F.J.
      • López-Armada M.J.
      ) showed that treatment of primary human chondrocytes with the respiratory inhibitors oligomycin or antimycin stimulated PTGS2 expression and PGE2 production. Second, Tomura et al. (
      • Tomura H.
      • Wang J.Q.
      • Liu J.P.
      • Komachi M.
      • Damirin A.
      • Mogi C.
      • Tobo M.
      • Nochi H.
      • Tamoto K.
      • Im D.S.
      • Sato K.
      • Okajima F.
      ) have shown that acidification of culture media results in an increase in PTGS2 expression and a corresponding increase in PGE2 synthesis. The up-regulation of the PG pathway is also consistent with the retrograde signaling hypothesis, whereby mitochondrial dysfunction results in increased cytosolic Ca2+, which stimulates calcineurin and activates NF-κB (
      • Butow R.A.
      • Avadhani N.G.
      ), a key transcriptional activator of PG biosynthetic genes. The up-regulation of PG biosynthetic genes could be adaptive because PGE2 inhibits apoptosis, up-regulates a number of prosurvival pathways, including phosphatidylinositol-3-OH-kinase/protein kinase B and extracellular signal-regulated kinase, and stimulates VEGF expression through activation of hypoxia-inducible factor 1α (
      • Greenhough A.
      • Smartt H.J.
      • Moore A.E.
      • Roberts H.R.
      • Williams A.C.
      • Paraskeva C.
      • Kaidi A.
      ). Thus, PGE2 biosynthesis could play a cytoprotective role in respiratory compromised cells.
      In previous studies, Cooper et al. (
      • Cooper M.P.
      • Qu L.
      • Rohas L.M.
      • Lin J.
      • Yang W.
      • Erdjument-Bromage H.
      • Tempst P.
      • Spiegelman B.M.
      ,
      • Cooper M.P.
      • Uldry M.
      • Kajimura S.
      • Arany Z.
      • Spiegelman B.M.
      ) have shown that LRPPRC interacts with PGC-1α to stimulate the expression of a subset of nuclear-encoded mitochondrial genes in mouse primary hepatocytes and brown fat cells. All shLRPPRC hairpins used in this study are predicted to silence mitochondria-targeted LRPPRC as well as LRPPRC targeted to the nucleus/cytosol. However, our GSEA analysis of LSFC cell lines did not detect any effect on expression of the nuclear-encoded mitochondrial genes that are targets of PGC-1α, including the OXPHOS genes (Fig. 2C). This discrepancy could be due to differences between cell types. Although our data strongly favor a role for LRPPRC in regulating mitochondrial mRNAs, an additional nucleus-specific function cannot be ruled out.
      In summary, we report the construction of stable cellular models of LSFC disease in an isogenic background and the discovery of a specific function of LRPPRC in mt-mRNA homeostasis. In addition, we identify nuclear-encoded pathways that could shed light on mitochondrial disease pathogenesis. Our approach overcomes a number of limitations of previous genomic profiling studies conducted on mitochondrial disease cells, where results were confounded by genetic heterogeneity and tissue diversity of samples (
      • Reinecke F.
      • Smeitink J.A.
      • van der Westhuizen F.H.
      ). Our engineered and validated cellular models of LSFC could serve as a valuable resource for understanding mitochondrial disease pathogenesis as well as for drug screening.

      Acknowledgments

      We thank Eric Shoubridge for providing MCH58 cell lines, Olga Goldberger for technical assistance, Oded Shaham for assistance in designing qRT-PCR assays, Serena Silver and Jennifer Grenier for assistance with RNA interference, and Joshua Baughman and Scott Vafai for valuable discussions and comments on this manuscript.

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