Cell Metabolism Short Article Mutations in MTFMT Underlie a Human Disorder of Formylation Causing Impaired Mitochondrial Translation Elena J. Tucker,1,3,15 Steven G. Hershman,4,5,6,15 Caroline Ko¨ hrer,7,15 Casey A. Belcher-Timme,4,5,6 Jinal Patel,6 Olga A. Goldberger,4,5,6 John Christodoulou,8,9,10 Jonathon M. Silberstein,11 Matthew McKenzie,12 Michael T. Ryan,13,14 Alison G. Compton,1 Jacob D. Jaffe,6 Steven A. Carr,6 Sarah E. Calvo,4,5,6 Uttam L. RajBhandary,7 David R. Thorburn,1,2,3,* and Vamsi K. Mootha4,5,6,* 1Murdoch Childrens Research Institute 2Genetic Health Services Victoria Royal Children’s Hospital, Melbourne, VIC 3052, Australia 3Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia 4Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA 5Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA 6Broad Institute, Cambridge, MA 02142, USA 7Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA 8Genetic Metabolic Disorders Research Unit, Children’s Hospital at Westmead, Sydney, NSW 2006, Australia 9Discipline of Paediatrics and Child Health 10Discipline of Genetic Medicine University of Sydney, Sydney, NSW 2006, Australia 11Department of Neurology, Princess Margaret Hospital for Children, Perth, WA 6008, Australia 12Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Melbourne, VIC 3168, Australia 13Department of Biochemistry 14ARC Centre of Excellence for Coherent X-Ray Science La Trobe University, Melbourne, VIC 3086, Australia 15These authors contributed equally to this work *Correspondence: [email protected] (D.R.T.), [email protected] (V.K.M.) DOI 10.1016/j.cmet.2011.07.010 SUMMARY INTRODUCTION The metazoan mitochondrial translation machinery is Of the 90 protein components of the oxidative phosphorylation unusual in having a single tRNAMet that fulfills the dual (OXPHOS) machinery, 13 are encoded by the mitochondrial DNA role of the initiator and elongator tRNAMet. A portion of (mtDNA) and translated within the organelle. Defects in mito- the Met-tRNAMet pool is formylated by mitochondrial chondrial protein synthesis lead to combined OXPHOS defi- methionyl-tRNA formyltransferase (MTFMT) to gen- ciency. Although the mtDNA encodes the ribosomal and transfer erate N-formylmethionine-tRNAMet (fMet-tRNAmet), RNAs, all remaining components of the mitochondrial transla- tional machinery are encoded by nuclear genes and imported which is used for translation initiation; however, the into the organelle. To date, mutations in more than ten different requirement of formylation for initiation in human nuclear genes have been shown to cause defective mitochon- mitochondria is still under debate. Using targeted drial translation in humans. However, molecular diagnosis by sequencing of the mtDNA and nuclear exons encod- sequencing these candidates in patients with defects in mito- ing the mitochondrial proteome (MitoExome), we chondrial translation is far from perfect (Kemp et al., 2011), identified compound heterozygous mutations in underscoring the need to identify additional pathogenic muta- MTFMT in two unrelated children presenting with tions underlying these disorders. Leigh syndrome and combined OXPHOS deficiency. Translation within metazoan mitochondria is reminiscent of Patient fibroblasts exhibit severe defects in mito- the bacterial pathway, initiating with N-formylmethionine (fMet) Met chondrial translation that can be rescued by exoge- (Kozak, 1983). Unlike bacteria, which encode distinct tRNA nous expression of MTFMT. Furthermore, patient molecules for translation initiation and elongation, metazoan mitochondria express a single tRNAMet that fulfills both roles fibroblasts have dramatically reduced fMet-tRNAMet (Anderson et al., 1981). After aminoacylation of tRNAMet, levels and an abnormal formylation profile of mito- a portion of Met-tRNAMet is formylated by mitochondrial chondrially translated COX1. Our findings demon- methionyl-tRNA formyltransferase (MTFMT) to generate fMet- MTFMT Met strate that is critical for efficient human tRNA . The mitochondrial translation initiation factor (IF2mt) mitochondrial translation and reveal a human has high affinity for fMet-tRNAMet, which is recruited to the ribo- disorder of Met-tRNAMet formylation. somal P site to initiate translation (Spencer and Spremulli, 2004). 428 Cell Metabolism 14, 428–434, September 7, 2011 ª2011 Elsevier Inc. Cell Metabolism MTFMT Mutations Impair Mitochondrial Translation 250 Here, we applied targeted exome sequencing to two unrelated A patients with Leigh syndrome and combined OXPHOS defi- Complex I:CS ciency to discover pathogenic mutations in MTFMT. Fibroblasts 200 Met Complex II:CS from these patients have impaired Met-tRNA formylation, )lortnoC %( ytivi %( )lortnoC peptide formylation, and mitochondrial translation. Despite Complex III:CS 150 studies in yeast suggesting that MTFMT is not essential for Complex IV:CS mitochondrial translation (Hughes et al., 2000; Li et al., 2000; Vial et al., 2003), we show that in humans this gene is required 100 t for efficient mitochondrial translation and function. c A 50 RESULTS 0 Mitochondrial Translation Is Impaired in Two Unrelated P1cousin(M) P1(Fb) P2(M)P1(M) P2(Fb) P2(L) Patients with Leigh Syndrome We studied two unrelated patients with Leigh syndrome and combined OXPHOS deficiency (Figure 1A). Clinical summaries for patient 1 (P1) and patient 2 (P2) are provided in the Supple- BC[35S] labelling immunoblot mental Results (available online). Patient fibroblasts had reduced Control P1 P2 kDa synthesis of most mtDNA-encoded proteins as assayed by Control P1 P2 [35S]-methionine labeling in the presence of inhibitors of cyto- 4646 ND5 solic translation (Figure 1B). This correlated with reduced steady COX1 state protein levels as detected by immunoblotting (Figure 1C), COX1 (CIV) and, at least for ND1, was not due to reduced mRNA (Figure S1). 30 Collectively, these data suggest a defect in translation of cytb ND1 (CI) mtDNA-encoded proteins. ND2 25 ND1 COX2 MTFMT COX3 (CIV) MitoExome Sequencing Identifies Mutations COX2 To elucidate the molecular basis of disease in P1 and P2, we 17 ATP6 performed next-generation sequencing of coding exons from SDHA (CII) 1034 nuclear-encoded mitochondrial-associated genes and the mtDNA (collectively termed the ‘‘MitoExome’’). DNA was 464 5 6 captured via an in-solution hybridization method (Gnirke et al., 2009) and sequenced on an Illumina GA-II platform (Bentley ND3 7 et al., 2008). Details are provided in the Supplemental Results and Table S1. ATP8/ We identified 700 single-nucleotide variants (SNVs) and ND4L short insertion or deletion variants (indels) in each patient relative 1 23 to the reference genome, and prioritized those that may underlie a severe, recessive disease (Figure 2A). We first filtered out likely Figure 1. Combined OXPHOS Deficiency Due to a Defect in Mito- benign variants present at a frequency of >0.005 in public data- chondrial Translation bases which left 20 variants in each patient. We then prioritized (A) Biochemical analysis of OXPHOS complexes relative to citrate synthase variants that were predicted to have a deleterious impact on (CS) in fibroblasts (Fb), muscle (M), or liver (L), expressed as a percent of mean protein function (Calvo et al., 2010), leaving 12 variants. from healthy controls. (B) SDS-PAGE analysis of 35S-methionine-labeled mtDNA-encoded proteins Focusing on genes that fit autosomal recessive inheritance, from control and patient fibroblasts. MtDNA-encoded subunits of complex I having either homozygous variants or two different variants in (ND1, ND2, ND3, ND5, ND4L), complex III (cytb), complex IV (COX1, COX2, the same gene, only one candidate gene, MTFMT, remained in COX3), and complex V (ATP6, ATP8) are shown. each patient (Figure 2A). (C) The gel in (B) was immunoblotted with antibodies against mtDNA-encoded We identified three distinct heterozygous variants in our ND1, COX1, COX2 and nuclear-encoded SDHA (complex II; loading control). patients (Figure 2B). Both patients harbor a c.626C / T mutation. The c.626C site is 20 bp upstream of the 30 end of exon 4 and is predicted to eliminate two overlapping exonic splicing enhancers (GTCAAG, TCAAGA) (Fairbrother et al., In contrast, the mitochondrial elongation factor (EF-Tumt) specif- 2002) and to generate an exonic splicing suppressor (GTTGTT) ically recruits Met-tRNAMet to the ribosomal A site to participate (Wang et al., 2004). Skipping of exon 4 results in a frameshift in polypeptide elongation. Synthesized proteins can then be and premature stop codon (p.R181SfsX5). The second mutation deformylated by a mitochondrial peptide deformylase (PDF) in P1 is a nonsense mutation (c.382C / T, p.R128X), while the and demethionylated by a mitochondrial methionyl aminopepti- second mutation in P2 changes a highly conserved serine to dase (MAP1D) (Serero et al., 2003; Walker et al., 2009). leucine in the catalytic core of MTFMT (c.374C / T, p.S125L) Cell Metabolism 14, 428–434, September 7, 2011 ª2011 Elsevier Inc. 429 Cell Metabolism MTFMT Mutations Impair Mitochondrial Translation A (Figure S2). An affected cousin of P1 also carries the c.382C / T P1 P2 and c.626C / T mutations. Total SNVs/Indels 662 801 As predicted by in silico analysis, the shared c.626C / T Rare SNVs/Indels 18 23 mutation caused skipping of exon 4 (Figure 2C). qRT-PCR Likely deleterious SNVs/Indels 13 12 analysis revealed that P1 had only 9% full-length