Lost in Translation: Genetic Defects Underlying Combined OXPHOS Complex I, III and IV Deficiencies Thesis Radboud University Nijmegen with a Summary in Dutch

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Paulien Smits Lost in translation: genetic defects underlying combined OXPHOS complex I, III and IV deficiencies Thesis Radboud University Nijmegen with a summary in Dutch

The research presented in this thesis was performed at the Department of Pediatrics, Radboud University Nijmegen Medical Centre, The Netherlands.

ISBN 978-94-6108-189-6

Cover: Representation of the main elements and processes required for the biogenesis of the OXPHOS complexes: nuclear and mitochondrial DNA, proteins synthesized in the cytoplasm (which are components of the OXPHOS system or proteins needed for mitochondrial translation), and mitochondrial translation. At all the different steps the 'translation' can go wrong, with an A being decoded into for instance an a, in the end resulting in an OXPHOS system deficiency. To make sure we will not be lost in translation, the puzzle of combined OXPHOS complex I, III and IV deficiencies needs to be solved.

Cover concept: Jordy van Emden & Paulien Smits Cover design & lay-out: Paul van Hoogstraten

Printed by: Gildeprint Drukkerijen Lo st in translation : genetic defects underlying combined OXPHOS complex I, III and IV deficiencies

Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen

Proefschrift

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het college van decanen in het openbaar te verdedigen op

vrijdag 2 september 2011 om 12.30 uur precies

door

Paulien Smits

geboren op 3 augustus 1981 te Krimpen aan den IJssel Promotoren: Prof. dr. L.P.W.J. van den Heuvel (UZ Leuven) Prof. dr. J.A.M. Smeitink

Manuscriptcommissie: Prof. dr. M.A. Huynen (voorzitter) Prof. dr. G.J.M. Pruijn Prof. dr. N.V.A.M. Knoers-van Slobbe (UMC Utrecht) Geluk is het enige dat zich vermenigvuldigt als je het deelt. Albert Schweitzer

Contents

C o n ten ts

Abbreviations 8

Chapter 1 General introduction and outline of this thesis 11

Chapter 2 Mitochondrial translation and beyond: processes implicated 19 in combined oxidative phosphorylation deficiencies

Chapter 3 Functional consequences of mitochondrial tRNA Trp and 69 tRNA Arg mutations causing combined OXPHOS defects

Chapter 4 Distinct clinical phenotypes associated with a mutation in 87 the mitochondrial translation elongation factor EFTs

Chapter 5 Mutation in subdomain G' of mitochondrial elongation 107 factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle

Chapter 6 Reconstructing the evolution of the mitochondrial 121 ribosomal proteome

Chapter 7 Sequence variants in four candidate genes (NIPSNAP1, 161 GbAs , CHCHD1 and METT11D1) in patients with combined oxidative phosphorylation system deficiencies

Chapter 8 Mutation in mitochondrial ribosomal protein MRPS22 leads 175 to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy

Chapter 9 General discussion and future perspectives 193

Summary 219

Samenvatting 225

Samenvatting voor leken 229

Dankwoord / Acknowledgements 237

Curriculum Vitae 243

List of publications 247 List of abbreviations

List of A bbreviations

ADP adenosine diphosphate ATP adenosine triphosphate BN blue native bp base pair BLAST basic local alignment search tool cDNA complementary DNA CI - CV complex I - complex V CoQ coenzyme Q or ubiquinone CO(X) cytochrome c oxidase CS citrate synthase CSF cerebrospinal fluid cyt cytochrome DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate ECG electrocardiogram EEG electroencephalogram EST expressed sequence tag FCS fetal calf serum gDNA genomic DNA GDP guanosine diphosphate GTP guanosine triphosphate HSP heavy strand promoter / hereditary spastic paraplegia / heat shock protein IGA in-gel activity kDa kilo Dalton LSP light strand promoter LSU large subunit MELAS mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes MERRF myoclonic epilepsy with ragged-red fibers MLASA mitochondrial myopathy and sideroblastic anemia MLPA multiplex ligation-dependent probe amplification MRI magnetic resonance imaging mRNA messenger RNA MRP mitochondrial ribosomal protein mt mitochondrial mtDNA mitochondrial DNA

8 List of abbreviations

NAD(H) nicotinamide adenine dinucleotide (reduced) ND NADH dehydrogenase or NADH CoQ oxidoreductase nDNA nuclear DNA NTP nucleoside triphosphate ORF open reading frame OXPHOS oxidative phosphorylation PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction PSI-BLAST position specific iterative BLAST RNA ribonucleic acid RP ribosomal protein rRNA ribosomal RNA RT-PCR reverse transcriptase PCR SD standard deviation SDS sodium dodecyl sulphate SNP single nucleotide polymorphism SSU small subunit TIM translocase of the inner mitochondrial membrane TOM translocase of the outer mitochondrial membrane tRNA transfer RNA VEP visual evoked potential

9 If you want to see a rainbow, you have got to put up with the rain. Dolly Parton C h apter 1 General introduction and outline of this thesis

H B Chapter 1 Energy is fundamental to life. For all our actions, from running to sleeping, and for the myriad of processes occurring in and outside our cells every second, energy is required. This energy is derived from food, mainly from carbohydrates and fat, and is generated in the form of adenosine triphosphate (ATP) during the following processes: glycolysis (i.e., the breakdown of glucose into pyruvate), the Krebs or citric acid cycle (i.e., breakdown of acetyl coenzyme A, which is synthesized from pyruvate, fatty acids or in lesser amounts from amino acids), and oxidative phosphorylation (Figure 1). Glycolysis takes place in the cell's cytoplasm, also in the absence of oxygen, whereas both the Krebs cycle and oxidative phosphorylation are aerobic processes occurring in the mitochondrion. Oxidative phosphorylation is by far the largest contributor to the ATP supply.

M itochondria Mitochondria, often called the powerhouses of the cell, are semiautonomous organelles present in varying numbers per cell, depending on the tissue type: from zero mitochondria in red blood cells to over a thousand in cells with high energy demands. Mitochondrial morphology ranges from small bean-shaped structures to highly branched tubular networks due to constant remodeling by fusion and fission. Reminiscent of its endosymbiotic origin, i.e., that it evolved from a bacterium that was engulfed by a host cell over a billion years ago, the mitochondrion is surrounded by a double membrane and has its own, circular genome. The outer mitochondrial membrane separates the cytosol of the cell from the intermembrane space, and the highly invaginated inner membrane envelops the mitochondrial matrix, which contains the mitochondrial genome and enzymes of the Krebs cycle, among others. Most genes from the prokaryotic genome have been lost or have been transferred to the nucleus of the eukaryotic cell during evolution. In human cells, nearly all genes that are required for mitochondrial functioning reside in the nuclear genome (nDNA); mitochondrial DNA (mtDNA) merely contains 37 genes, coding for rRNAs, tRNAs and several constituents of the oxidative phosphorylation (OXPHOS) system.

OXPHOS SYSTEM The respiratory or electron transport chain is composed of four enzymatic complexes and two electron carriers, ubiquinone and cytochrome c, and together with a fifth complex it makes up the OXPHOS system (Figure 1).1 The five complexes of the OXPHOS system are multiprotein structures, consisting of 89 proteins in total, and constitute the only metabolic pathway that is under dual genetic control. The OXPHOS system is embedded in the inner mitochondrial membrane and executes the final biochemical steps in energy production. NADH

12 General introduction and outline of this thesis and succinate, produced during glycolysis, fatty acid oxidation and the Krebs cycle, are oxidized by complex I and complex II, respectively, of the OXPHOS system. The hereby liberated electrons are transferred via the electron carriers and complex III to complex IV, where they are delivered to the final electron acceptor oxygen, reducing it to water. The energy released during electron transport is utilized by complexes I, III and IV to translocate protons across the mitochondrial inner membrane, generating an electrochemical proton gradient. This gradient serves as the driving force for the backflow of protons from the intermembrane space to the matrix compartment via complex V, ATP synthase. Complex V harnesses the free energy to phosphorylate ADP, using inorganic phosphate (Pi), to form ATP.

CYTOSOL

I Compiei; 1 1 Il iv] v| Carbohydrates Lactate I Compte» I [Compta | Compia*

Figure 1. The mitochondrial oxidative phosphorylation system. Schematic representation of oxidative phosphorylation and other metabolic processes, adapted from ref. 2. Thick arrows represent the direction of electron movement and dotted arrows indicate the proton flux. For each OXPHOS complex, the number of subunits encoded by the nDNA or mtDNA is depicted at the top. The import complexes needed for translocation of nDNA-encoded proteins across the outer and inner mitochondrial membranes are not shown. Abbreviations: OMM, outer mitochondrial membrane; IS, intermembrane space; IMM, inner mitochondrial membrane; CI-V, complex I-V; Ub, ubiquinone; CytC, cytochrome c.

M itochondrial d is o r d e r s Due to the mitochondrion's central role in cell metabolism, impairment in mitochondrial function is associated with a wide range of human disorders. The term 'mitochondrial disorder' generally refers to diseases resulting from defects in the OXPHOS system, which constitute the majority of disorders caused

13 Ü Chapter 1 by mitochondrial dysfunction. Since the first description of a patient suffering from a mitochondrial disorder by Luft et al in 1962,3 mitochondrial disorders have turned out to be the most frequent group of inborn errors of metabolism with an estimated incidence of 1 in 5000 live births.4 Because of the complexity of the OXPHOS system - i.e., its bigenomic control, its structural organization and difference herein between tissues - and its intricate regulation, defects in a multitude of structures involved in OXPHOS system functioning can lead to disorders with extremely heterogeneous clinical manifestations.5,6 Mitochondrial diseases vary in their age of onset (from birth to adulthood), course (from static to rapidly progressive), and number and type of tissues affected (from any single organ to multisystemic). Typically, multiple tissues with high energy demands such as brain, skeletal muscle and heart are involved and although symptoms can be mild, children commonly show a severe and progressive phenotype that often leads to early death. Furthermore, similar clinical manifestations can be caused by distinct genetic and biochemical defects and, conversely, the exact same mutation or OXPHOS disturbance can result in different symptomatology in different patients. The myriad of phenotypes and the fact that no single clinical feature is specific complicates clinical diagnosis of mitochondrial disorders. Also treatment is a challenge since no cure has been found as yet, except for specific cases such as ubiquinone (coenzyme Q) supplementation in ubiquinone biosynthesis defects.7,8 Until research into new therapeutic strategies - some of them quite promising - bear fruit, symptom relief is the only hope for the majority of patients. Fortunately, various symptomatic treatment options are available and can be very effective. Preventing mitochondrial disorders through genetic counseling and prenatal diagnosis is particularly important given the lack of a cure. Due to the dual genetic origin (nDNA and mtDNA) of the OXPHOS system, autosomal recessive and dominant, X-linked and maternal modes of inheritance can occur, in addition to sporadic (de novo) mutations. Prenatal diagnosis is largely based on biochemical measurements, which are reliable in only a limited number of cases.9 With the progress in our understanding of the molecular bases of mitochondrial disease, more specific and accurate prenatal diagnosis at the molecular level should become increasingly feasible.

O b j e c t iv e s , a p p r o a c h a n d o u t l in e o f t h is t h e s is Despite substantial advances in elucidation of the genes and pathogenic mechanisms responsible for mitochondrial disorders,10,11 in the majority of patients the genetic defect cannot be identified. Consequently, further research into the genetic origin of mitochondrial disease is called for. Biochemically, mitochondrial defects are classified based on the enzyme(s) affected and whether one or more OXPHOS complexes are involved - isolated or combined deficiencies, respectively.

14 General introduction and outline of this thesis

Nearly 30% of all OXPHOS deficiencies in fresh muscle biopsies analyzed at the Nijmegen Center for Mitochondrial Disorders consist of combined deficiencies.12 Whenever de activities of multiple enzymes are diminished, the molecular cause is not likely to be found in genes encoding building blocks of the OXPHOS system. In these cases, the defect is suspected to affect processes indirectly involved in the formation of the OXPHOS system: mtDNA maintenance, mitochondrial transcription or translation including posttranscriptional or -translational processes, transport of proteins across mitochondrial membranes, mitochondrial membrane biogenesis, and assembly of the OXPHOS system. The main objective of the research presented in this thesis was to expand our knowledge of the underlying molecular causes of combined OXPHOS system defects. We focused our investigations on deficiencies of complexes I, III and IV, aiming to find novel nuclear gene defects. To date, numerous mutations associated with combined OXPHOS deficiencies have been reported, most in mtDNA but the number of nDNA mutations is increasing rapidly, yet many cases remain unresolved. Gaining more insight into the localization of the genetic defects in such patients will not only contribute to a thorough understanding of the OXPHOS system and processes related to its functioning, but will also improve possibilities of pre- and postnatal diagnosis and hopefully therapy in the future. The general approach that was used in the present study is the following. First of all, patients with decreased enzyme activities of the complexes I, III and IV in fibroblasts and/or muscle tissue were included in the study. Normal complex II activity was a prerequisite for selection. This is because our research was primarily directed toward discovering molecular causes of mitochondrial translation defects and complex II, being composed of subunits encoded solely by the nuclear genome, should not be affected by impairments in mitochondrial translation. The activity of complex V was often not known and therefore not included as a criterion. Secondly, mutations in the mtDNA were excluded by sequencing the entire mitochondrial genome in fibroblast DNA of the 33 patients who were selected. Subsequently, nuclear genes were screened for mutations: 1) genes known to be implicated in combined complex I, III and IV deficiencies, namely genes coding for polymerase gamma, mitochondrial translation factors mtEFTs, mtEFTu and mtEFG1, and mitochondrial ribosomal proteins (MRPs) MRPS16 and MRPS22; 2) candidate genes for combined OXPHOS defects, namely genes encoding the 6 remaining mitochondrial translation factors, 7 more MRPs, 2 potential mtDNA maintenance proteins, mitochondrial inner membrane transport protein OXA1L, and 7 mitochondrial transcription factors. Additionally, the rate of mitochondrial protein synthesis was determined in 18 out of a total of 33 patients. The results were used either to direct mutational analysis or to indicate the pathogenicity of a mutation. Furthermore, homozygosity mapping was performed for 7 patients.

15 Ü Chapter 1 This aided in the detection of the genetic defect in 1 patient (see chapter 8), but was uninformative in the other cases because of the lack of DNA from family members, resulting in large homozygous regions and consequently still many candidate genes. Due to the multitude of proteins (in)directly involved in mitochondrial translation in particular and the biogenesis of the OXPHOS system in general, selection of genes for mutational screening in patients with combined OXPHOS deficiencies is not an easy task. Chapter 2 of this thesis gives an overview of mitochondrial protein synthesis and other processes required for a functional OXPHOS system, highlighting proteins involved in the biogenesis or maintenance of multiple OXPHOS complexes and implicated in mitochondrial disorders. MtDNA sequencing in our patient cohort revealed two novel mitochondrial tRNA mutations responsible for progressive multisystemic diseases. These mutations and their functional consequences are described in chapter 3. The next two chapters deal with mutations in the nuclear genes coding for mitochondrial translation elongation factors mtEFTs (chapter 4) and mtEFG1 (chapter 5). These studies emphasize the heterogeneity and complexity of mitochondrial disorders. Chapter 4 reports a striking difference in clinical presentation of an identical genetic defect in two unrelated patients, and chapter 5 demonstrates the importance of a thorough biochemical analysis of fibroblasts in addition to muscle tissue as OXPHOS enzyme deficiencies are not always manifested in muscle tissue. Chapter 4 concerns a different patient cohort than the one described above; assistance was provided in the research during a traineeship and in the publication during the current PhD. In chapter 6, we investigated the evolution of the mitochondrial ribosomal proteome in a diverse set of eukaryotic species using extensive homology searches with the aim to 1) gain insight into the evolutionary history of MRPs and 2) aid prioritization of MRPs as candidates for their involvement in combined OXPHOS defects. This comparative genomics analysis revealed two new human MRPs. These two MRPs, together with two other candidate genes that were selected based on their potential function in mtDNA maintenance, were screened for mutations in our group of patients with OXPHOS complex I, III and IV deficiencies. Chapter 7 contains the results of this study, i.e. many non-pathogenic sequence variations. The second mutation in MRPS22 and its pathogenic role in the patient's mitochondrial disorder is reported in chapter 8. This finding underscores that candidate genes should not be prioritized based solely on their evolutionary conservation because proteins lacking bacterial homologs, such as MRPS22, have been shown to be implicated in mitochondrial disorders. Finally, chapter 9 reflects on all results obtained and discusses directions for future research with respect to combined complex I, III and IV OXPHOS deficiencies.

16 General introduction and outline of this thesis

R e f e r e n c e s 1. Saraste M: Oxidative phosphorylation at the fin de siecle. Science 1999; 283: 1488-1493.

2. Fernandez-Moreno MA, Bornstein B, Petit N, Garesse R: The pathophysiology of mitochondrial biogenesis: towards four decades of mitochondrial DNA research. Mol Genet Metab 2000; 71: 481-495. 3. Luft R, Ikkos D, Palmieri G, Ernster L, Afzelius B: A case of severe hypermetabolism of nonthyroid origin with a defect in the maintenance of mitochondrial respiratory control: a correlated clinical, biochemical, and morphological study. J Clin Invest 1962; 41: 1776-1804. 4. Schaefer AM, Taylor RW, Turnbull DM, Chinnery PF: The epidemiology of mitochondrial disorders--past, present and future. Biochim Biophys Acta 2004; 1659: 115-120. 5. Munnich A, Rustin P: Clinical spectrum and diagnosis of mitochondrial disorders. Am J Med Genet 2001; 106: 4-17. 6. Zeviani M, Di Donato S: Mitochondrial disorders. Brain 2004; 127: 2153-2172. 7. Dimauro S, Hirano M, Schon EA: Approaches to the treatment of mitochondrial diseases. Muscle Nerve 2006; 34: 265-283. 8. Koene S, Smeitink J: Mitochondrial medicine: entering the era of treatment. J Intern Med 2009; 265: 193-209. 9. Niers L, van den Heuvel L, Trijbels F, Sengers R, Smeitink J: Prerequisites and strategies for prenatal diagnosis of respiratory chain deficiency in chorionic villi. J Inherit Metab Dis 2003; 26: 647-658. 10. Tuppen HA, Blakely EL, Turnbull DM, Taylor RW: Mitochondrial DNA mutations and human disease. Biochim Biophys Acta 2010; 1797: 113-128. 11. Ugalde C, Moran M, Blazquez A, Arenas J, Martin MA: Mitochondrial disorders due to nuclear OXPHOS gene defects. Adv Exp Med Biol 2009; 652: 85-116. 12. Loeffen JL, Smeitink JA, Trijbels JM et al: Isolated complex I deficiency in children: clinical, biochemical and genetic aspects. Hum Mutat 2000; 15: 123-134.

17 This report, by its very length, defends itself against the risk of being read. Winston Churchill C h apter 2 Mitochondrial translation and beyond: processes implicated in combined oxidative phosphorylation deficiencies

Paulien Smits, Jan A.M. Smeitink and Lambert P. van den Heuvel

Journal of Biomedicine and Biotechnology 2010; Article ID 737385: 24 pages Chapter 2

Chapter A b s t r a c t Mitochondrial disorders are a heterogeneous group of often multi-systemic and 2 early fatal diseases, which are amongst the most common inherited human diseases. These disorders are caused by defects in the oxidative phosphorylation (OXPHOS) system, which comprises five multi-subunit enzyme complexes encoded by both the nuclear and the mitochondrial genomes. Due to the multitude of proteins and intricacy of the processes required for a properly functioning OXPHOS system, identifying the genetic defect that underlies an OXPHOS deficiency is not an easy task, especially in the case of combined OXPHOS defects. In the present communication we give an extensive overview of the proteins and processes (in)directly involved in mitochondrial translation and the biogenesis of the OXPHOS system and their roles in combined OXPHOS deficiencies. This knowledge is important for further research into the genetic causes, with the ultimate goal to effectively prevent and cure these complex and often devastating disorders.

20 Mitochondrial translation and beyond

1. I ntroduction Chapter Mitochondria are essential organelles that are present in virtually all eukaryotic cells. They originated from an ancestral alpha-proteobacterial endosymbiont.1 2 The primary function of the mitochondrion is ATP production via the oxidative phosphorylation (OXPHOS) pathway. Additionally, mitochondria have been found to perform crucial roles in many other metabolic, regulatory and developmental processes. Mitochondrial dysfunction can therefore result in a variety of diseases, including common multi-factorial disorders such as diabetes23 and Parkinson's disease.4 Furthermore, mitochondria are implicated in the normal aging process.5,6 The term mitochondrial disorder usually refers to diseases that are caused by disturbances in the OXPHOS system. This is a heterogeneous group of often multisystemic and early fatal diseases, which are amongst the most common inherited human diseases.7 The OXPHOS system is embedded in the mitochondrial inner membrane and consists of five multiprotein enzyme complexes (I-V) and two electron carriers.8 The main function of the system is the coordinated transport of electrons and protons, resulting in the production of ATP. The great complexity of the OXPHOS system, which comprises almost 90 proteins encoded by both the nuclear and the mitochondrial genomes, explains the heterogeneity in clinical phenotypes that are associated with genetic defects in oxidative phosphorylation. Approximately 67% of the OXPHOS disorders diagnosed at our centre consist of isolated enzyme deficiencies, whereas in 33% of the cases multiple enzyme complexes show lowered activities.9 Due to the dual genetic control, the defect can be located on the nuclear (n) as well as the mitochondrial (mt) DNA. Isolated OXPHOS deficiencies are generally caused by mutations in structural genes (encoding subunits of the OXPHOS system) or in genes encoding proteins involved in the assembly of a specific OXPHOS enzyme complex.1011 For combined deficiencies the situation is more complicated. Most mutations associated with combined OXPHOS defects have been reported in mtDNA-encoded transfer (t) and ribosomal (r) RNAs.12 Additionally, nDNA-encoded proteins required for the replication and integrity of mtDNA, such as polymerase y and thymidine kinase, are implicated in combined deficiencies.13 Recently, mutations in nine different nuclear gene products involved in mitochondrial protein synthesis were reported: elongation factors mtEFG1, mtEFTs and mtEFTu, small ribosomal subunit proteins MRPS16 and MRPS22, aspartyl- and arginyl-tRNA synthetases, and tRNA-modifying enzymes PUS1 and TRMU.14-25 These findings defined a new class of gene defects underlying combined OXPHOS disorders. In general, when multiple OXPHOS enzymes are affected, the genetic defect is presumed not to be located in genes encoding OXPHOS subunits, but rather in genes needed for

21 Chapter 2

Chapter mtDNA maintenance, mitochondrial transcription or translation including post- transcriptional or -translational processes, import of nDNA-encoded proteins into 2 the mitochondrion or mitochondrial membrane biogenesis. Given the number of proteins involved in these processes, a comprehensive overview of the processes and proteins that could be implicated in combined OXPHOS deficiencies is important for further research into the cause of these complex diseases. This review will focus on the mammalian mitochondrial translation and its role in mitochondrial disorders. However, other processes implicated in combined OXPHOS deficiencies, in particular combined OXPHOS deficiencies with normal complex II activities, and indirectly related to mitochondrial translation will also be outlined. We will first describe which processes are required before protein synthesis can take place in the mammalian mitochondrion and which components are needed for these processes. Second, the current state of knowledge of the mitochondrial translation machinery and the mechanism of translation, including regulation and functions of translation factors beyond protein synthesis, will be discussed. Third, we will cover post-translational processes with a functional OXPHOS system as end result. Fourth, we will give a non-exhaustive overview of the mutations that have been reported to impair mitochondrial protein synthesis and result in OXPHOS deficiencies. And finally, prospects of research into the pathogenesis of mitochondrial disorders will be mentioned.

2. Requirements before m itochondrial translation can take place For translation in the mitochondrion to be able to take place, a number of conditions have to be fulfilled. First of all, mtDNA has to be present, maintained, replicated and transcribed. Additionally, nuclear-encoded proteins required for the proper functioning of the mitochondrion have to be imported from the cell cytoplasm. These processes will now be discussed successively. Figure 1 gives an overview of the major processes (in)directly involved in the biogenesis of the OXPHOS system and covered in this review.

2.1. Human mtDNA The mitochondrial genome is a double-stranded, circular molecule of 16,659 base pairs. It is a highly compact genome that lacks introns and contains only one major non-coding region (the displacement or D-loop) and 37 genes, which code for 22 tRNAs, 2 rRNAs and 13 polypeptide subunits of the OXPHOS complexes I, III, IV and V.26 These genes are located on both strands of the mtDNA molecule, the heavy (H) and light (L) strand. MtDNA has a mutation rate 10-20 times that of nDNA,27-29 which is thought to be caused by the lack of protective histones, slightly limited DNA repair and proximity to damaging reactive oxygen species (ROS)

22 Mitochondrial translation and beyond generated at the inner membrane. The unique features of mitochondrial genetics are essential for understanding the etiology and pathogenesis of mitochondrial disorders:30 (a) mtDNA is maternally inherited; (b) cells typically contain hundreds of mitochondria and thousands of mtDNA molecules (polyplasmy); (c) mutations can affect all mtDNA copies in an individual (homoplasmy) or only some copies resulting in the coexistence of two or more mtDNA genotypes within a single cell, organ or individual (heteroplasmy); (d) in case of heteroplasmy, a minimum percentage of mutated mtDNAs has to be present in a cell for the OXPHOS system to malfunction (threshold effect) and this threshold level varies widely between different tissues; (e) during mitosis both normal and mutant mtDNA are randomly distributed to the daughter cells, which can result in changing mutational loads during the life of the patient and different mutational loads in different cells and tissues (mitotic segregation).

(TYMP, RRM2B)

MtDNA transcription

Mt translation (mtEFG1, mtEFTs, mtEFTu, MRPS16+S22, PUS1, TRMU, DARS2, RARS2)

Mt protein processing and quality control (SPG7)

(TAZ, OPA1, MFN2, DNM1L)

Figure 1. Schematic overview of the processes involved in mitochondrial translation and the biogenesis of the OXPHOS system. Before translation can take place in the mitochondrion, the mtDNA needs to be maintained, replicated and transcribed and numerous nDNA-encoded proteins have to be imported into the mitochondrion for these processes and for mitochondrial translation itself (see Figure 2 for more details on mitochondrial translation and its components depicted here). For the formation of the OXPHOS system, the nDNA- and mtDNA-encoded subunits need to be synthesized, imported, inserted into the inner membrane and assembled into enzyme complexes. The 13 mRNAs depicted refer to 9 monocistronic and 2 dicistronic transcripts. Proteins implicated in mitochondrial disorders are mentioned in brackets (also see Table 1). CI-CV = complex I-V of the OXPHOS system; TIM and TOM = translocase of the inner and outer mitochondrial membranes.

23 Chapter 2

Chapter 2.2. Maintenance and replication of mtDNA Unlike nDNA, which replicates only once during cell division, mtDNA is continuously 2 replicated, independent of the cell cycle and also in non-dividing cells such as skeletal muscle fibers and central neurons.31 Replication is generally thought to take place via a strand-asynchronous mechanism involving two unidirectional, independent origins.3233 Synthesis starts at the origin of H-strand replication (OH) in the D-loop and proceeds along the parental L-strand to produce a full daughter H-strand. When the second origin (OL) at two-thirds of the way around the genome is reached, DNA synthesis of the L-strand initiates in the opposite direction. Recently, however, a bidirectional mode of mtDNA replication has been proposed.3435 This second mechanism involves a coupled leading- and lagging- strand synthesis and is reported to exist along with the strand-asynchronous mechanism. MtDNA replication is achieved by a number of nuclear-encoded proteins. First of all, the only DNA polymerase present in mammalian mitochondria: polymerase y. Polymerase y is a heterotrimer consisting of a catalytic subunit with proof-reading ability (POLG) and two identical accessory subunits (POLG2) that bind DNA and increase the processivity of POLG. Second, Twinkle is a 5' to 3' DNA helicase that unwinds double-stranded mtDNA and thereby plays a role in mtDNA maintenance and regulation of mtDNA copy number. Third, the mitochondrial single-stranded binding protein (mtSSB) is thought to maintain the integrity of single-stranded regions of DNA at replication forks and to stimulate the activity of Twinkle and polymerase y. Additionally, several topoisomerases regulate supercoiling of mtDNA. Furthermore, mtDNA ligase III is involved in replication as well as repair of the mitochondrial genome. For replication initiation, RNA primers are needed. An as yet unidentified mtDNA primase likely provides the RNA primer for L-strand synthesis, whereas the mitochondrial transcription machinery is involved in RNA primer formation for H-strand synthesis (see Section 2.3). RNase mitochondrial RNA processing endonuclease (RNase MRP) and endonuclease G are implicated in processing of the precursor RNA primers for H-strand replication. Last, RNase H1 has been proposed to be involved in replication of the mitochondrial genome by removal of the RNA primers. Ligase III, RNase MRP and RNase H1 are not specific mitochondrial proteins; they are also located in the cell nucleus. For a more detailed description of mitochondrial replication see a review by Graziewicz et al.36 Furthermore, various other proteins are indirectly involved in the maintenance of mtDNA either through protecting the mitochondrial genome, repairing mtDNA damage, or supplying nucleotide pools. Firstly, the protection of the mitochondrial genome. MtDNA is packaged into protein-DNA complexes called nucleoids, which are believed to be the units of mtDNA transmission and inheritance.37,38

24 Mitochondrial translation and beyond

Among the nucleoid components are proteins involved in the maintenance, Chapter replication and/or transcription of mtDNA, such as mtSSB, Twinkle, POLG and TFAM (mitochondrial transcription factor A). Secondly, mtDNA repair is crucial to 2 avoid accumulation of damage to the rapidly replicating mitochondrial genome. Mitochondria possess multiple repair mechanisms, but these are beyond the scope of this review (detailed descriptions of mtDNA repair have been published previously).3940 Thirdly, a proper balance of the mitochondrial (deoxy)nucleoside triphosphate ((d)NTP) pools, accomplished by mitochondrial transport proteins and salvage pathway enzymes, is also essential for mtDNA maintenance and replication (for an overview of mitochondrial dNTP metabolism and all proteins involved see reference 41). Shortage and/or imbalance of the dNTP pool could affect the efficiency and accuracy of mitochondrial replication. Additionally, disturbances of the NTP pool may interfere with replication by affecting the synthesis of the RNA primers necessary for replication initiation. This could subsequently lead to deletions in the mtDNA or depletion (i.e., reduced copy number) of the mitochondrial genome. A number of mitochondrial disorders are caused by defects in the mitochondrial dNTP metabolism due to mutations in the following nine nuclear genes: DGUOK, TK2, TYMP, SLC25A4, SLC25A3, SUCLG1, SUCLA2, RRM2B, and MPV17 (for reviews see references134243).Mitochondrial deoxyguanosine kinase (DGUOK) and thymidine kinase (TK2) catalyze the first step in the salvage pathways of pyrimidine and purine deoxynucleosides, respectively. In the salvage pathway, deoxynucleosides are activated by stepwise phosphorylation leading to formation of the dNTPs. The cytoplasmic enzyme thymidine phosphorylase (TYMP) catalyzes the reversible phosphorylation of thymidine and thereby regulates the availability of thymidine for DNA synthesis. Additionally, adenine nucleotide translocators (ANT) are needed to regulate the concentration of adenine nucleotides by exchanging ATP for ADP in and out of the mitochondrial matrix; SLC25A4 (ANT1) is the heart and skeletal muscle specific isoform. The mitochondrial phosphate carrier SLC25A3 transports inorganic phosphate into the mitochondrial matrix. SUCLG1 and SUCLA2 encode the a- and ß-subunits of the Krebs cycle enzyme succinate-CoA ligase (SUCL). Defects in these genes could lead to mtDNA depletion through decreased activity of the mitochondrial nucleotide diphosphate kinase (NDPK), which functions in the last step of the mitochondrial dNTP salvage pathway and associates with SUCL. The p53R2 subunit (encoded by RRM2B) of the cytosolic enzyme ribonucleotide reductase is required for de novo deoxyribonucleotide synthesis in nonproliferating cells, thereby supplying dNTPs for nDNA repair and mtDNA synthesis. Finally, the mitochondrial inner membrane protein MPV17 is also involved in dNTP metabolism, with defects causing mtDNA depletion; the exact function of MPV17 remains to be elucidated, however. In addition to disturbed homeostasis of mitochondrial dNTP pools, mtDNA instability can

25 Chapter 2

Chapter naturally be caused by mutations in genes affecting mtDNA replication directly. Mutations have been reported in POLG, POLG2, and C10orf2 (Twinkle), with POLG 2 being the most important contributor.13 2.3. Transcription of mtDnA Transcription originates from three promoters: two H strand promoters (HSP1 and HSP2) and one L strand promoter (LSP). The HSP1 and LSP are located in the D-loop, whereas HSP2 is located downstream of HSP1 close to the 5' end of the 12S rRNA gene. Transcription from both the HSP2 and LSP generates polycistronic molecules covering nearly the entire H or L strand, corresponding to 12 protein- coding genes, 14 tRNA genes and 2 rRNA genes (HSP2) or 1 protein-coding gene and 8 tRNA genes (LSP). Additionally, transcription from the LSP produces the RNA primers necessary for initiating mtDNA replication of the H-strand. Transcripts derived from HSP1, on the other hand, contain mainly the 12S and 16S rRNAs. The basic human mitochondrial transcription machinery consists of three components: mtRNA polymerase (POLRMT), TFAM and either mitochondrial transcription factor B1 (TFB1M) or B2 (TFB2M). Promoter recognition and transcription initiation require the simultaneous presence of these three factors, however, the precise contribution of each has not yet been fully determined.4445 Termination of transcription appears to be regulated by multiple termination factors, but the exact mechanism and all factors involved still need to be elucidated.4546 Termination sites have been identified for transcription from HSP1 and HSP2; the two proteins binding to the HSP2 termination site await identification. Even though the mitochondrial transcription termination factor MTERF1 (or MTERF) has been shown to bind the HSP1 termination site (at the 3' end of the 16S rRNA gene) and be required for HSP1 transcription termination in vitro, it seems to block L strand transcription more effectively than transcription from the H strand; the precise function of the protein in vivo is unclear. Recently, MTERF1 was found to be essential for transcription initiation in vitro, which led to the hypothesis that MTERF1 regulates and promotes transcription at HSP1 by forming a loop between the MTERF1 initiation and termination binding sites. This way it helps favor a higher synthesis rate of rRNAs compared to mRNAs from the H strand. Furthermore, MTERF1 appears to modulate mtDNA replication pausing; its dual role could be important for coordination of replication and transcription.47 Three homologs of MTERF1 and thus potential mitochondrial transcription termination factors have been identified: MTERF2 (or MTERFD3 or MTERFL), MTERF3 (or MTERFD1) and MTERF4 (or MTERFD2).48 MTERF2 is proposed to regulate cell growth through modulation of mitochondrial transcription.49 MTERF2 shows the opposite expression pattern in response to serum compared to MTERF1, suggesting that they have divergent roles. Additionally, MTERF2 was found to be present in nucleoids, displaying non

26 Mitochondrial translation and beyond

sequence-specific DNA-binding activity.50 Contrastingly, Wenz et al demonstrated Chapter specific binding of MTERF2 to the HSP promoter region.51 Furthermore, MTERF2 knock-out resulted in decreased mitochondrial transcription and mRNA levels. 2 MTERF3, on the other hand, has been shown in mouse in vivo and in human in vitro to function as a negative regulator of transcription initiation through interaction with the mtDNA promoter region.52 Moreover, MTERF3 knock-down in Drosophila led to a decreased rate of mitochondrial protein synthesis, possibly through down- regulation of TFB1M (see Section 3.3 for more information on TFB1M's function in translation).4653 In summary, the current view is as follows: MTERF1 through 3 share a common binding site in the D-loop; both MTERF1 and 2 promote transcription initiation, whereas MTERF3 inhibits it; all three factors are needed to maintain optimal transcript levels and thereby ensure proper functioning of the OXPHOS system. The role of MTERF4 has not been investigated in detail, however, it appears to bind mtDNA in the D-loop region and form a stable homodimer with a putative RNA methyltransferase.54 Processing of the polycistronic primary transcripts is thought to require four enzymes. The tRNA genes mark most of the junctions between mitochondrial protein-coding and rRNA genes. According to the tRNA punctuation model, the secondary structures of the tRNA sequences provide the signals for endonucleolytic excision of the tRNAs, yielding most of the tRNAs, mRNAs and rRNAs.55 This initial processing step is performed by the mitochondrial RNase P (5'-end endonucleolytic cleavage) and tRNase Z (3'-end cleavage) enzymes. Maturation of the excised tRNAs is completed by addition of a CCA triplet to their 3'-end, which is catalyzed by an ATP(CTP):tRNA nucleotidyltransferase. After post-transcriptional modification and correct folding of the tRNAs, the amino acid can be attached to the CCA triplet by the corresponding aminoacyl-tRNA synthetase. The tRNA acceptor stem and the anticodon play important roles for the recognition of the tRNA by the appropriate synthetase. During or immediately after cleavage of the tRNAs, the rRNAs and mRNAs are polyadenylated by a mitochondrial poly(A) polymerase. This post-transcriptional modification creates the stop codons for some mRNAs and may also be necessary for stabilization of some RNAs (for a review see Montoya et al).56 In this way, 9 monocistronic and 2 dicistronic mRNA transcripts are formed. Another important post-transcriptional process is RNA degradation, which is required to control RNA levels and eliminate processing by-products and aberrant transcripts.57 Nevertheless, the players involved and the exact mechanism have yet to be revealed. SUV3 and polynucleotide phosphorylase are possible candidates.5859 Until now, no mutations causing mitochondrial disease have been reported in genes coding for proteins involved in mitochondrial transcription. Nonetheless, a mutation in the MTERF1 binding site (in the tRNALeu(UUR) gene) is associated with the

27 Chapter 2

Chapter mitochondrial disorder MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). This mutation reduces the binding affinity for 2 MTERF1 and results in a drastic impairment of transcription termination in vitro, however, in vivo the transcription termination defect could not be confirmed.6061 Furthermore, TFB1M modifies the phenotypic expression of the deafness- associated 1555A>G mtDNA mutation (see Section 3.3). Additionally, post- transcriptional modification of tRNAs is disturbed in patients with mitochondrial myopathy and sideroblastic anemia (MLASA) due to a mutation in pseudouridylate synthase PUS1.1417 PUS1 converts uridine into pseudouridine in cytosolic as well as mitochondrial tRNAs. Recently, mutations in TRMU, a mitochondria-specific enzyme that is required for the 2-thiolation on the wobble position of the tRNA anticodon, were detected in patients with acute liver failure in infancy.24 Previously, TRMU was identified as another nuclear modifier gene for the mitochondrial 12S rRNA mutation 1555A>G.62 See Section 5.2 for more details on defects in the latter two genes.

2.4. Import of nDnA encoded proteins Most mitochondrial proteins, including all proteins involved in mitochondrial translation, are encoded in the nucleus and therefore have to be transported to and imported into the mitochondrion. Cytosolic chaperones, such as heat shock proteins Hsp70 and Hsp90, guide the precursor proteins to translocation channel receptors on the mitochondrial surface, keep the proteins unfolded to prevent aggregation and enable entrance into the translocation channels. Alternatively, preproteins seem also to be imported into mitochondria in a co- translational manner.63-67 These proteins, produced on ribosomes bound to the outer mitochondrial membrane, are almost exclusively of prokaryotic origin.6869 The cytoplasmic and mitochondrial translation machineries are suggested to be localized in close proximity on either side of the mitochondrial membranes, thereby allowing efficient assembly of the OXPHOS system.70 Import and sorting of nDNA-encoded proteins into mitochondria is achieved by translocases in the outer and inner mitochondrial membrane.71,72 The translocase of the outer mitochondrial membrane (TOM complex) forms the entrance for basically all nuclear-encoded mitochondrial proteins. After passing through the TOM complex, the preproteins can follow various routes depending on their targeting signals: (1) outer membrane proteins are integrated into the membrane by the sorting and assembly machinery (SAM complex); (2) translocation to the intermembrane space is mediated by the TIM23 complex (translocase of the inner mitochondrial membrane) or by components in the intermembrane space; (3) matrix proteins reach their destination through the TIM23 channel; (4) for inner membrane proteins there are three pathways, (a) insertion by the TIM22 complex,

28 Mitochondrial translation and beyond

(b) lateral integration after arrest at the TIM23 complex, and (c) import into the Chapter matrix via TIM23 followed by export to the inner membrane (see Section 4.2 for more information on the export to the inner membrane). After translocation, 2 mitochondrial targeting sequences are removed proteolytically and the proteins fold into their functional structures. Two syndromes have been related to defects in mitochondrial import. Mutations in the gene for the translocase subunit TIMM8A (or DDP1) have been reported to result in the deafness dystonia syndrome or Mohr-Tranebjaerg syndrome (MTS).7374 TIMM8A is part of a small alternative TIM complex that is involved in the import of TIMM23, an essential constituent of the TIM23 complex, and a few other proteins.71 Remarkably, no impairment of the OXPHOS system has been observed.7375 The exact mechanism by which TIMM8A mutations affect mitochondrial function remains to be clarified. Dilated cardiomyopathy with ataxia (DCMA) is caused by a mutation in DNAJC19, which is thought to be a homolog of yeast Tim14.76 Tim14 is an essential component of the TIM23 complex,77 suggesting that the underlying disease mechanism is defective import of nDNA-encoded proteins into the mitochondrion. However, this has not been verified yet.

3. M itochondrial translation Although most of the proteins present in mitochondria are encoded by the nDNA, a few are encoded by the mtDNA and are synthesized by the separate mitochondrial translation system. The human mitochondrial genome codes for 22 tRNAs, 2 rRNAs and 13 polypeptide subunits of the enzyme complexes I, III, IV and V.26 Whereas the components and mechanisms of translation are well characterized for bacterial and eukaryotic cytoplasmic systems, far less is known about mitochondrial protein synthesis due to the lack of a proper in vitro mitochondrial translation system. For a review on mammalian mitochondrial protein synthesis, insertion and disorders associated with these processes see Pérez-Martínez et al.78 The mitochondrial translation more closely resembles its prokaryotic than its eukaryotic cytoplasmic counterpart. However, the protein-synthesizing system of mitochondria has a number of interesting characteristics not observed in prokaryotes or the eukaryotic cell cytoplasm. First of all, mitochondria use a genetic code that has several distinct differences from the universal code.79 For example, human mitochondria use the universal arginine codons AGG and AGA, in addition to UAA and UAG, for termination. Furthermore, UGA serves as a codon for tryptophan rather than as a stop codon. Additionally, AUA has been reassigned to Met rather than serving as an Ile codon. Secondly, also the mitochondrial mRNAs have unusual features: they contain no or very few 5' untranslated nucleotides,80

29 Chapter 2

Chapter are uncapped,81 and contain a poly(A) tail that immediately follows or even forms part of the stop codon.55 The small subunit of mitochondrial ribosomes 2 (mitoribosomes) appears to bind these mRNAs tightly in a sequence-independent manner and in the absence of initiation factors or initiation tRNA,82 unlike the prokaryotic83 and eukaryotic cytoplasmic84 systems. Thirdly, mitochondria use a simplified decoding mechanism that allows translation of all codons with only 22 tRNAs instead of the 31 predicted by Crick's wobble hypothesis.2685 Fourthly, mammalian mitochondria use a single tRNAMet for both the initiation and elongation phases (depending on the presence or absence of a formyl group, resp.), whereas not only in the prokaryotic and eukaryotic cytoplasmic translation systems but also in the mitochondria of most lower eukaryotes two specialized tRNAMet species exist.86 In this section, we will cover in subsequent paragraphs the components of the protein synthesis machinery, the translation process steps, its regulation, and additional roles of mitochondrial translation proteins outside the translation process.

3.1. The mitochondrial translation machinery The basic mitochondrial translation machinery comprises mtDNA-encoded rRNAs and tRNAs as well as many proteins coded for by the nuclear genome: (1) initiation, elongation and termination translation factors; (2) mitochondrial ribosomal proteins (MRPs); (3) mitochondrial aminoacyl-tRNA synthetases and methionyl-tRNA transformylase. These components will be described in more detail successively. First of all, the translation factors. The exact functions of the mitochondrial translation factors will be discussed in Section 3.2. Bacterial translation initiation involves three factors: IF1, IF2, and IF3. Whereas IF1 and IF2 are considered to be universal and essential initiation factors, IF3 orthologs have not been found in archaea or the cytoplasm of eukaryotes.87,88 Surprisingly, the mitochondrial translation machinery consists of two initiation factors orthologous to prokaryotic IF289 and IF390 and despite extensive searches, no IF1 ortholog has been detected.91 Recently, mitochondrial IF2 (mtIF2) was demonstrated to perform functions of both bacterial IF1 and IF2; a conserved 37 amino acid insertion in mtIF2 seems to have assumed the role of IF1, facilitating the bond between mtIF2 and the mitoribosome and the formation of the initiation complex.92 All three prokaryotic elongation factors have also been found in human mitochondria: mtEFTu, mtEFTs and mtEFG.93-95 In contrast to most bacteria, which have merely one EFG protein that acts during both the elongation and termination phases of the translation process, mitochondria contain two EFG homologs, mtEFG1 and mtEFG2,93 that are 35% identical.91 The importance of mtEFG1 for mitochondrial protein synthesis has been demonstrated by mtEFG1 defects in patients with a mitochondrial

30 Mitochondrial translation and beyond

disorder152225 and its translocation activity was shown in vitro.96 Even though Chapter expression levels of mtEFG2 are greatest in skeletal muscle, heart and liver,93 three tissues with high metabolic energy rates, the functional significance of 2 mtEFG2 for mitochondrial translation is not entirely clear. Notably, deletion of the mtEFG2 ortholog in yeast (MEF2) does not lead to impaired mitochondrial protein synthesis and respiratory defects, as is the case for MEF1, the mtEFG1 yeast ortholog.97 Moreover, complementation of mtEFG1 defects through overexpression of mtEFG2 could not be attained,1525 indicating that mtEFG2 might not play a role in the translocation step of mitochondrial translation. This was recently confirmed by Tsuboi et al, who demonstrated that mtEFG1 specifically catalyzes translocation, whereas mtEFG2 has an essential function in ribosome recycling and lacks translocation activity.98 Thus the dual role of prokaryotic EFG is distributed between mtEFG1 and mtEFG2. Bacteria contain four factors responsible for translation termination: the three release factors RF1-3 and the ribosome recycling factor RRF.99 The release factors are divided into two classes, with class I factors (RF1 and RF2) promoting codon-specific hydrolysis of peptidyl-tRNA and class II factors (RF3) lacking specificity but stimulating the activity of class I factors and their dissociation from the ribosome. Bacteria utilize three stop codons; both class I release factors recognize UAA, whereas UAG and UGA are decoded only by RF1 or RF2, respectively. In the eukaryotic cytosol two release factors, RF1 and RF3 orthologs, are required for the termination step with just one class I factor recognizing all three stop codons.100 A factor equivalent to bacterial RRF appears to be absent. The termination process in mitochondria, on the other hand, has not yet been fully elucidated. Two release factors, mtRF1 and mtRF1a (or HMRF1L), and a recycling factor (mtRRF) have been identified and partly characterized.101-104 Being involved in ribosome recycling instead of the elongation phase, mtEFG2 should be added to this list and be renamed mtRRF2.96 MtRF1 was proposed to be a member of the class I release factors based on bioinformatic analyses,103 but recently this factor failed to exhibit release factor activity in vitro and in vivo.101102 Nonetheless, the newly identified release factor mtRF1a was shown to terminate translation at UAA and UAG codons, analogous to bacterial RF1. Thus the question whether a mitochondrial release factor exists that recognizes the other two mitochondrial stop codons, AGG and AGA, which are found in just two of the mitochondrial transcripts, remains unresolved. The fact that release activity was not observed for mtRF1 could be attributed to the use of bacterial and yeast systems that naturally do not terminate with these codons.102 Therefore, the possibility remains that mtRF1 is indeed a mitochondrial release factor. Alternatively, AGA and AGG might not be used as stop codons since there is no experimental data supporting this and remarkably, in rat and mouse mitochondria the codons AGA and AGG are unassigned and UAA appears

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Chapter to be the single stop codon utilized.105106 The terminal AGA and AGG codons could be edited post-transcriptionally, creating UAG stop codons, which could 2 then be decoded by mtRF1a.101 Whether mitochondria contain a class II factor equivalent to bacterial RF3 is unclear. Secondly, the mitoribosomes that are made up of rRNAs and MRPs and comprise two subunits, the small (SSU or 28S) and the large (LSU or 39S) subunit. The human mitoribosome consists of 2 rRNAs (12S and 16S) and around 81 MRPs.107 Mammalian mitoribosomes differ markedly from bacterial, cytosolic and even from other mitochondrial ribosomes.108 They lack nearly half the rRNA present in bacterial ribosomes, resulting in a sedimentation coefficient of 55S compared with 70S in bacteria. Nevertheless, mitoribosomes contain a correspondingly higher protein content due to enlargement of proteins and recruitment of numerous extra proteins, causing a greater molecular mass and size than bacterial ribosomes. Most of these enlarged and supernumerary proteins do not seem to compensate for the missing rRNA segments since they occupy new positions in the mitoribosome,109 suggesting that they perform mitochondria-specific functions. Third, the mitochondrial tRNAs. In general, human mitochondrial tRNAs deviate from the canonical tRNAs, but still fold into mostly classical cloverleaf secondary structures and presumably also into L-shaped tertiary structures.110 Mitochondrial tRNAs are shorter than bacterial or eukaryotic cytoplasmic tRNAs, have large variations in the size of the D- and T-loops, and lack multiple conserved nucleotides that are involved in classical tertiary interactions creating the L-shape, which possibly results in a weaker tertiary structure. Post-transcriptional base modification appears to be more important for the proper tertiary structure and functioning of mitochondrial tRNAs compared with cytosolic tRNAs.111 Certain mitochondrial tRNAs will consequently be completely non-functional when they lack the post-transcriptional modification resulting in an aberrant structure.112 Furthermore, modifications can improve tRNA specificity and its recognition by mRNA codons and to a lesser extent by aminoacyl-tRNA synthetases.113114 In total, 19 mitochondrial aminoacyl-tRNA synthetases have been identified, of which two are encoded by the same gene as the cytosolic enzyme.115 Only the gene for mitochondrial glutaminyl-tRNA synthetase has not been found yet. After aminoacylation, the tRNAMet needs to be formylated by methionyl-tRNA transformylase to initiate mitochondrial translation. Although the core components of the mitochondrial translation machinery have been identified, many more factors are likely to be involved, directly or indirectly, and have yet to be discovered. Recently, Davies et al showed that pentatricopeptide repeat domain protein 3 (PTCD3) associates with the mitoribosomal SSU and is necessary for mitochondrial protein synthesis.116 The precise function of PTCD3 remains to be clarified, however.

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3.2. The mitochondrial translation process Chapter The basic model of protein synthesis is derived from studies in bacteria (see references87,99 117 for reviews on the processes involved in protein synthesis). Our 2 understanding of the mechanisms of mitochondrial translation is based on this model and additional studies in mitochondria. Protein synthesis is divided into three phases: initiation, elongation and termination. The exact starting mechanism of the translation process in mitochondria is poorly understood. Due to the unusual characteristics of mitochondrial mRNAs, neither the Shine-Dalgarno sequence observed in prokaryotes nor the 7-methylguanlyate cap structure found in the eukaryotic cell cytoplasm can facilitate ribosome binding and direct the ribosome to the start codon. It is thought that the mRNA entry gate on the SSU of the mitoribosome has evolved in such way that it recognizes the unique mitochondrial mRNAs with their unstructured 5' sequences.109118 Many questions remain concerning the precise sequence of events during the initiation phase in mammalian mitochondria. In the current model, mitochondrial translation factor mtIF3 catalyzes the dissociation of the mitoribosome into its two component subunits (Figure 2, step 1), which may be an active rather than a passive process, thereby permitting the assembly of the initiation complex while preventing premature binding of the LSU.90119 Possibly, complete subunit dissociation is not essential for initiation of translation, however, the subunit interface must become accessible for fMet-tRNAMet and mRNA binding. It has been postulated that the first step in initiation complex formation is sequence-independent binding of mRNA to the SSU (Figure 2, step 2).82120 MtIF3 is thought to assist the mRNA to bind the SSU so that the start codon (AUG) is correctly positioned at the peptidyl (P) site of the mitoribosome. Both fMet-tRNA and mtIF2 can bind weakly to the SSU in the absence of mRNA and mtIF3 is hypothesized to prevent or correct the premature binding of these components.119 The binding of fMet-tRNAMet to the SSU requires mtIF2, which is markedly enhanced by GTP (Figure 2, step 3).121122 Recombining of the LSU with the SSU (Figure 2, step 4) probably stimulates the dissociation of mtIF3 (Figure 2, step 5).123 Additionally, GTP hydrolysis on mtIF2 is triggered by the LSU, leading to its release from the complex (Figure 2, step 6). The initiation phase is now complete and translation can proceed with the elongation phase. The basic steps in the elongation phase are the same in bacteria and mitochondria, however, the equilibrium dissociation constants for interactions between mtEFTu and its ligands differ considerably between the prokaryotic and mitochondrial systems.124,125 The relative ratios of the elongation factors are important for efficient translation.21,25 126 These ratios differ between tissues and can be adapted in response to dysfunction of one of the elongation factors.25 Elongation factor mtEFTu forms a ternary complex with GTP and an

33 Chapter 2

Chapter 2

•GTP Figure 2. Diagram of human mitochondrial protein synthesis. The three phases of mitochondrial translation - initiation, elongation and termination - and all translation factors involved are represented in this figure. See Section 3.2 in the text for a detailed description of all steps (numbered in boxes) of the mitochondrial translation process. Initiation, elongation and termination factors are represented by green, purple and red ovals, respectively. GTP and GDP are shown in yellow and beige circles, respectively.

aminoacylated tRNA (Figure 2, step 7). It is proposed to be critical for translational accuracy through surveillance of aminoacyl-tRNAs for misacylation.127 MtEFTu protects the tRNA from hydrolysis and, after a proofreading step, carries it to the mitoribosomal aminoacyl or acceptor (A) site for the decoding of mRNA by codon- anticodon interactions on the SSU (Figure 2, step 10). When the codon-anticodon recognition occurs, GTP hydrolysis on mtEFTu is stimulated by the mitoribosome, resulting in the release of mtEFTu-GDP (Figure 2, step 8). The nucleotide exchange

34 Mitochondrial translation and beyond

protein mtEFTs converts mtEFTu-GDP in active mtEFTu-GTP (Figure 2, step 9). Chapter Following the release from mtEFTu, the 3' end of the aminoacyl-tRNA moves into the peptidyl transferase center of the LSU where peptide bond formation is 2 catalyzed, adding one amino acid to the growing peptide (Figure 2, step 11). The elongation factor mtEFG1 with bound GTP catalyzes the translocation step by conformational changes in both mtEFG1 and the mitoribosome, during which the A and P site tRNAs move to the P and exit (E) sites of the mitoribosome and mRNA is advanced by one codon (Figure 2, step 12). Subsequently, the tRNA leaves the mitoribosome via the E site (Figure 2, step 13) and a new elongation cycle can start (Figure 2, step 14). Whether the mitoribosome contains an actual E site is uncertain. The bovine mitoribosomal structure and a comparative analysis of ribosome sequences revealed that the mitoribosomal E site deviates substantially from the prokaryotic and eukaryotic cytosolic situations.109128 129 Based on these findings, the E site has been suggested to be very weak or even absent in the mitoribosome. Moreover, the mitoribosomal polypeptide exit tunnel is markedly different, allowing premature exposure of the nascent polypeptide to the mitochondrial matrix or membrane before reaching the conventional exit site.109 Whether all or some nascent polypeptide chains emerge prematurely from the peculiar mitoribosomal exit tunnel is currently unknown. The third and last step in protein synthesis, termination, begins when a stop codon (UAA, UAG, AGA or AGG) is encountered in the A site. A mitochondrial release factor, mtRF1a or possibly also mtRF1 or an as yet unidentified protein, recognizes the stop codon (Figure 2, step 15) and causes the protein that is attached to the last tRNA molecule in the P site to be released (Figure 2, step 16). The ester bond between the tRNA and the nascent polypeptide is hydrolyzed, presumably by the peptidyl transferase center on the LSU triggered by the release factor, and this process is catalyzed by GTP. After release of the newly synthesized protein, mtRRF and mtEFG2-GTP together enable the mitoribosomal subunits, tRNA and mRNA to dissociate from each other (Figure 2, step 17), making the components available for a new round of protein synthesis. GTP hydrolysis is required for the release of mtRRF and mtEFG2-GDP from the LSU (Figure 2, step 18).96

3.3. Regulation of mitochondrial translation In yeast, nuclear-encoded, gene-specific activation factors are required for mitochondrial translation initiation.130131 Currently, translational activation factors have been found for (nearly) all eight yeast mtDNA-encoded proteins and synthesis of some proteins, for example Cox3, depends on multiple activation factors.130-132 These activators bind to the 5' untranslated leader (5'-UTL) sequences of the mRNA, probably to assist in positioning the mitoribosomes over the initiation

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Chapter codon. All translational activator proteins studied so far are integral membrane proteins or bound to the mitochondrial inner membrane, suggesting that they are 2 also involved in tethering mitochondrial translation to the inner membrane. This way they can promote cotranslational insertion of newly synthesized proteins and subsequent assembly into the OXPHOS complexes. Thus the yeast mitochondrial translational activators regulate not only the levels of mitochondrially synthesized gene products, but also the location of mitochondrial translation. The mechanisms of human mitochondrial translation regulation are poorly understood. Human mitochondrial mRNAs lack 5'-UTL sequences and until recently no clear evidence was found for the existence of mRNA-specific translation activators, which suggests that modulation of mitochondrial protein synthesis in humans involves other strategies than in yeast.133 Genome-wide linkage analysis and chromosome transfer in a patient presenting with Leigh syndrome due to an isolated complex IV deficiency resulted in the identification of a human translational activator of the complex IV subunit COXI: CCDC44 or TACO1.134 A homozygous base insertion, creating a premature stop codon, led to severely decreased levels of TACO1 in patient fibroblasts. Consequently, only a small amount of COXI was synthesized despite normal concentrations of the COXI transcript, compromising complex IV assembly and activity. Remarkably, deletion of the TACO1 ortholog in yeast produced no respiration or mitochondrial translation defect. In contrast to the human situation, TACO1 is apparently not essential for respiration in yeast. Furthermore, another potential translational activator for COXI has been identified in humans: a member of the pentatricopeptide (PPR) family, leucine- rich PPR-motif containing protein (LRPPRC, also known as LRP130). The PPR motif has been found in proteins that interact with RNA, such as POLRMT, which contains two PPR motifs in the amino-terminal domain (ATD).135 LRPPRC has been postulated to be a homolog of Pet309,136 the yeast mitochondrial translational activator for COX1.137 Mutations in LRPPRC lead to the neurodegenerative disorder Leigh Syndrome French-Canadian type (LSFC), with a deficiency of complex IV of the OXPHOS system.136 LRPPRC appears to play a role in the translation and/or stability of COXI and COXIII mRNAs, similar to yeast Pet309.138 In addition to its role as translational activator, Pet309 might be involved in coupling mitochondrial transcription to translation through interaction with Nam1,139 a protein that is postulated to stabilize and direct mRNAs to the mitochondrial inner membrane for translation140 and that binds to the ATD of yeast mtRNA polymerase.135 LRPPRC has been suggested to function together with heterogeneous nuclear ribonucleoprotein K (hnRNP K) and POLRMT in coupling the mitochondrial transcription and translation machineries in a manner analogous to the yeast system.141 However, the situation is more complex than depicted here,

36 Mitochondrial translation and beyond

encompassing additional proteins for which homology between yeast and human Chapter has not been identified yet. Moreover, LRPPRC binds not only mitochondrial but also nuclear mRNAs, indicating that it could be involved in coordinating nuclear 2 and mitochondrial gene expression.142 Nolden et al proposed a negative-feedback loop mechanism for regulation of mitochondrial translation.143 The ATP-dependent m-AAA protease plays an important role in quality control of mitochondrial inner membrane proteins. One of the substrates of this enzyme is the ribosomal protein MRPL32. Processing of MRPL32 by the m-AAA protease results in a tight association of MRPL32 with the inner membrane and allows completion of mitoribosome assembly in close proximity to the inner membrane. Maturation of MRPL32 seems to be required for mitochondrial translation since synthesis of mtDNA-encoded proteins was substantially impaired in cells lacking the m-AAA protease. Regulation of translation via the m-AAA protease could for instance take place when nuclear and mitochondrial gene expression are unbalanced. Excess respiratory subunits and other nonnative substrates of the m-AAA protease may then accumulate and compete with MRPL32 for binding to the protease. This will hamper MRPL32 processing, ribosome assembly, and finally mitochondrial translation. Accordingly, the amount of respiratory subunits available to the m-AAA protease decreases and MRPL32 processing increases again. The importance of this regulation process has been demonstrated by loss-of-function mutations in paraplegin, a subunit of the human m-AAA protease, which result in the neurodegenerative disorder hereditary spastic paraplegia.144 Possibly, TFB1M and TFB2M are involved in a retrograde pathway regulating mitochondrial biogenesis and function.145 146 Overexpression of TFB2M resulted in increased TFB1M levels and consequently an increase in mitochondrial biogenesis. In addition to their transcriptional stimulatory activity, TFB1M and TFB2M have rRNA methyltransferase activity. Thus these factors are indirectly involved in mitochondrial protein synthesis via their ability to methylate the mitochondrial 12S rRNA, which is important for mitoribosome activity. TFB1M has been identified as a nuclear modifier of the 1555A>G mutation in the 12S rRNA gene that causes nonsyndromic or aminoglycoside antibiotic-induced deafness.147 Presumably, altered or lack of methylation due to malfunctioning TFB1M can diminish the effect of the 1555A>G mutation on mitoribosome conformation. In vivo studies in mice revealed that TFB1M is an essential rRNA methyltransferase, needed for stability of the mitoribosomal SSU, that does not directly modulate transcription, whereas TFB2M is suggested to have transcriptional activation as its primary function.148 Therefore, differential expression of these two factors could modulate not only transcription, but also replication (via the transcription factor activity) and translation (via the rRNA methyltransferase activity), and in this manner ensure a

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Chapter balance between the amounts of mitochondrial transcripts and fully assembled mitoribosomes.145,148 2 Mitochondria are under general nuclear control through transcription factors, such as nuclear respiratory factors 1 and 2 (NRF-1 and -2).149 These factors coordinate the expression of the nuclear and mitochondrial gene products required for oxidative phosphorylation and other essential mitochondrial functions. They act directly on nuclear genes coding for OXPHOS subunits as well as various nuclear genes encoding proteins involved in mtDNA replication, mitochondrial transcription or translation, by which they exert indirect control over expression of mitochondrial genes. Transcription factor CREB (cAMP response element-binding protein) promotes transcription of mitochondrial (in addition to nuclear) genes after its import into the mitochondrion directly.150

3.4. Functions of the mitochondrial translation machinery beyond translation Initiation factor mtIF2 might be involved in mitochondrial mRNA degradation and apoptosis. The endoribonuclease RNase L is an important player in apoptosis induced by interferons (IFNs).151 After binding its IFN-induced activator, RNase L degrades single-stranded RNAs, leading to inhibition of protein synthesis. RNase L was shown to interact with mtIF2 and thereby modulate the stability of mitochondrial mRNAs, which appears to be essential for IFNa-induced apoptosis.152 RNase L is brought into association with the mitochondrial mRNA during their translation through interaction with mtIF2. In the presence of IFNa, RNase L becomes activated and degrades mRNA, which can eventually result in apoptosis. An excess level of mtIF2 could hold RNase L away from the mRNAs, preventing their degradation and thus also inhibiting IFNa-induced apoptosis. In addition to its function in the elongation phase of the mitochondrial translation, mtEFTu has been reported to act as a chaperone.153 It plays a role in protein quality control in mitochondria, as has been found for its cytosolic and prokaryotic counterparts. MtEFTu interacts with unfolded proteins, especially with misfolded, newly synthesized polypeptides, and is hypothesized to recruit these proteins to a mitochondrial protease complex for their degradation. This protease complex presumably consists of the homologs of the bacterial GroEL/ES (Hsp60 class) chaperone and ClpA/ClpP protease systems, with which EFTu can interact via the heat shock protein Hsp31.154 Recently, human mitochondrial ribosomal protein MRPL12 was demonstrated to bind to POLRMT, which enhances transcription.155 Free MRPL12, that is MRPL12 not incorporated in the mitoribosome, appears to interact with POLRMT and consequently might coordinate the rate of mitochondrial transcription with the rate of mitoribosomal biogenesis. When the import rate of MRPs is exceeding the rate of mtDNA-encoded rRNA expression, free MRPL12 will accumulate and

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associate with POLRMT. Subsequently, the rate of mtDNA transcription increases, Chapter which rebalances the system. Furthermore, mitochondrial ribosomal proteins MRPS29 and MRPS30 seem 2 to be bifunctional proteins.156 These two proteins are the proapoptotic proteins death-associated protein 3 (DAP3) and programmed cell death protein 9 (PDCD9 or p52), respectively. Whether MRPS29 and/or MRPS30 are released from the mitoribosome and exported to the cytosol during apoptosis or whether they carry out their proapoptotic role while still associated with the mitoribosome is unknown. Possibly, inner membrane-associated mitoribosomes affect the mitochondrial permeability transition pores via MRPS29 and MRPS30 and thereby induce apoptosis.

4 . P o s t -translational p r o c e s s e s r e q u ir e d f o r f u n c t io n a l o x p h o s c o m p l e x e s After translation has taken place, the mtDNA-encoded and imported nDNA- encoded proteins need to be incorporated into the inner membrane to form a functional OXPHOS system. Mitochondria contain chaperones, proteases and assembly factors for particular OXPHOS complexes to aid in this process.

4.1. Quality control by chaperones and proteases The entire chaperone system represents a mechanism for quality control that determines the fate of all mitochondrial proteins: proteolytic degradation or folding and assembly. Chaperones from the Hsp60 and Hsp70 class bind to and stabilize (partially) unfolded or newly synthesized or imported proteins, thereby preventing their aggregation and facilitating their proper folding.157 Hsp100/Clp family chaperones are involved in the re-solubilization of protein aggregates and unfolding of misfolded proteins, resulting in either refolding by other chaperones or degradation by proteases. Excess or non-native proteins are degraded into peptides by ATP-dependent proteases and subsequently into amino acids by oligopeptidases.158 Mammalian mitochondria have three major ATP-dependent proteases: Lon, Clp-like, and other AAA proteases. Both Lon protease and proteins from the Clp family are located in the matrix and contain proteolytic as well as chaperone activities. Two membrane-bound ATP-dependent AAA- proteases, active on the intermembrane space side (i-AAA) or on the matrix side (m-AAA), are responsible for quality control of inner membrane proteins. Additionally, as mentioned in Section 3.3, they regulate mitochondrial biogenesis through selective processing of mitochondrial proteins, for example MRPL32. The significance of these AAA proteases is demonstrated by the severe defects due to dysfunction of one of the AAA proteases found in several species. The prohibitin complex, consisting of Phb1 and Phb2 and also located in the inner membrane, is

39 Chapter 2

Chapter thought to act as a chaperone that stabilizes mitochondrially synthesized OXPHOS subunits against degradation by AAA proteases.159,160 While the exact functions 2 of the prohibitin complex remain poorly understood, its role in mitochondrial biogenesis and metabolism has been corroborated by numerous findings, such as different prohibitin expression levels depending on metabolic demand and lack of prohibitin leading to reduced mitochondrial membrane potential and instability of mtDNA-encoded OXPHOS subunits.160

4.2. Protein insertion into the inner membrane A subset of mitochondrial inner membrane proteins that are synthesized in the cytosol are imported into the mitochondrial matrix prior to their export to the inner membrane; all other nuclear-encoded proteins are integrated directly into the inner membrane during import into the mitochondrion (also see Section 2.4). All proteins synthesized by the human mitochondrial translation system are destined for the inner membrane and become inserted through the same export machinery as used by nDNA-encoded proteins. At least half of the mitoribosomes are associated with the inner membrane161 and it has been proposed that only membrane-bound mitoribosomes are translationally active.143 It appears that mitochondrial gene products become inserted into the inner membrane as they are undergoing synthesis on mitoribosomes, that is in a co-translational fashion.162-166 However, the relative contributions of co- and post-translational insertion and the exact mechanisms are unknown. The inner membrane protein Oxa1 plays an important role in insertion of both mitochondrial- and nuclear-encoded proteins from the mitochondrial matrix into the inner membrane.167,168 Recently, the interaction of Oxa1 with the mitoribosome was concluded to involve at least the two yeast LSU proteins Mrp20 and Mrpl40, orthologs of bacterial ribosomal proteins L23 and L24 respectively, which are located close to the polypeptide exit tunnel.169170 Furthermore, mitoribosomal protein MRPL45 was postulated to function in the co-translational insertion of mtDNA-encoded proteins into the inner membrane.107 The yeast ortholog of MRPL45, Mba1, associates with the inner membrane and is involved in protein insertion into the membrane in a concerted action with Oxa1, possibly by positioning the mitoribosomal exit tunnel at the right location for insertion.164,171 Nonetheless, mitoribosomes remain partially anchored to the mitochondrial inner membrane in absence of MRPL45 and Oxa1, indicating that additional factors are involved in the membrane association.164 One of these factors could be LETM1, leucine zipper EF-hand- containing transmembrane protein 1, located in the chromosomal region that is deleted in patients suffering from Wolf-Hirschhorn syndrome.172 It is the homolog of yeast inner membrane protein Mdm38, which has been proposed to function in an Oxa1-independent transport pathway across the inner membrane.173 LETM1

40 Mitochondrial translation and beyond

was found to associate with MRPL36 and could thereby anchor the mitoribosome Chapter to the inner membrane.174 Overexpression of LETM1 resulted in inhibition of mitochondrial biogenesis and ATP production. Conversely, LETM1 knockdown 2 caused mitochondrial swelling, loss of tubular networks and disassembly of OXPHOS complexes I, III, and IV.175 Much of the exact functions of LETM1 remain to be clarified, however, such as its role in cell viability and tumorigenesis, in addition to its potential interaction with the mitoribosome.174-176 Coupling mitochondrial protein synthesis to insertion of the protein into the inner membrane will be advantageous for the efficient formation of OXPHOS complexes. As mentioned previously, transcription seems to be coupled to the translation system as well. A similar process called transertion, the coupled transcription-translation-insertion of proteins into and through membranes, is found in bacteria.177,178 Linking of these processes generates hyperstructures, which are assemblies of different types of (macro)molecules that form an organizational level intermediate between genes/proteins and whole cells.179 Thus the mtDNA, transcription and translation machineries may be dynamically connected to the inner membrane into hyperstructures at assembly sites for the OXPHOS system.

4.3. Assembly of OXPHOS complexes Each OXPHOS complex has a specific assembly pathway, which involves chaperones that are not part of the functional complex but are implicated in its formation: the assembly factors. Up till now, 22 assembly factors have been identified and the list is still growing (see reference180 for an overview, including defects in assembly): eleven for complex I (NDUFA12L or B17.2L,181 NDUFAF1 or CIA30,82 NDUFAF2,181183 NDUFAF3 or C3ORF60,184 NDUFAF4 or C6ORF66,185 Ecsit,186 C8ORF38,187 C20ORF7,188189 and possibly the CIA84 ortholog PTCD1,190 AIF191 and IND1),192 one for complex II (SDHAF1),193 one for complex III (BCS1L),194 six for complex IV (SURF1, COX10, COX15, SCO1, SCO2, and supposedly LRPPRC),138195 and four for complex V (ATP11, ATP12,196 and possibly ATP23197 and OXA1L).198 Naturally, structural proteins can have additional functions in the assembly of the particular OXPHOS complex. The organization of the OXPHOS system is more intricate than separately assembled complexes that are arranged in sequence in the inner mitochondrial membrane. Two models for the organization of the mitochondrial respiratory chain have been proposed: (1) the "fluid-state" or "random collision" model, which has been the preferred description, where all OXPHOS complexes diffuse individually in the membrane and electron transfer depends on the random collision of the complexes and electron carriers; (2) the "solid-state" model, which was proposed over 50 years ago and has recently received more attention, where the complexes together form large supramolecular structures termed supercomplexes or

41 Chapter 2

Chapter respirasomes.199,200 The most plausible scenario, however, is a combination of these two models: the "plasticity" model.201 In this model, single complexes ("fluid- 2 state" model) and different types of supercomplexes ("solid-state" model) coexist in the inner membrane. Complex I, for instance, is mainly found in association with complex III in various supercomplexes that additionally contain the electron carriers coenzyme Q and cytochrome c, complex IV, and sometimes complex II or V, and are able to respire. On the other hand, most of the complexes II and IV are present as individual entities. How the supercomplexes are assembled is currently not known, but the significance of this arrangement for the stability of the different complexes is certain. This is emphasized by the finding that primary defects in, for example, complex III can lead to secondary instability of another complex, such as complex I, through improper supercomplex formation.202 More often, though, a mutation results merely in an isolated deficiency of the particular complex. Recently, defects in Tafazzin, a protein required for the metabolism of the inner membrane phospholipid cardiolipin, was shown to affect complex I/ III2/IV supercomplex stability.203 The cardiolipin deficiency resulted in weakened interactions between complexes I, III, and IV, unstable supercomplexes, and decreased levels and activities of the complexes themselves, ultimately causing Barth syndrome. Thus combined OXPHOS deficiencies can also be caused by defects in the assembly of supercomplexes. For proper assembly of the OXPHOS system, mitochondrial fusion and fission events are crucial since they control mitochondrial morphology and thereby also mitochondrial function. Disruption of fusion or fission primarily affects two key functions of mitochondria: respiration and regulation of apoptosis.204,205 Defects in fusion proteins MFN2 (mitofusin 2) and OPA1 (optic atrophy 1), for instance, cause a reduction in membrane potential and OXPHOS enzyme activities and are associated with the neurodegenerative diseases Charcot-Marie-Tooth type 2A and dominant optic atrophy, respectively. Additionally, down-regulating the expression of DNML1 (dynamin 1-like, also called DRP1 or DLP1), a protein involved in mitochondrial and peroxisomal fission, led to loss of mtDNA and a decrease in mitochondrial respiration.206 In a patient with a DNML1 deficiency, however, no mitochondrial morphology abnormalities or impairment in respiratory function could be detected, despite elevated lactate levels.206,207

5. M u t a t io n s t h a t im p a ir mitochondrial translation a n d r e s u l t in mitochondrial d is o r d e r s Given the multitude of proteins and complexity of the processes that are required for a properly functioning OXPHOS system, it is not surprising that in many patients with a mitochondrial disorder the underlying molecular genetic defect

42 Mitochondrial translation and beyond

has not yet been identified. Nonetheless, since the discovery of the first mtDNA Chapter mutations associated with mitochondrial disorders in 19 8 8,208,209 numerous mutations in mtDNA and nDNA have been reported and the list is still expanding 2 (for an overview see e.g., reference43). In each of the previous sections we have briefly mentioned the relevant genes implicated in mitochondrial disorders, with the exception of genes of the mitochondrial translation process. Here we will discuss in more detail the mutations found in this class of genes. Table 1 gives an overview of the genes implicated in combined OXPHOS deficiencies. These genes are also depicted in Figure 1.

5.1. MtDNA mutations As already stated in the introduction, the majority of mutations associated with combined OXPHOS deficiencies and a mitochondrial translation defect are located in the mitochondrial genome. Approximately 150 mutations, of which a large percentage awaits proper determination of their pathological significance (see reference210 for a scoring system), have been reported in mitochondrial tRNA genes and a few in rRNA genes.12 It is beyond the scope of this review to discuss these mutations in detail; overviews of mitochondrial tRNA mutations and their molecular and clinical consequences have been published before.210-212 The tRNALeu(UUR) gene forms a hotspot for pathogenic mutations with nearly 30 different mutations, but in all tRNA genes, mutations have been detected now. A pathogenic tRNA gene mutation is expected to lead to a combined OXPHOS defect through a decreased rate of mitochondrial protein synthesis. The exact complexes that show a deficiency differ for each mutation, partly depending on which tRNA is affected and the percentages of the corresponding amino acid in the different OXPHOS subunits. The pathogenic mechanisms involved in the translation defect due to a tRNA mutation are numerous and frequently multiple events are involved; potential effects are: impaired transcription termination, impaired tRNA maturation, defective post-transcriptional modification of the tRNA, effect on tRNA structure (e.g., global structural weakness or conformational alteration), decreased tRNA stability (found for all mutations investigated), reduced aminoacylation, decreased binding to translation factor mtEFTu or the mitoribosome, and disturbed codon reading.211 However, cases are known where mitochondrial translation was not or only slightly affected despite clear impairment of the OXPHOS system.e.g., references220,221 Possibly, this is due to toxic effects of premature translation products generated by the absence of the correctly functioning tRNA.222 These peptides could interfere with the assembly of the OXPHOS complexes or exert their toxic effect through interactions with other (non)mitochondrial components, while a quantitative deficit in mitochondrial protein synthesis cannot be detected.

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Table 1. Genes involved in the biogenesis or maintenance of multiple OXPHOS complexes Chapter and implicated in mitochondrial disorders Affected process Gene Protein (function) References 2 Combined OXPHOS deficiencies with normal complex II activities0 MtDNA replication POLG Polymerase y catalytic subunit [13,213] POLG2 Polymerase y accessory subunit [13,214] C10orf2 Twinkle (mtDNA helicase) [13] DGUOK Deoxyguanosine kinase [13] TK2 Thymidine kinase 2 [13] TYMP Endothelial cell growth factor 1 (thymidine phosphorylase) [13] Nucleotide synthesis SLC25A4 Adenine nucleotide translocator 1 [13] and transport SLC25A3 Solute carrier family 25 member [43,215]c 3 (phosphate transporter) SUCLG1 Succinate-CoA ligase a-subunit [43,216] SUCLA2 Succinate-CoA ligase ß-subunit [13,216] RRM2B Ribonucleotide reductase M2 B [13,217] MPV17 Mt inner membrane protein [13] 22 mitochondrial tRNA genes [12] 2 mitochondrial rRNA genes [12] GFM1 Mt translation elongation factor G1 [15] TSFM Mt translation elongation factor Ts [21] TUFM Mt translation elongation factor Tu [22] Mt translation MRPS16 Mt ribosomal protein S16 [18] MRPS22 Mt ribosomal protein S22 [19] PUS1 Pseudouridine synthase 1 [14] TRMU tRNA 5-methylaminomethyl- [24] 2-thiouridylate methyltransferase DARS2 Mt aspartyl-tRNA synthetase 2 [20]c RARS2 Mt arginyl-tRNA synthetase 2 [16] Other combined OXPHOS deficienciesb TIMM8A Translocase of inner mt membrane 8 [74]c Mt protein import homolog A (small TIM complex subunit) DNAJC19 DnaJ homolog, subfamily C, member 19 [76] (TIM23 complex subunit) TAZ Tafazzin (cardiolipin metabolism) [203,218] Mt membrane biogenesis OPA1 Optic atrophy 1 (mt fusion) [205] and maintenance MFN2 Mitofusin 2 (mt fusion) [205] DNM1L Dynamin 1-like [207]c (mt and peroxisomal fission) Mt protein processing SPG7 Spastic paraplegia 7 or paraplegin [144,219] and quality control (m-AAA protease subunit)

" Based on the function of the affected proteins, a combined complex I, III, IV and V deficiency would be expected, however, not always do all these enzyme complexes display decreased activities. b All OXPHOS complexes are expected to malfunction based on the function of the affected proteins; nonetheless, large variations have been found in the exact OXPHOS complexes involved. c The OXPHOS complexes showed normal activities.

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The best-studied mitochondrial tRNA mutations are 3243A>G in tRNALeu(UUR) Chapter (MT-TL1) and 8344A>G in tRNALys (MT-TK). The 3243A>G mutation is one of the most common mutations and causes a range of clinical phenotypes, of 2 which MELAS is the most prevalent.223 There is controversy over the pathogenic mechanism of the 3243A>G mutation: both loss-of-function (due to poor aminoacylation, reduced stability or lack of wobble-base U hypermodification) and gain-of-function (due to lack of the hypermodification) of the mutant tRNA have been proposed.224 The post-transcriptional taurine modification at the anticodon wobble position is needed to restrict decoding to leucine UUR codons. Loss of this modification leads to varying degrees of mitochondrial translation malfunctioning in different cellular backgrounds through a combination of a decoding defect of UUG (and UUA) codons (loss-of-function) and amino acid misincorporation (gain-of-function).126,225 Additionally, the 3243A>G mutation was shown to diminish 16S rRNA transcription termination and alter processing of the primary transcript,61,226 but these effects are likely to contribute less to the disease etiology than the previously mentioned mechanisms. The 8344A>G mutation is associated with MERRF (myoclonic epilepsy with ragged-red fibers). It has also been reported to affect aminoacylation and taurine modification of the wobble-base U, the latter which abolishes codon-anticodon pairing on the mitoribosomes for both tRNALys codons.225,227 This generates a marked decrease in mitochondrial protein synthesis that is most pronounced in proteins with a high lysine content and is believed to result from premature translation termination. Most rRNA mutations have been reported in the 12S rRNA gene (MT-RNR1) and all of these are associated with nonsyndromic sensorineural hearing loss or aminoglycoside-induced deafness, with the 1555A>G mutation forming one of the most common causes.12,228 This mutation is located in the decoding site of the mitoribosomal SSU and results in a secondary rRNA structure that more closely resembles the corresponding region of the bacterial 16S rRNA, impairing mitochondrial protein synthesis and facilitating interaction with aminoglycoside antibiotics, which again exacerbates the translation defect. The mutation alone does not lead to disease, only in combination with modulators such as the aminoglycosides, mitochondrial haplotypes and nuclear modifier genes (e.g., TFB1M, as already mentioned in Section 3.3). In the 16S rRNA gene (MT-RNR2) merely 3 mutations have been found: 2835C>T, 3093C>G, and 3196G>A.12 These mutations are thought to be associated with Rett syndrome, MELAS, and Alzheimer and Parkinson disease, respectively.229-231 Nevertheless, further investigations are necessary to determine their pathogenicity.

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Chapter 5.2. nDNA mutations Up till now, mutations in nine nuclear genes implicated in mitochondrial protein 2 synthesis have been associated with mitochondrial disorders. The first report was a homozygous missense mutation in the tRNA modifying gene PUS1,14 and shortly thereafter homozygous mutations were detected in the genes for elongation factor mtEFG115 and mitoribosomal protein MRPS16.18 Subsequently, four compound heterozygous mutations in GFM1 (coding for mtEFG1)22,25 and homozygous mutations in TUFM (encoding mtEFTu),22 TSFM (coding for mtEFTs),21 MRPS22,19 PUS1 again,17 and in the arginine tRNA synthetase gene (RARS2)16 were found, bringing the total number of mutations on 12. Additionally, several compound heterozygous mutations have been reported in the gene for mitochondrial aspartyl-tRNA synthetase (DARS2).2023 Recently, 9 mutations were identified in the gene for another tRNA modifying gene, TRMU.24 All patients harboring these mutations show combined OXPHOS deficiencies, with (near) normal complex II activities, and a clear defect in mitochondrial translation (the latter was not tested for the MRPS22, RARS2 and DARS2 mutations). The one exception is DARS2: surprisingly, Blue-native PAGE as well as spectrophotometric measurements revealed normal OXPHOS enzyme activities.20 The clinical features differ substantially between all patients and even between patients that carry the same mutation, but generally the mutations result in severe and early-fatal diseases. As already mentioned in Section 2.3, defects in the tRNA-modifying enzymes PUS1 and TRMU can result in mitochondrial disease. PUS1 converts uridine into pseudouridine at several cytoplasmic and mitochondrial tRNA positions and thereby improves translation efficiency in the cytosol as well as the mitochondrion.17 Thus it is not part of the translation machinery, but it is required for protein synthesis due to its function in post-transcriptional modification of tRNAs. Pseudouridylation is the most frequently found modification in tRNAs,232 however, the exact function is not entirely clear. The marked variability in the severity of the MLASA syndrome, despite the presence of an identical PUS1 mutation, could partly be explained by the dual localization of PUS1.17 A defect in PUS1 therefore impairs both cytosolic and mitochondrial translation, resulting in corresponding clinical symptoms that can vary due to individual differences in compensation mechanisms in both cell compartments. Bykhovskaya et al suggested that compensatory changes in transcript levels of ribosomal proteins can overcome the lack of pseudouridylation of tRNAs and that pleiotropic effects of PUS1 on non-tRNA substrates involved in transcription and iron metabolism are a major cause of the disease phenotype.233 Notably, complex II can be affected slightly in addition to the other OXPHOS complexes that all contain mtDNA- encoded subunits.17 This could be a primary effect of the decrease in cytosolic

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translation or it could be a secondary effect of the mitochondrial translation Chapter deficit, leading to disruption of the mtDNA-dependent complexes, which can subsequently cause destabilization of the entire OXPHOS system. TRMU (tRNA 2 5-methylaminomethyl-2-thiouridylate methyltransferase) is responsible for the 2-thio modification of the wobble-base of the mitochondrial tRNALys, tRNAGln and tRNAGlu. Defects in this enzyme result in reduced steady-state levels of these three tRNAs and consequently impaired mitochondrial protein synthesis.24,62 The 2-thiouridylation is reported to be critical for effective codon-anticodon interaction and ribosome binding.234,235 Concerning the mitochondrial translation factors, defects have only been found in proteins involved in the elongation phase, in all elongation factors except for mtEFG2, which appears to function in termination instead elongation. In GFM1, mutations have been reported in nearly all protein domains, leading to severe hepato(encephalo)pathy.15,22,25 All mutations result in a marked global translation defect, with the strongest deficit in the three complex IV subunits and the two complex I subunits ND5 and ND6. Both subunits of complex V (ATP6 and ATP8) show normal or even increased synthesis rates, possibly caused by more efficient protein synthesis of bicistronic compared to monocistronic mRNAs, which would then also explain the near normal mitochondrial translation levels of ND4 and ND4L. This variable pattern in translation impairment was also found for a mutation in TSFM,21 which will be described below. Surprisingly, tissues are selectively affected by GFM1 mutations, in spite of its ubiquitous expression, with liver being most severely affected and heart hardly showing a defect.25 This tissue specificity appears to result from differences among tissues in the relative ratios of the elongation factors and in adaptive changes herein in response to dysfunction. For example, transcription of TUFM was upregulated in cardiac tissue in patients with a GFM1 mutation and overexpression of either TUFM or TSFM in control and patient fibroblasts impaired mitochondrial translation. Overexpression of GFM2, on the contrary, did not have a clear effect on protein synthesis in either control or patient cells.1525 Remarkably, mtEFTu or mtEFG2, but not mtEFTs or mtEFG1, can partially suppress the combined OXPHOS system defect caused by the 3243A>G mutation in tRNALeu(UUR).126 These observations evidence that efficient mitochondrial translation partly depends on appropriate ratios of the elongation factors. A homozygous mutation in TUFM was shown to be responsible for rapidly progressive encephalopathy.22 The mutation, located in the tRNA-binding region of mtEFTu, hampers the formation of the ternary complex with GTP and an aminoacylated tRNA, resulting in a severe decrease in mitochondrial protein synthesis.22,236 Notably, a homozygous mutation in TSFM led to encephalomyopathy in one patient and hypertrophic cardiomyopathy in another.21 This could be due to individual differences in relative abundance of

47 Chapter 2

Chapter the translation factors and compensatory mechanisms in the various tissues. Alternatively, as yet unknown genetic modifiers of the mitochondrial translation 2 machinery could be involved. Steady-state levels of not only mtEFTs but also mtEFTu were reduced, and overexpression of either factor rescued the OXPHOS deficiency and translation defect. The most likely explanation for these findings is that the mutation, situated in a subdomain of mtEFTs that interacts with mtEFTu, destabilizes the mtEFTu-mtEFTs complex and promotes turnover of its components. Additional mtEFTu or mtEFTs would then stabilize the complex. Of all 81 human MRPs, mutations have been found in merely two of them: MRPS16 and MRPS22.1819 Both defects resulted in a marked decrease in the 12S rRNA transcript level, probably caused by impaired assembly of the mitoribosomal small subunit, generating unincorporated and instable 12S rRNA. MRPS16 is evolutionary highly conserved, however, MRPS22 is only present in metazoa.107 Recently, both proteins were shown to be important for assembly of the SSU.123 In fibroblasts from patients with a MRPS16 or MRPS22 mutation, the level of MRPS11 was significantly reduced, whereas considerable amounts of MRPS2 were present. Furthermore, MRPS16 was barely detectable in the MRPS22-mutated patient. The presence of MRPS22 was not determined in these patients. On the other hand, near normal levels of MRPL13, MRPL15123 as well as 16S rRNA18,19 were found. These observations indicate that both MRPS16 and MRPS22 are essential for assembly and stability of the SSU. A lack of these MRPs results in the failure to assemble part of the mitoribosome, containing at least MRPS11, MRPS16, MRPS22 and 12S rRNA, and subsequent degradation of its components. Both a macromolecular complex containing MRPS2 and the mitoribosomal large subunit can still be formed in the absence of a functional SSU, suggesting that the assembly of the mitoribosome is a process consisting of relatively independent subassembly steps. Mutations in the mitochondrial arginyl- and aspartyl-tRNA synthetases (RARS2 and DARS2) are associated with severe encephalopathy with pontocerebellar hypoplasia and LBSL (leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation), respectively.16,20,23 In both genes, intronic mutations that affect splicing were detected. The absence of extracerebral symptoms might be explained by a potential difference in abundance of splicing factors between brain and the unaffected tissues, enabling synthesis of small amounts of wild-type transcript of the synthetases in most tissues. Alternatively, the vulnerability of the brain for aminoacyl-tRNA synthetase defects could be due to the high expression of mitochondrial tRNAs in this tissue.237 The tRNAArg transcript is scarcely present, but almost fully acylated, in patient fibroblasts harboring a RARS2 mutation. Presumably, the little available wild-type RARS2 can aminoacylate a small portion of the tRNAArg molecules and the uncharged

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transcripts then become unstable. This will impair mitochondrial protein synthesis, Chapter which has only been confirmed in yeast.238 In contrast, DARS2 mutations do not seem to affect mitochondrial translation and likewise do not result in defects 2 of the OXPHOS complexes, notwithstanding a clear reduction in aminoacylation activity.20 The reason for this is currently not understood. Besides these nine gene products, numerous proteins are indirectly involved in mitochondrial translation, as should be evident from the current review, and defects in these proteins could undoubtedly also interfere with the translation process. For example, lack of the protease paraplegin (see Section 3.3 for information on its function) results in impaired mitochondrial translation in yeast and in a hereditary spastic paraplegia (HSP) mouse model.143 Nonetheless, HSP patients with mutations in paraplegin (HSP7) do not show consistent OXPHOS enzyme deficiencies;239-241 often only or mainly complex I is affected, while a combined defect would be expected. The selective involvement of certain neurons could in this case be rationalized by tissue-specific differences in the expression of m-AAA protease subunits and their assembly into proteolytic complexes, which vary in their subunit composition depending on subunit availability.242 This is analogous to the importance of elongation factor ratios for efficient mitochondrial translation and tissue-specific variability herein.

6 . F u t u r e p r o s p e c t s We have provided an extensive overview of the proteins and processes (in)directly involved in mitochondrial translation and the biogenesis of the OXPHOS system. Even though our understanding of the mechanisms implicated in mitochondrial disease has increased rapidly over the last two decades, it is far from complete. Due to the multitude of proteins and intricacy of the processes needed for a properly functioning OXPHOS system, identifying the genetic defect that underlies an OXPHOS deficiency is not an easy task. The shortage of large or consanguineous families as well as the substantial clinical and genetic heterogeneity of mitochondrial disorders complicate the search by limiting the available strategies. For instance, techniques such as linkage analysis and homozygosity mapping that form powerful tools in combination with whole-genome experimental data sets136,243 often cannot be applied and mutation chips are currently only available for the mtDNA. Consequently, the molecular basis of many OXPHOS disorders remains unresolved. In the future, molecular genetic diagnosis of patients suspected to suffer from a mitochondrial disorder might no longer require extensive investigations that integrate information from clinical phenotype, family history, brain imaging and laboratory findings to direct the laborious tasks of screening known candidate genes and, when this is unsuccessful, searching for new genetic causes.43 Instead, recent progress in the development of next-generation DNA sequencing technologies,

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Chapter which are much cheaper and faster than the conventional approach of polymerase chain reaction followed by capillary sequencing, indicates that within the next few 2 decades high-throughput sequencing could become a feasible option for mutation detection.244 245 These methods are anticipated to eventually enable sequencing of the entire human genome for under $1000 within a day, allowing their routine clinical use and accelerating the discovery of novel disease genes. Exome sequencing, that is the targeted sequencing of all protein-coding regions, offers an alternative to whole-genome sequencing by facilitating direct identification of the causative gene at a fraction of the costs.246,247 However, much remains to be achieved, for example proper bioinformatic tools to deal with the tremendous amounts of data, before such technologies can be readily applied to elucidate the genetic etiology of OXPHOS deficiencies and other disorders. Systems biology techniques will keep increasing our knowledge of the mechanisms underlying complex diseases and in combination with high-throughput sequencing these approaches will advance disease-gene discovery even more. Integrative analysis of functional data is useful especially for gaining insight into the scarcely understood field of modifier genes, which are thought to account for part of the clinical variability seen in mitochondrial diseases. Systematic mapping of genetic interactions revealed a class of modifier or "hub" genes that are proposed to enhance the phenotypic consequences of mutations in many different genes, the "specifier" genes that define the specific disorders, and thus serve as global modifier genes in multiple mechanistically unrelated disorders.248 Hopefully, these and other important findings will lead to the discovery of additional modifier genes implicated in mitochondrial disorders. Progress in mutation detection, both in specifier and modifier genes, is crucial for extending the possibilities for genetic counseling, prenatal diagnosis, and interventions to prevent transmission now and to cure these serious disorders in the future. Currently, no effective therapy is available; the various existing treatment strategies are mainly supportive.249250 Gene therapy might offer a solution since it allows for curative treatment without the need for a clear genotype-phenotype correlation, which is often lacking in mitochondrial disorders. Although the development is still in its infancy for both mtDNA and nDNA gene therapy and many challenges are to be overcome, promising results have been obtained in cell cultures and animal models, providing hope for a cure in the not-too-distant future. Thus rapid advances in technologies and consequently in our understanding of the pathogenesis of OXPHOS defects should lead to the ultimate goal of effectively preventing and curing these often devastating disorders.

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A cknowledgements Chapter This work was supported by the European Union's Sixth Framework Program, contract number LSHMCT-2004-005260 (MITOCIRCLE). 2

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158. Koppen M, Langer T: Protein degradation within mitochondria: versatile activities of Chapter AAA proteases and other peptidases. Crit Rev Biochem Mol Biol 2007; 42: 221-242. 2 159. Nijtmans LG, Artal SM, Grivell LA, Coates PJ: The mitochondrial PHB complex: roles in mitochondrial respiratory complex assembly, ageing and degenerative disease. Cell Mol Life Sci 2002; 59: 143-155. 160. Artal-Sanz M, Tavernarakis N: Prohibitin and mitochondrial biology. Trends Endocrinol Metab 2009; 20: 394-401. 161. Liu M, Spremulli L: Interaction of mammalian mitochondrial ribosomes with the inner membrane. J Biol Chem 2000; 275: 29400-29406. 162. Hell K, Herrmann JM, Pratje E, Neupert W, Stuart RA: Oxa1p, an essential component of the N-tail protein export machinery in mitochondria. Proc Natl Acad Sci USA 1998; 95: 2250-2255. 163. Hell K, Neupert W, Stuart RA: Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA. EMBO J 2001; 20: 1281-1288. 164. Ott M, Prestele M, Bauerschmitt H, Funes S, Bonnefoy N, Herrmann JM: Mba1, a membrane-associated ribosome receptor in mitochondria. EMBO J 2006; 25: 1603-1610. 165. Sanchirico ME, Fox TD, Mason TL: Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs. EMBO J 1998; 17: 5796-5804. 166. Watson K: The organization of ribosomal granules within mitochondrial structures of aerobic and anaerobic cells of Saccharomyces cerevisae. J Cell Biol 1972; 55: 721-726. 167. Stuart R: Insertion of proteins into the inner membrane of mitochondria: the role of the Oxa1 complex. Biochim Biophys Acta 2002; 1592: 79-87. 168. Bonnefoy N, Fiumera HL, Dujardin G, Fox TD: Roles of Oxa1-related inner-membrane translocases in assembly of respiratory chain complexes. Biochim Biophys Acta 2009; 1793: 60-70. 169. Jia L, Dienhart M, Schramp M, McCauley M, Hell K, Stuart RA: Yeast Oxa1 interacts with mitochondrial ribosomes: the importance of the C-terminal region of Oxa1. EMBO J 2003; 22: 6438-6447. 170. Jia L, Kaur J, Stuart RA: Mapping of the Saccharomyces cerevisiae Oxa1-mitochondrial ribosome interface and identification of MrpL40, a ribosomal protein in close proximity to Oxa1 and critical for oxidative phosphorylation complex assembly. Eukaryot Cell 2009; 8: 1792-1802. 171. Preuss M, Leonhard K, Hell K, Stuart RA, Neupert W, Herrmann JM: Mba1, a novel component of the mitochondrial protein export machinery of the yeast Saccharomyces cerevisiae. J Cell Biol 2001; 153: 1085-1096. 172. Schlickum S, Moghekar A, Simpson JC et al: LETM1, a gene deleted in Wolf-Hirschhorn syndrome, encodes an evolutionarily conserved mitochondrial protein. Genomics 2004; 83: 254-261.

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173. Frazier AE, Taylor RD, Mick DU et al: Mdm38 interacts with ribosomes and is a component Chapter of the mitochondrial protein export machinery. J Cell Biol 2006; 172: 553-564. 174. Piao L, Li Y, Kim SJ et al: Association of LETM1 and MRPL36 contributes to the 2 regulation of mitochondrial ATP production and necrotic cell death. Cancer Res 2009; 69: 3397-3404. 175. Tamai S, Iida H, Yokota S et al: Characterization of the mitochondrial protein LETM1, which maintains the mitochondrial tubular shapes and interacts with the AAA-ATPase BCS1L. J Cell Sci 2008; 121: 2588-2600. 176. Dimmer KS, Navoni F, Casarin A et al: LETM1, deleted in Wolf-Hirschhorn syndrome is required for normal mitochondrial morphology and cellular viability. Hum Mol Genet 2008; 17: 201-214. 177. Lynch AS, Wang JC: Anchoring of DNA to the bacterial cytoplasmic membrane through cotranscriptional synthesis of polypeptides encoding membrane proteins or proteins for export: a mechanism of plasmid hypernegative supercoiling in mutants deficient in DNA topoisomerase I. J Bacteriol 1993; 175: 1645-1655. 178. Vos-Scheperkeuter GH, Witholt B: Co-translational insertion of envelope proteins: theoretical consideration and implications. Ann Microbiol (Paris) 1982; 133A: 129-138. 179. Trinei M, Vannier JP, Beurton-Aimar M, Norris V: A hyperstructure approach to mitochondria. Mol Microbiol 2004; 53: 41-53. 180. Fernandez-Vizarra E, Tiranti V, Zeviani M: Assembly of the oxidative phosphorylation system in humans: what we have learned by studying its defects. Biochim Biophys Acta 2009; 1793: 200-211. 181. Ogilvie I, Kennaway NG, Shoubridge EA: A molecular chaperone for mitochondrial complex I assembly is mutated in a progressive encephalopathy. J Clin Invest 2005; 115: 2784-2792. 182. Vogel RO, Janssen RJ, Ugalde C et al: Human mitochondrial complex I assembly is mediated by NDUFAF1. FEBS J 2005; 272: 5317-5326. 183. Hoefs SJ, Dieteren CE, Rodenburg RJ et al: Baculovirus complementation restores a novel NDUFAF2 mutation causing complex I deficiency. Hum Mutat 2009; 30: E728-E736. 184. Saada A, Vogel RO, Hoefs SJ et al: Mutations in NDUFAF3 (C3ORF60), encoding an NDUFAF4 (C6ORF66)-interacting complex I assembly protein, cause fatal neonatal mitochondrial disease. Am J Hum Genet 2009; 84: 718-727. 185. Saada A, Edvardson S, Rapoport M et al: C6ORF66 is an assembly factor of mitochondrial complex I. Am J Hum Genet 2008; 82: 32-38. 186. Vogel RO, Janssen RJ, van den Brand MA et al: Cytosolic signaling protein Ecsit also localizes to mitochondria where it interacts with chaperone NDUFAF1 and functions in complex I assembly. Genes Dev 2007; 21: 615-624. 187. Pagliarini DJ, Calvo SE, Chang B et al: A mitochondrial protein compendium elucidates complex I disease biology. Cell 2008; 134: 112-123. 63 Chapter 2

188. Gerards M, Sluiter W, van den Bosch BJ et al: Defective complex I assembly due to Chapter C20orf7 mutations as a new cause of Leigh syndrome. J Med Genet 2009; 47: 507-512. 2 189. Sugiana C, Pagliarini DJ, McKenzie M et al: Mutation of C20orf7 disrupts complex I assembly and causes lethal neonatal mitochondrial disease. Am J Hum Genet 2008; 83: 468-478. 190. Vogel RO, Smeitink JA, Nijtmans LG: Human mitochondrial complex I assembly: a dynamic and versatile process. Biochim Biophys Acta 2007; 1767: 1215-1227. 191. Vahsen N, Cande C, Briere JJ et al: AIF deficiency compromises oxidative phosphorylation. EMBO J 2004; 23: 4679-4689. 192. Bych K, Kerscher S, Netz DJ et al: The iron-sulphur protein Ind1 is required for effective complex I assembly. EMBO J 2008; 27: 1736-1746. 193. Ghezzi D, Goffrini P, Uziel G et al: SDHAF1, encoding a LYR complex-II specific assembly factor, is mutated in SDH-defective infantile leukoencephalopathy. Nat Genet 2009; 41: 654-656. 194. de Lonlay P, Valnot I, Barrientos A et al: A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure. Nat Genet 2001; 29: 57-60. 195. Pecina P, Houstkova H, Hansikova H, Zeman J, Houstek J: Genetic defects of cytochrome c oxidase assembly. Physiol Res 2004; 53: S213-S223. 196. Wang ZG, White PS, Ackerman SH: Atp11p and Atp12p are assembly factors for the F(1)-ATPase in human mitochondria. J Biol Chem 2001; 276: 30773-30778. 197. Zeng X, Neupert W, Tzagoloff A: The metalloprotease encoded by ATP23 has a dual function in processing and assembly of subunit 6 of mitochondrial ATPase. Mol Biol Cell 2007; 18: 617-626. 198. Stiburek L, Fornuskova D, Wenchich L, Pejznochova M, Hansikova H, Zeman J: Knockdown of human Oxa1l impairs the biogenesis of F1Fo-ATP synthase and NADH:ubiquinone oxidoreductase. J Mol Biol 2007; 374: 506-516. 199. Dudkina NV, Sunderhaus S, Boekema EJ, Braun HP: The higher level of organization of the oxidative phosphorylation system: mitochondrial supercomplexes. J Bioenerg Biomembr 2008; 40: 419-424. 200. Schon EA, Dencher NA: Heavy breathing: energy conversion by mitochondrial respiratory supercomplexes. Cell Metab 2009; 9: 1-3. 201. Acin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, Enriquez JA: Respiratory active mitochondrial supercomplexes. Mol Cell 2008; 32: 529-539. 202. Schagger H, de Coo R, Bauer MF, Hofmann S, Godinot C, Brandt U: Significance of respirasomes for the assembly/stability of human respiratory chain complex I. J Biol Chem 2004; 279: 36349-36353. 203. McKenzie M, Lazarou M, Thorburn DR, Ryan MT: Mitochondrial respiratory chain supercomplexes are destabilized in Barth Syndrome patients. J Mol Biol 2006; 361: 462-469.

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204. Chen H, Chan DC: Emerging functions of mammalian mitochondrial fusion and fission. Chapter Hum Mol Genet 2005; 14: R283-R289. 205. Liesa M, Palacin M, Zorzano A: Mitochondrial dynamics in mammalian health and 2 disease. Physiol Rev 2009; 89: 799-845. 206. Parone PA, Da Cruz S, Tondera D et al: Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA. PLoS One 2008; 3: e3257. 207. Waterham HR, Koster J, van Roermund CW, Mooyer PA, Wanders RJ, Leonard JV: A lethal defect of mitochondrial and peroxisomal fission. N Engl J Med 2007; 356: 1736-1741. 208. Holt IJ, Harding AE, Morgan-Hughes JA: Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 1988; 331: 717-719. 209. Wallace DC, Singh G, Lott MT et al: Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 1988; 242: 1427-1430. 210. Scaglia F, Wong LJ: Human mitochondrial transfer RNAs: role of pathogenic mutation in disease. Muscle Nerve 2008; 37: 150-171. 211. Florentz C, Sohm B, Tryoen-Toth P, Putz J, Sissler M: Human mitochondrial tRNAs in health and disease. Cell Mol Life Sci 2003; 60: 1356-1375. 212. Zifa E, Giannouli S, Theotokis P, Stamatis C, Mamuris Z, Stathopoulos C: Mitochondrial tRNA mutations: clinical and functional perturbations. RNA Biol 2007; 4: 38-66. 213. Chan SS, Copeland WC: DNA polymerase gamma and mitochondrial disease: understanding the consequence of POLG mutations. Biochim Biophys Acta 2009; 1787: 312-319. 214. Longley MJ, Clark S, Yu Wai MC et al: Mutant POLG2 disrupts DNA polymerase gamma subunits and causes progressive external ophthalmoplegia. Am J Hum Genet 2006; 78: 1026-1034. 215. Mayr JA, Merkel O, Kohlwein SD et al: Mitochondrial phosphate-carrier deficiency: a novel disorder of oxidative phosphorylation. Am J Hum Genet 2007; 80: 478-484. 216. Ostergaard E, Christensen E, Kristensen E et al: Deficiency of the alpha subunit of succinate-coenzyme A ligase causes fatal infantile lactic acidosis with mitochondrial DNA depletion. Am J Hum Genet 2007; 81: 383-387. 217. Bourdon A, Minai L, Serre V et al: Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat Genet 2007; 39: 776-780. 218. Hauff KD, Hatch GM: Cardiolipin metabolism and Barth Syndrome. Prog Lipid Res 2006; 45: 91-101. 219. Rugarli EI, Langer T: Translating m-AAA protease function in mitochondria to hereditary spastic paraplegia. Trends Mol Med 2006; 12: 262-269. 220. Janssen GMC, Maassen JA, van Den Ouweland JM: The diabetes-associated 3243 mutation in the mitochondrial tRNA(Leu(UUR)) gene causes severe mitochondrial dysfunction

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Chapter without a strong decrease in protein synthesis rate. J Biol Chem 1999; 274: 29744-29748. 221. Toompuu M, Tiranti V, Zeviani M, Jacobs HT: Molecular phenotype of the np 7472 2 deafness-associated mitochondrial mutation in osteosarcoma cell cybrids. Hum Mol Genet 1999; 8: 2275-2283. 222. Jacobs HT: Disorders of mitochondrial protein synthesis. Hum Mol Genet 2003; 12: R293-R301. 223. Finsterer J: Genetic, pathogenetic, and phenotypic implications of the mitochondrial A3243G tRNALeu(UUR) mutation. Acta Neurol Scand 2007; 116: 1-14. 224. Jacobs HT, Holt IJ: The np 3243 MELAS mutation: damned if you aminoacylate, damned if you don't. Hum Mol Genet 2000; 9: 463-465. 225. Kirino Y, Yasukawa T, Ohta S et al: Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease. Proc Natl Acad Sci USA 2004; 101: 15070-15075. 226. King MP, Koga Y, Davidson M, Schon EA: Defects in mitochondrial protein synthesis and respiratory chain activity segregate with the tRNA(Leu(UUR)) mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes. Mol Cell Biol 1992; 12: 480-490. 227. Enriquez JA, Chomyn A, Attardi G: MtDNA mutation in MERRF syndrome causes defective aminoacylation of tRNA(Lys) and premature translation termination. Nat Genet 1995; 10: 47-55. 228. Xing G, Chen Z, Cao X: Mitochondrial rRNA and tRNA and hearing function. Cell Res 2007; 17: 227-239. 229. Cardaioli E, Dotti MT, Hayek G, Zappella M, Federico A: Studies on mitochondrial pathogenesis of Rett syndrome: ultrastructural data from skin and muscle biopsies and mutational analysis at mtDNA nucleotides 10463 and 2835. J Submicrosc Cytol Pathol 1999; 31: 301-304. 230. Hsieh RH, Li JY, Pang CY, Wei YH: A novel mutation in the mitochondrial 16S rRNA gene in a patient with MELAS syndrome, diabetes mellitus, hyperthyroidism and cardiomyopathy. J Biomed Sci 2001; 8: 328-335. 231. Shoffner JM, Brown MD, Torroni A et al: Mitochondrial DNA variants observed in Alzheimer disease and Parkinson disease patients. Genomics 1993; 17: 171-184. 232. Rozenski J, Crain PF, McCloskey JA: The RNA Modification Database: 1999 update. Nucleic Acids Res 1999; 27: 196-197. 233. Bykhovskaya Y, Mengesha E, Fischel-Ghodsian N: Pleiotropic effects and compensation mechanisms determine tissue specificity in mitochondrial myopathy and sideroblastic anemia (MLASA). Mol Genet Metab 2007; 91: 148-156. 234. Ashraf SS, Sochacka E, Cain R, Guenther R, Malkiewicz A, Agris PF: Single atom modification (O-->S) of tRNA confers ribosome binding. RNA 1999; 5: 188-194.

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235. Yarian C, Marszalek M, Sochacka E et al: Modified nucleoside dependent Watson- Chapter Crick and wobble codon binding by tRNALysUUU species. Biochemistry 2000; 39: 13390-13395. 2 236. Valente L, Shigi N, Suzuki T, Zeviani M: The R336Q mutation in human mitochondrial EFTu prevents the formation of an active mt-EFTu.GTP.aa-tRNA ternary complex. Biochim Biophys Acta 2009; 1792: 791-795. 237. Dittmar KA, Goodenbour JM, Pan T: Tissue-specific differences in human transfer RNA expression. PLoS Genet 2006; 2: e221. 238. Tzagoloff A, Shtanko A: Mitochondrial and cytoplasmic isoleucyl-, glutamyl- and arginyl-tRNA synthetases of yeast are encoded by separate genes. Eur J Biochem 1995; 230: 582-586. 239. Arnoldi A, Tonelli A, Crippa F et al: A clinical, genetic, and biochemical characterization of SPG7 mutations in a large cohort of patients with hereditary spastic paraplegia. Hum Mutat 2008; 29: 522-531. 240. Atorino L, Silvestri L, Koppen M et al: Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia. J Cell Biol 2003; 163: 777-787. 241. Wilkinson PA, Crosby AH, Turner C et al: A clinical, genetic and biochemical study of SPG7 mutations in hereditary spastic paraplegia. Brain 2004; 127: 973-980. 242. Koppen M, Metodiev MD, Casari G, Rugarli EI, Langer T: Variable and tissue-specific subunit composition of mitochondrial m-AAA protease complexes linked to hereditary spastic paraplegia. Mol Cell Biol 2007; 27: 758-767. 243. Tiranti V, D'Adamo P, Briem E et al: Ethylmalonic encephalopathy is caused by mutations in ETHE1, a gene encoding a mitochondrial matrix protein. Am J Hum Genet 2004; 74: 239-252. 244. Ansorge WJ: Next-generation DNA sequencing techniques. N Biotechnol 2009; 25: 195-203. 245. Tucker T, Marra M, Friedman JM: Massively parallel sequencing: the next big thing in genetic medicine. Am J Hum Genet 2009; 85: 142-154. 246. Ng SB, Turner EH, Robertson PD et al: Targeted capture and massively parallel sequencing of 12 human exomes. Nature 2009; 461: 272-276. 247. Porreca GJ, Zhang K, Li JB et al: Multiplex amplification of large sets of human exons. Nat Methods 2007; 4: 931-936. 248. Lehner B, Crombie C, Tischler J, Fortunato A, Fraser AG: Systematic mapping of genetic interactions in Caenorhabditis elegans identifies common modifiers of diverse signaling pathways. Nat Genet 2006; 38: 896-903. 249. DiMauro S, Mancuso M: Mitochondrial diseases: therapeutic approaches. Biosci Rep 2007; 27: 125-137. 250. Koene S, J. Smeitink J: Mitochondrial medicine: entering the era of treatment. J Intern Med 2009; 265: 193-209. 67 Translation is the art of failure. Umberto Eco

The original is unfaithful to the translation. Jorge Luis Borges C h apter 3 Functional consequences of mitochondrial tRNATrp and tRNAArg mutations causing combined OXPHOS defects

• t ì *

Paulien Smits, Sandy Mattijssen, Eva Morava, Mariël A.M. van den Brand, Frans C.A. van den Brandt, Frits A. Wijburg, Ger J.M. Pruijn, Jan A.M. Smeitink, Leo G.J. Nijtmans, Richard J.T. Rodenburg and Lambert P. van den Heuvel

European Journal of Human Genetics 2010; 18(3): 324-329 Chapter 3

A b s t r a c t Combined oxidative phosphorylation (OXPHOS) system deficiencies are a group of mitochondrial disorders that are associated with a range of clinical phenotypes and genetic defects. They occur in approximately 30% of all OXPHOS disorders Chapter and around 4% are combined complex I, III and IV deficiencies. In this study we present two mutations in the mitochondrial tRNATrp (MT-TW) and tRNAArg (MT-TR) 3 genes, m.5556G>A and m.10450A>G, respectively, which were detected in two unrelated patients showing combined OXPHOS complex I, III and IV deficiencies and progressive multisystemic diseases. Both mitochondrial tRNA mutations were almost homoplasmic in fibroblasts and muscle tissue of the two patients and not present in controls. Patient fibroblasts showed a general mitochondrial translation defect. The mutations resulted in lowered steady-state levels and altered conformations of the tRNAs. Cybrid cell lines showed similar tRNA defects and impairment of OXPHOS complex assembly as patient fibroblasts. Our results show that these tRNATrp and tRNAArg mutations cause the combined OXPHOS deficiencies in the patients, adding to the still expanding group of pathogenic mitochondrial tRNA mutations.

70 Mitochondrial tRNATrp and tRNAArg mutations

I ntroduction Combined oxidative phosphorylation (OXPHOS) system deficiencies, in which two or more enzyme complexes show a decrease in activity, occur in around 30% of all OXPHOS disorders diagnosed at our center, and approximately 4% Chapter consist of combined complex I, III and IV defects.1 Candidate genes for these deficiencies are, among others, all genes encoding proteins and RNAs involved in mitochondrial translation. Protein synthesis in the mitochondrion, which is 3 responsible for the production of 13 polypeptides of the OXPHOS complexes I, III, IV and V, is executed by mitochondrial DNA (mtDNA)-encoded rRNAs and tRNAs and a multitude of nuclear-encoded factors. Although tRNA-encoding genes make up only 9% of the entire mitochondrial genome, over 40% of all point mutations reported in the mtDNA are located in tRNA genes.2 In contrast, rRNA sequences cover 15% of the mtDNA, but mutations in the two rRNAs account for only 4% of all mtDNA mutations. In addition, in recent years mutations in various nuclear-encoded proteins involved in the mitochondrial translation process have been found and this group of gene defects is expanding rapidly.3 This study describes two mutations, m.5556G>A in the tRNATrp and m.10450A>G in the tRNAArg gene, that lead to combined OXPHOS deficiencies and shows their pathogenicity.

M a t e r i a l s a n d m e t h o d s Case reports Patient T. This female child was born at term as the first child of healthy unrelated parents. She had feeding problems from birth and gastroesophageal reflux disease was suspected. From the age of 7 months, a regression of psychomotor functioning was noted and she became somnolent. In addition, she developed a severe constipation. There was failure to thrive; weight decreased from 0 to -2.5 SD, length from 0 to -1 SD and head circumference from +1 to -1 SD from the age of 1 month to the age of 10 months. Physical examination at the age of 10 months showed an irritable child with marked generalized hypotonia. She could not sit without support and showed no intentional hand function. Fundoscopy was normal. Gavage feeding was initiated. Laboratory studies revealed a metabolic acidosis. Plasma lactate was 12.3 mmol/l (N: <2.1 mmol/l) with an increased lactate/pyruvate ratio and CSF lactate was 12.5 mmol/l (N: <1.4 mmol/l). Additional laboratory studies, including liver function tests, were normal. A muscle biopsy was performed at the age of 11 months. Immunohistochemistry and electron microscopy were normal. She became comatose and died at the age of 13 months because of respiratory insufficiency. Autopsy or additional studies were not performed.

71 Chapter 3

Patient A. This male patient was born at 37 weeks of gestation with a birth weight of 2.118 kg (< -2.5 SD), length 44.5 cm (< -2.5 SD) and head circumference 32.4 cm (-2 SD), as a first child of healthy, non-consanguineous parents. He was admitted to the neonatal unit for severe generalized hypotonia, respiratory insufficiency, Chapter feeding difficulty and anemia. He was further evaluated at the age of 4 months for severe psychomotor retardation, growth retardation, visual dysfunction, epilepsy 3 (West syndrome) and chronic lactic acidemia. He received a gastric tube placement and started on nutritional intervention. A cranial MRI reported hypoplastic corpus callosum and abnormal signal intensity of the basal ganglia, including the globus pallidus and the nucleus caudatus. A follow-up cranial MRI showed progressive cerebral and cerebellar atrophy. EEG and VEP were severely abnormal. EEG showed generalized seizure activity; no burst suppression pattern was present. The VEP showed severe central visual perception loss. Metabolic studies showed markedly elevated lactate in both blood (3.6-7 mmol/l, N: <2.1 mmol/l) and CSF (3.8 mmol/l, N: <1.4 mmol/l), hyperalaninemia (860 Mmol/l, N: <460 Mmol/l) and increased urinary lactate and Krebs cycle intermediates. Additional laboratory studies were normal. At the age of 8 months, at the time of the surgical muscle biopsy, he had severe microcephaly; other growth parameters were normal. No internal organ dysfunction was noted. He had severe generalized hypotonia and hyporeflexia. The psychomotor development of the patient remained at the level of a 3-month- old baby. He had no visual contact with the surroundings and was suffering from intractable Blitz-Nick-Salaam (BNS)-type seizures. Using histological analysis, a homogenously decreased staining of COX activity was confirmed with a residual activity of 15-25%. No fiber-type disproportion, lipid storage or ragged red fibers were detected. The patient is currently 4.5 years old, has no acoustic or visual contact, is severely retarded and fully tube fed.

Cell culture Skin fibroblasts were cultured in M199 medium (Gibco, Breda, The Netherlands) supplemented with 10% fetal calf serum (FCS) and antibiotics. Transmitochondrial cybrid cell lines were constructed as described elsewhere.4 The cybrids were grown in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 mg/ml glucose and supplemented with 10% FCS, 110 Mg/ml pyruvate, and antibiotics; for the mtDNA-less 143B206 cells this medium was additionally supplemented with 50 Mg/ml uridine and 100 Mg/ml bromodeoxyuridine. The human osteosarcoma cell line 143B (TK-) was cultured in Eagle's minimum essential medium (EMEM) with Earle's BSS supplemented with 10% FCS and 15 Mg/ml bromodeoxyuridine.

72 Mitochondrial tRNATrp and tRNAArg mutations

Enzyme measurements The activities of the OXPHOS enzyme complexes were measured in skin fibroblasts and muscle tissue of the patients as described previously.5,6

Molecular genetic analysis Chapter DNA was isolated from cultured skin fibroblasts, muscle tissue or blood using salt extraction.7 The entire mtDNA of the patients was amplified and sequenced 3 according to the protocol of Taylor et al8 with minor adaptations. Percentage heteroplasmy was quantified using the Pyrosequencing technology (Biotage AB, Uppsala, Sweden). Pyrosequencing was performed on the PSQ96 MA platform according to the protocol of the manufacturer. Primer sequences are available upon request.

Analysis of mitochondrial protein synthesis In vitro pulse labeling of mitochondrial translation was performed as described elsewhere,9 with a few adaptations. In short, cells were labeled for 60 min at 37 °C in methionine-free DMEM with 10% dialyzed FCS, 200 MCi/ml [35S]methionine (Tran35S-Label, MP Biomedicals, Eindhoven, The Netherlands) and 100 Mg/ ml emetine, and subsequently chased for 10 min in regular DMEM with 10% FCS. Total cellular protein (100 Mg) was resuspended and incubated for 10 min in PBS containing 2% lauryl maltoside. Unsolubilized material was removed by centrifugation at 10 000 g for 10 min and loading buffer was added to the supernatant to a final concentration of 100 mM Tris-HCl (pH 6.8), 20% glycerol, 1% SDS, 0.02% Coomassie blue G-250 and 1% mercaptoethanol. Samples were run on 16% polyacrylamide gels,10 which were subsequently scanned on a FLA5100 (Fujifilm Life Science, Düsseldorf, Germany) and analyzed using ImageQuant software (GE Healthcare, Diegem, Belgium). Equal protein loading was confirmed using Coomassie blue staining (data not shown).

Blue-native PAGE and complex I in-gel activity assay Blue-native PAGE was used for separation of the OXPHOS complexes on 5-15% polyacrylamide-gradient gels as described before.11 After electrophoresis of 40 Mg protein from fibroblast and cybrid cells, gels were further processed for in-gel activity assays and western blotting. Assembly of the OXPHOS complexes was analyzed using monoclonal antibodies against subunits of complexes I, III, V (Molecular Probes, Leiden, The Netherlands), II, and IV (MitoSciences, Eugene, OR, USA).

73 Chapter 3

Northern blot analysis Total RNA samples (10 Mg) isolated from cultured fibroblast and cybrid cell lines using RNA-Bee (Tel-Test) were separated on a 15% denaturing UREA-PAGE gel and transferred to a Hybond-N membrane (Amersham Pharmacia Biotech, Chapter Roosendaal, The Netherlands) using electroblotting. For the conformational analysis, total RNA samples were separated for 24 h at 4 °C on a non-denaturing 3 15% polyacrylamide gel. Denaturing was performed by placing the gel for 10 min in 0.2 M NaOH/ 0.5 M NaCl. Before electroblotting to a Hybond-N membrane (Amersham Pharmacia Biotech), neutralization was carried out by washing twice for 10 min in 5xTBE and twice for 10 min in 1xTBE. After hybridization with the tRNAArg probe, the membrane was washed and subsequently rehybridized with the tRNATrp probe. For the detection of tRNATrp and tRNAArg, templates for hybridization probes were generated by PCR using oligonucleotides containing the T7 RNA polymerase promoter sequence in the reverse primer. Primer sequences are available upon request. For in vitro transcription, the gel-purified PCR template was incubated for 2 h at 37 °C in 60 ml of buffer containing 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 10 mM dithiotreitol, 1 mM each of ATP, CTP and UTP, 0.1 mM GTP, 60mCi [a-32P]GTP and 45 U T7 RNA polymerase. Imaging was performed using a PhosphorImager (Molecular Imager FX, Bio Rad, Veenendaal, The Netherlands); signal intensities for tRNATrp and tRNAArg were normalized to each other for each sample.

R e s u l t s Enzyme measurements The biochemical assays of OXPHOS enzyme activities showed clear combined complex I, III and IV deficiencies in patients T and A (Table 1). The activities measured as percentage of the lowest control value ranged from 15 to 59% in

Table 1. OXPHOS enzyme activities in skeletal muscle and fibroblasts Activities (mU/U citrate synthase) in Skeletal muscle Fibroblasts Complex Patient T Patient A Control range Patient T Patient A Control range I 26 31 70 - 251 39 39 100 - 310 II 387 455 335 - 749 774 544 520 - 845 III 1306 1870 2200 - 6610 571 1383 1320 - 2610 II+III 386 420 300 - 970 140 218 110 - 470 IV 322 157 810 - 3120 102 223 680 - 1190

Samples were supernatants obtained after 600g centrifugation of skeletal muscle or mitochondrial- enriched fractions from fibroblasts. The decreased activities as compared with controls are in bold.

74 Mitochondrial tRNATrp and tRNAArg mutations

Table 2. Percentages of mutant mtDNA as determined using pyrosequencing Subject Sample m.5556G>A m.10450A>G Patient Fibroblasts 92 92 Skeletal muscle 93 93 Cybrid clone 1 90 91 Chapter Cybrid clone 2 93 90 Cybrid clone 3 96 88 3 Mother Blood 0 0 Fibroblasts ND 18 Abbreviation: ND, not determined. patient T and from 19 to 44% in patient A, excluding complex III activity, which was not decreased considerably in patient A.

Molecular genetic analysis Sequence analysis of the entire mitochondrial genome revealed 18 (patient T) and 39 (patient A) known polymorphic variants compared with the revised Cambridge Reference Sequence,12 and two mutations: a G-to-A transition at position m.5556 in the tRNATrp gene (MT-TW) in patient T and an A-to-G transition at position m.10450 in the tRNAArg gene (MT-TR) in patient A. The levels of the mutations were nearly homoplasmic in both patients, in muscle tissue as well as cultured fibroblasts (Table 2). In the blood of the mothers the mutations could not be detected, whereas in fibroblasts of the mother of patient A the m.10450A>G mutation was present with 18% heteroplasmy. The two mutations were not reported before and were found neither in 84 healthy control subjects and in 31 patients with a combined OXPHOS complex I, III and IV deficiency (tested by PCR) nor in haplogroup-matched controls, 499 controls of haplogroup H for tRNATrp and 121 controls of haplogroup T for tRNAArg (database search).13 Position m.5556 is located in the variable region of the tRNATrp structure and position m.10450 is located in the T stem of tRNAArg (Figures 1a and b). The nucleotide mutated in tRNATrp is entirely conserved in mammals14 and also in several lower species (Figure 1c). The mutation in tRNAArg, in contrast, affects a base that is conserved in many mammalia,14 but not in lower species (Figure 1d).

Analysis of mitochondrial protein synthesis As shown in Figure 2, both patients showed a marked reduction in radioactive labeling as compared with controls, indicating that the rate of mitochondrial protein synthesis is decreased. The total incorporation of [35S]methionine was 45% (patient T) and 57% (patient A) of that in controls; however, different polypeptides were affected to variable extents (Table 3). In patient T, the rate of mitochondrial

75 Chapter 3

A b A CC C C G A A-■ U U-A G -C G -C A--U G -C Chapter A-■ U U-A A-■ U A--U U-A u.. U U A- ' A U U C A U A UAUAC UA 3 1 U iii A i i i i A A A A L A A AG.. C a A u U UG UAUG . u U • u u uG A i i i i U \ C a A A A C CA 5 5 5 6 G > A G...... aAA 10450A>G A-U A-U G-C A-U U-A G-C A-U C A U C U A U A ÜC A U C G I \ Patient CAAT Patient TGTG Human CAGT Human TATG Bovine AAGC Bovine TATG Mouse A AG A Mouse TATG Zebrafish CAGT Zebrafish TGCG Nematode TA AT Nematode TGGT Yeast TCAT Yeast ATAG Bacteria GTGT Bacteria GCAG

Figure 1. Schematic representation of the tRNATrp (a) and tRNAArg (b) cloverleaf structures, showing the location of the mutations. Comparison of the sequences of the variable region of the tRNATrp gene (c) and the T-stem of the tRNAArg gene (d) across different species; the mutated bases are depicted in bold. translation ranged from 22 to 126% and the three subunits of complex IV (COI- COIII) were most severely affected. In patient A, synthesis of COI showed by far the strongest decrease (12%) and the other polypeptides were affected only minimally with incorporation rates of 68% or higher. In both patients, the synthesis levels of the two ATP synthase subunits (ATP6 and ATP8; the latter migrated in a band together with ND4L) were comparable to or even higher than the levels in controls.

Blue-native PAGE and complex I in-gel activity assay To determine whether the combined OXPHOS deficiencies in the patients were indeed caused by a mtDNA defect, we analyzed the assembly of all OXPHOS complexes in fibroblasts and cybrid cell lines using blue-native PAGE analysis. Patient fibroblasts showed reduced levels of fully assembled complexes I, III (only in patient T), IV and V as well as decreased complex I activities (Figure 3), which is consistent with the deficiencies detected in the enzyme measurements (Table 1). This pattern was also found in the different cybrid clones, proving the mitochondrial origin of the OXPHOS defect. In addition to the fully assembled

76 Mitochondrial tRNATrp and tRNAArg mutations complex V, two subcomplexes were observed in most samples with raised levels in the patient samples. These subcomplexes are often increased in patients with a defect in the mtDNA, but sometimes they are entirely absent. This was the case for the experiment with fibroblasts from patient A and the control, which was probably caused during the preparation of the samples. Chapter

Northern blot analysis 3 To analyze the effect of both mutations on tRNA stability, steady-state levels of tRNATrp and tRNAArg were determined using northern blot hybridization in cultured fibroblasts and cybrid cell lines of the two patients. The amounts of mutant tRNA were markedly decreased (Figure 4): in patient T, the tRNATrp levels were reduced to 29% (fibroblasts) and 33% (cybrids), and in patient A the tRNAArg levels were reduced to 29% (fibroblasts) and 50% (cybrids) (after normalization to the tRNAArg or tRNATrp levels, respectively, and relative to average control values). In addition, native gel electrophoresis provided insights into whether the mutations affect tRNA structure. Whereas on a denaturing gel the tRNA mutants migrated at the same rate as the wild type (see steady-state data, Figure 4), both tRNA mutations resulted in aberrant migration patterns under

Figure 2. In vitro labeling of mitochondrial translation products. Fibroblasts from the patients (PT and PA) and controls (C1 and C2) were labeled with [35S]methionine in the presence of emetine. The mitochondrial translation products are indicated on the left of the autoradiogram. COI-COIII, subunits of cytochrome c oxidase; cyt b, cytochrome b subunit; ND1-6, subunits of NADH CoQ oxidoreductase; ATP6-8, ATP synthase subunit 6 and 8. Chapter 3

Table 3. Level of mitochondrial protein synthesis Polypeptide Patient T Patient A COI 28 12 Cytb/ND2 56 86 Chapter ND1 35 82 COIII/COII 22 68 3 ATP6 124 163 ND6/ND3 82 89 ND4L/ATP8 126 94 The values represent percentages of the average incorporation of [35S]methionine measured in two controls. Abbreviations: COI-COIII, subunits of cytochrome c oxidase; cyt b, cytochrome b subunit; ND1-6, subunits of NADH CoQ oxidoreductase; ATP6-8, ATP synthase subunits 6 and 8.

non-denaturing conditions (see conformation data, Figure 4), indicating that they primarily affect the secondary or tertiary structure of the tRNAs. The mutation in tRNATrp led to retardation of the electrophoretic mobility of the residual tRNATrp compared with controls. The analysis of tRNAArg revealed the presence of two different conformations with accelerated mobility. As the cybrid clones showed similar steady-state levels and migration patterns as found in fibroblasts of the patients, these results are again consistent with the mitochondrial origin of the OXPHOS defects.

Figure 3. Blue-native PAGE and complex I in-gel activity assay. Fibroblasts and cybrid clones from patient T (PT) and A (PA) and controls (C1-3 and 143B) were analyzed. The blue-native PAGE gels were immunoblotted with antibodies directed against specific individual subunits to assess the amount of each of the five fully assembled OXPHOS complexes. Complex II was used as a loading control. IGA, in-gel activity.

78 Mitochondrial tRNATrp and tRNAArg mutations

Chapter 3

Figure 4. Steady-state levels and conformation of tRNATrp and tRNAArg. Fibroblasts and cybrid clones from patient T (PT) and A (PA) and controls (C1-3 and 143B) were analyzed using high-resolution northern blot under denaturing (steady-state) and native (conformation) conditions. Signal intensities for tRNATrp and tRNAArg were normalized to each other for each sample, as shown in the column charts (only relevant steady-state levels are depicted). The asterisks indicate the position of altered conformations of mutant tRNATrp and mutant tRNAArg.

D i s c u s s i o n In this study we clearly show that the m.5556G>A and m.10450A>G mutations are pathogenic and are the cause of the combined OXPHOS disorders found in the two patients. This conclusion is based on the following evidence: (1) the mutations are not present in healthy controls or in asymptomatic maternal relatives (or with a low mutant load), (2) the mutations affect evolutionary conserved bases (applies more to the tRNATrp mutation than the mutation in the tRNAArg gene), (3) patient fibroblasts have a general mitochondrial translation defect, (4) tRNA steady-state levels and conformation are affected by the mutations and (5) transmitochondrial cybrids show similar OXPHOS complex and tRNA defects as patient fibroblasts. Although the mutation in the tRNATrp gene was not found in the blood of the patient's mother, we cannot conclude that it is a de novo mutation. Mutation load can vary greatly between tissues causing the mutation to be undetectable in, for instance, blood, as was the case for the tRNAArg mutation. In patient A the mutation was present at 92% heteroplasmy, whereas his mother had a mutant load of only 18% in fibroblasts, which is apparently below the threshold level for this mutation and therefore does not result in clinical disease. Approximately 70% of pathogenic mutations occur in the stems of the

79 Chapter 3

tRNA cloverleaf structure.1516 Furthermore, the disruption of Watson-Crick base pairing is an important characteristic of pathogenic mutations located in the stem structures.15 The mutation in the tRNAArg gene affects base 50 in the T stem and disrupts Watson-Crick base pairing, which makes it a likely candidate for the Chapter disease-causing mutation. However, a wobble base pair can still be formed with the mutant base. The disturbance of Watson-Crick base pairing could affect the 3 secondary structure of the tRNAArg, leading to misfolding and instability of the mutant tRNA. This was confirmed using northern blotting, which showed reduced tRNAArg steady-state levels and the existence of two aberrant conformations in the absence of the normal tRNA. In contrast, the site that is mutated in the tRNAArg gene is conserved merely in mammalia. Although most pathogenic tRNA mutations are found at highly conserved sites, there are exceptions, indicating that evolutionary conservation is not required for a mutation to be pathogenic.1617 The m.5556G>A mutation is located at position 46 in the variable region of tRNATrp, which is not a common site for pathogenic mutations.15 However, this base is highly conserved. In addition, a potential tertiary interaction between nucleotides 13-22-46 in tRNATrp has been described.14 This interaction is present in all mammalian tRNA families and is part of a set of three tertiary interactions that are thought to be minimally required for proper functioning of the mitochondrial tRNAs. Nucleotide 46 is N7-methylated in many tRNAs of different species.18 This post-transcriptional modification, together with hydrogen bonding to the bases G22 and C13, results in a positive charge, which could be necessary for the L-shaped three-dimensional tRNA structure and/or for protein recognition.19 In general, the role of modified bases for the maintenance of the tRNA tertiary structure is of higher importance in mitochondrial tRNAs than in their cytosolic counterparts.20 Certain mitochondrial tRNAs will consequently be completely non-functional when lacking the post-transcriptional modification. Northern blot analysis revealed a clear decrease in the amount of mutant tRNATrp as well as an altered conformation, indicating that the mutation might indeed interfere with the tertiary interaction and thereby disturb the three-dimensional tRNA structure and tRNA stability. Although the MITOMAP database lists the m.5556G>A transition as an unpublished polymorphism,21 our results clearly show that this nucleotide change is a pathogenic mutation in our patient. This underscores the importance of functional studies in defining the pathogenicity of tRNA mutations. Seven pathogenic mutations have been reported in the mitochondrial tRNATrp gene.2 None of these mutations are located near the m.5556G>A mutation that we found. However, the m.5532G>A mutation22 affects base 22, which is supposedly involved in the tertiary interaction with base 46 (m.5556) mentioned above. Unfortunately, the effect of this point mutation on tRNA stability and conformation has not been analyzed. A common feature of most tRNATrp mutations is a profound complex

80 Mitochondrial tRNATrp and tRNAArg mutations

IV defect.22-28 In some cases complex IV is selectively decreased, in other cases complex I is affected to a similar extent and occasionally complex III is involved as well. Biochemical analysis revealed a combined complex I, III and IV deficiency in patient T, in which complex IV was most severely affected (on average, 28% of control levels) followed by complex I (38%) and last by complex III (51%). Chapter The results of the in vitro translation assay also supported this as the synthesis rates of COI and COIII/COII showed the strongest decrease as compared with 3 control rates. The fact that complex IV is more susceptible to tRNATrp mutations could be explained by the higher percentage of tryptophan in the subunits COI and COIII as compared with other complex subunits, as has been proposed before.26,27 Nonetheless, a correlation between the relative amount of tryptophan in individual subunits and the reduction of their mitochondrial translation rates in our patient was not evident, which has also been observed by Sacconi et al for the m.5545C>T mutation.24 They found COI and COIII translation to be most severely disturbed, but in contrast to our results ATP6 showed a profoundly decreased synthesis as well. Increased synthesis of ATP6 and ATP8, as we observed for our tRNATrp and tRNAArg mutations, has been reported before29-32 and has been suggested to be caused by the fact that bicistronic mRNAs can be translated more efficiently than monocistronic mRNAs, thereby giving the translation of ATP6 and ATP8 an advantage.32 Many patients with a tRNATrp mutation, including our patient, show developmental delay, gastrointestinal symptoms and failure to thrive.22,24,25'28 In addition, visual defects, deafness, epilepsy and neurodegenerative disease are frequently observed in other patients.22-28,33 In the mitochondrial tRNAArg gene only two pathogenic mutations have been reported so far: m.10406G>A and m.10438A>G.2 In the patient with the m.10438A>G mutation, no clear OXPHOS deficiency was found, only a minor decrease in complex IV activity.34 In contrast, the m.10406G>A mutation caused a marked reduction in complex IV activity, and complex I and III were also affected.35 This is consistent with our findings, except that complex III was hardly impaired in patient A. The clinical phenotypes of the three patients carrying tRNAArg mutations are variable; the only common features are muscle weakness and brain abnormalities. Furthermore, visual dysfunction and progressive psychomotor retardation were observed both in our patient and for the m.10438A>G mutation.34 The clinical presentation of the m.10406G>A mutation is considerably different with Asperger syndrome and normal cognitive function.35 Our patient manifested the most severe symptoms in a progressive multisystem disorder. The variability in clinical phenotypes could be explained by the fact that all three mutations are located in different domains of the tRNA cloverleaf structure. A distinct correlation seems not to exist between clinical presentation and the specific tRNA that is mutated.16,36 For our m.10450A>G mutation, the disproportionate

81 Chapter 3

decrease in synthesis of some polypeptides (mainly COI synthesis was impaired) is not correlated with the arginine content of the polypeptides. The effects on translation have not been analyzed for the other tRNAArg mutations. In conclusion, we show that the mutations in the mitochondrial tRNATrp and Chapter tRNAArg genes are pathogenic. The effect of the mutations on the tertiary (tRNATrp) or secondary (tRNAArg) structure will likely lead to misfolding and instability, 3 resulting in increased degradation and possibly also in impaired aminoacylation. Consequently, the scarcity and malfunctioning of the tRNAs cause a combined OXPHOS deficiency through reduced rates of mitochondrial protein synthesis. This gives rise to a progressive multisystem disease in both patients, marked by failure to thrive, lactic acidosis, feeding difficulties, gastrointestinal problems, generalized muscle hypotonia, progressive psychomotor retardation and microcephaly.

A cknowledgements This work was supported by the European Union's Sixth Framework Program, contract number LSHMCT-2004-005260 (MITOCIRCLE), and in part by the Council for Chemical Sciences (NWO-CW) of the Netherlands Organization for Scientific Research. We thank M Zeviani for providing one of the cybrid control cell lines.

82 Mitochondrial tRNATrp and tRNAArg mutations

R e f e r e n c e s 1. Loeffen JL, Smeitink JA, Trijbels JM et al: Isolated complex I deficiency in children: clinical, biochemical and genetic aspects. Hum Mutat 2000; 15: 123-134. 2. MITOMAP: A Human Mitochondrial Genome Database. http://www.mitomap.org, Chapter 2009. 3. Spinazzola A, Zeviani M: Disorders from perturbations of nuclear-mitochondrial 3 intergenomic cross-talk. J Intern Med 2009; 265: 174-192. 4. King MP, Attardi G: Mitochondria-mediated transformation of human rho(0) cells. Methods Enzymol 1996; 264: 313-334. 5. Janssen AJ, Smeitink JA, van den Heuvel LP: Some practical aspects of providing a diagnostic service for respiratory chain defects. Ann Clin Biochem 2003; 40: 3-8. 6. Smeitink J, Sengers R, Trijbels F, van den Heuvel L: Human NADH:ubiquinone oxidoreductase. J Bioenerg Biomembr 2001; 33: 259-266. 7. Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215. 8. Taylor RW, Taylor GA, Durham SE, Turnbull DM: The determination of complete human mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations. Nucleic Acids Res 2001; 29: E74. 9. Boulet L, Karpati G, Shoubridge EA: Distribution and threshold expression of the tRNA(Lys) mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF). Am J Hum Genet 1992; 51: 1187-1200. 10. Schagger H, von Jagow G: Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 1991; 199: 223-231. 11. Calvaruso MA, Smeitink J, Nijtmans L: Electrophoresis techniques to investigate defects in oxidative phosphorylation. Methods 2008; 46: 281-287. 12. Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N: Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 1999; 23: 147. 13. Ingman M, Gyllensten U: mtDB: human mitochondrial genome database, a resource for population genetics and medical sciences. Nucleic Acids Res 2006; 34: D749-D751. 14. Helm M, Brule H, Friede D, Giege R, Putz D, Florentz C: Search for characteristic structural features of mammalian mitochondrial tRNAs. RNA 2000; 6: 1356-1379. 15. McFarland R, Elson JL, Taylor RW, Howell N, Turnbull DM: Assigning pathogenicity to mitochondrial tRNA mutations: when 'definitely maybe' is not good enough. Trends Genet 2004; 20: 591-596. 16. Scaglia F, Wong LJ: Human mitochondrial transfer RNAs: role of pathogenic mutation in disease. Muscle Nerve 2008; 37: 150-171. 83 Chapter 3

17. Florentz C, Sissler M: Disease-related versus polymorphic mutations in human mitochondrial tRNAs. Where is the difference? EMBO Rep 2001; 2: 481-486. 18. Sprinzl M, Horn C, Brown M, Ioudovitch A, Steinberg S: Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res 1998; 26: 148-153. Chapter 19. Agris PF, Sierzputowska-Gracz H, Smith C: Transfer RNA contains sites of localized positive charge: carbon NMR studies of [13C]methyl-enriched Escherichia coli and 3 yeast tRNAPhe. Biochemistry 1986; 25: 5126-5131. 20. Helm M: Post-transcriptional nucleotide modification and alternative folding of RNA. Nucleic Acids Res 2006; 34: 721-733. 21. Mkaouar-Rebai E, Chahnez T, Fakhfakh F: MITOMAP mtDNA Sequence Data. http://www.mitomap.org/cgi-bin/tbl15gen.pl#20070724006, 2007. 22. Maniura-Weber K, Taylor RW, Johnson MA et al: A novel point mutation in the mitochondrial tRNA(Trp) gene produces a neurogastrointestinal syndrome. Eur J Hum Genet 2004; 12: 509-512. 23. Anitori R, Manning K, Quan F et al: Contrasting phenotypes in three patients with novel mutations in mitochondrial tRNA genes. Mol Genet Metab 2005; 84: 176-188. 24. Sacconi S, Salviati L, Nishigaki Y et al: A functionally dominant mitochondrial DNA mutation. Hum Mol Genet 2008; 17: 1814-1820. 25. Santorelli FM, Tanji K, Sano M et al: Maternally inherited encephalopathy associated with a single-base insertion in the mitochondrial tRNATrp gene. Ann Neurol 1997; 42: 256-260. 26. Silvestri G, Rana M, DiMuzio A, Uncini A, Tonali P, Servidei S: A late-onset mitochondrial myopathy is associated with a novel mitochondrial DNA (mtDNA) point mutation in the tRNA(Trp) gene. Neuromuscul Disord 1998; 8: 291-295. 27. Silvestri G, Mongini T, Odoardi F et al: A new mtDNA mutation associated with a progressive encephalopathy and cytochrome c oxidase deficiency. Neurology 2000; 54: 1693-1696. 28. Tulinius M, Moslemi AR, Darin N et al: Leigh syndrome with cytochrome-c oxidase deficiency and a single T insertion nt 5537 in the mitochondrial tRNATrp gene. Neuropediatrics 2003; 34: 87-91. 29. Antonicka H, Sasarman F, Kennaway NG et al: The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1. Hum Mol Genet 2006; 15: 1835-1846. 30. Coenen MJ, Antonicka H, Ugalde C et al: Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency. N Engl J Med 2004; 351: 2080-2086.

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31. Sasarman F, Antonicka H, Shoubridge EA: The A3243G tRNALeu(UUR) MELAS mutation causes amino acid misincorporation and a combined respiratory chainassembly defect partially suppressed by overexpression of EFTu and EFG2. Hum Mol Genet 2008; 17: 3697-3707. 32. Smeitink JA, Elpeleg O, Antonicka H et al: Distinct clinical phenotypes associated with Chapter a mutation in the mitochondrial translation elongation factor EFTs. Am J Hum Genet 2006; 79: 869-877. 3 33. Nelson I, Hanna MG, Alsanjari N, Scaravilli F, Morgan-Hughes JA, Harding AE: A new mitochondrial DNA mutation associated with progressive dementia and chorea: a clinical, pathological, and molecular genetic study. Ann Neurol 1995; 37: 400-403. 34. Uusimaa J, Finnila S, Remes AM et al: Molecular epidemiology of childhood mitochondrial encephalomyopathies in a Finnish population: sequence analysis of entire mtDNA of 17 children reveals heteroplasmic mutations in tRNAArg, tRNAGlu, and tRNALeu(UUR) genes. Pediatrics 2004; 114: 443-450. 35. Pancrudo J, Shanske S, Coku J et al: Mitochondrial myopathy associated with a novel mutation in mtDNA. Neuromuscul Disord 2007; 17: 651-654. 36. Schon EA, Bonilla E, DiMauro S: Mitochondrial DNA mutations and pathogenesis. J Bioenerg Biomembr 1997; 29: 131-149.

85 Science never solves a problem without creating ten more. George Bernard Shaw C h apter 4 Distinct clinical phenotypes associated with a mutation in the mitochondrial translation elongation factor EFTs

Ml*

Jan A.M. Smeitink,* Orly Elpeleg,* Hana Antonicka, Heleen Diepstra, Ann Saada, Paulien Smits, Florin Sasarman, Gert Vriend, Jasmine Jacob-Hirsch, Avraham Shaag, Gideon Rechavi, Brigitte Welling, Jürgen Horst, Richard J.T. Rodenburg, Lambert P. van den Heuvel and Eric A. Shoubridge

* Joined first authorship

American Journal of Human Genetics 2006; 79(5): 869-877 Chapter 4

A b s t r a c t The 13 polypeptides encoded in mitochondrial DNA (mtDNA) are synthesized in the mitochondrial matrix on a dedicated protein-translation apparatus that resembles that found in prokaryotes. Here we have investigated the genetic basis for a mitochondrial protein-synthesis defect associated with a combined oxidative phosphorylation enzyme deficiency in two patients, one of whom presented with encephalomyopathy and the other with hypertrophic cardiomyopathy. Sequencing of candidate genes revealed the same homozygous mutation (C997T) in both patients in TSFM, a gene coding for the mitochondrial translation elongation factor EFTs. EFTs functions as a guanine nucleotide exchange factor for EFTu, another translation elongation factor that brings aminoacylated transfer RNAs to the ribosomal A site as a ternary complex with guanosine triphosphate. The mutation predicts an Arg333Trp substitution at an evolutionarily conserved site in a subdomain of EFTs that interacts with EFTu. Molecular modeling showed that the substitution disrupts local subdomain structure and the dimerization interface. The steady-state levels of EFTs and EFTu in patient fibroblasts were reduced by 75% and 60% respectively, and the amounts of assembled complexes I, IV and V were reduced by 35-91% compared with the amounts in controls. These phenotypes and the translation defect were rescued by retroviral expression of either EFTs or EFTu. These data clearly establish mutant EFTs as the cause of disease in these patients. The fact that the same mutation is associated with distinct clinical phenotypes suggests the presence of genetic modifiers of the mitochondrial translation apparatus.

88 Mitochondrial elongation factor Ts mutation

I ntroduction Defects in mitochondrial oxidative phosphorylation (OXPHOS) are estimated to occur in approximately ~1 in 5,000 live births.1 They can be classified biochemically as either isolated or combined deficiencies of any of the five OXPHOS complexes.2 Genetic defects leading to combined deficiencies of complexes I, III, IV, and V can be caused by mutations in mtDNA or in the components of the mitochondrial translation apparatus encoded by nuclear genes.3 Synthesis of the 13 mitochondrial-encoded proteins occurs on a dedicated mitochondrial translation apparatus similar to that found in prokaryotes and requires, in addition to the tRNAs and ribosomal RNAs (rRNAs) encoded in mtDNA, the concerted action of several translation factors and a large number of mitochondrial ribosomal (mitoribosome) proteins, all of which are encoded by nuclear genes. Two mammalian initiation factors, IF2 and IF3; four elongation factors, EFTu, EFTs, EFG1, and EFG2 (reviewed by Spremulli et al);4 one release factor, RF1; and one ribosomal recycling factor, RRF,5 have been cloned and sequenced. Mitoribosomes are distinct from both prokaryotic and eukaryotic cytosolic ribosomes; they are 55S particles composed of a small (28S) and a large (39S) subunit and contain a much higher protein:RNA ratio than that of the bacterial 70S ribosome.6 Although the vast majority of components of the mitochondrial translation system are nuclear encoded, most mutations associated with mitochondrial translation defects have been reported in mtDNA-encoded tRNAs and rRNAs.7,8 However, we recently reported mutations in the nuclear-encoded translation elongation factor EFG19,10 and in MRPS16, a protein of the small ribosomal subunit,11 in patients with autosomal recessive mitochondrial translation defects. Encouraged by these findings, we investigated a cohort of 16 patients with combined OXPHOS enzyme defects expressed in both muscle and fibroblasts in which mtDNA sequencing and complex II activity-measurement results were normal. We describe the first mutation in the mitochondrial elongation factor EFTs (encoded by TSFM [MIM 604723]) associated with a fatal mitochondrial encephalomyopathy in one pedigree and with fatal hypertrophic cardiomyopathy in another, and demonstrate the molecular basis for the defect.

89 Chapter 4

M a t e r i a l s a n d m e t h o d s Case reports Patient 1, a boy, was born at term as the second child of consanguineous Turkish parents by cesarean section performed on maternal indication. Birth weight (3,140 g), length (50 cm), and head circumference (36 cm) were age appropriate. Muscular hypotonia, sucking weakness, and a severe lactic acidosis (pH 6.8; base excess -26; lactic acid 42 mM [control <2.1 mM]) were the initial clinical signs and symptoms, followed by rhabdomyolysis with creatine kinase values up to 7,252 U/ liter (control <248 U/liter). The initial lactate:pyruvate (L:P) ratio was 92 (control 12 15). During life, lactic acid dropped to values between 4 and 5.5 mM, with L:P ratios ~25. Artificial ventilation was needed because of persistent dyspnea. On the 3rd day of life, generalized convulsions became obvious. The electroencephalogram showed a discontinuous pattern with bifrontal symmetrical sharper spikes. Repeated ultrasound imaging of the brain showed reduced gyri on day 4, plexus bleeding with enlarged ventricles on day 6, and abnormal signal intensity of the thalami on day 9. Echocardiographic examination on day 6 revealed a persistent ductus arteriosus Botalli, with left-to-right shunting, and a persistent foramen ovale. Septum thickness and contractility were age appropriate. On the basis of the suspicion of a mitochondrial disorder, a fibroblast culture was established and a skeletal muscle (m. vastus lateralis dextra) biopsy sample was obtained for measurements of the OXPHOS enzymes. Despite intensive treatment, the patient died of progressive encephalomyopathy and respiratory failure at age 7 wk. Patient 2, a girl, was the third child of second cousins once removed whose parents are of Kurdish Jewish origin. Two older children were healthy. The mother reported a paucity of fetal movements throughout the pregnancy. Delivery was vaginal, and birth weight was 2,600 g. Initial examination results were normal, and the patient was breast-fed. At age 36 h, apathy, irregular breathing, and severe muscular hypotonia were noted. Laboratory investigation revealed severe metabolic acidosis (pH 6.93; HCO3 -4.6; base excess -26). Serum lactate level was increased (17.6 mM; control <2.4 mM), as were blood ketone levels (3-OH- butyrate 4,803 |jM; acetoacetate 257 |jM; control values <200 |jM and <100|jM, respectively) and serum ammonia levels (268 |jM; control <32 |jM). Blood count and liver enzyme levels were normal. Echocardiographic examination disclosed severe concentric hypertrophic cardiomyopathy with normal contractility. Apart from generalized muscle hypotonia, neurological examination, including fundoscopy and brain CT scan, were normal. Skin biopsy for fibroblast culture and muscle biopsy from the right quadriceps were performed at age 2 wk; histochemical analysis of the muscle revealed a generally decreased cytochrome c oxidase (COX) stain, whereas the succinate dehydrogenase stain was normal. The patient was

90 Mitochondrial elongation factor Ts mutation given treatment with carnitine, sodium-dichloroacetate, thiamine, idebenone, and riboflavin, but the lactate level remained elevated, and cardiac function gradually deteriorated. Her death at age 7 wk was preceded by severe hyponatremia with a low urinary output of 2 d. An open-liver biopsy was performed 20 min after her death. Southern-blot analysis of the muscle, liver, and fibroblast DNA revealed normal size and abundance of mtDNA. Northern-blot analysis showed that the steady-state levels of several mitochondrial transcripts (ND6, COXI, COXII, and 12S rRNA) were normal. Sequencing of the 22 mitochondrial tRNA genes failed to identify a pathogenic mutation.

Human studies Informed consent was obtained, and research studies were approved by the Helsinki committee of Hadassah-Hebrew University Medical Center, the Radboud University Medical Centre Nijmegen, and the Montreal Neurological Institute Institutional Review Boards.

Cell culture Primary human skin fibroblasts were immortalized with a retrovirus expressing the E7 gene of type 16 human papilloma virus and a retroviral vector expressing the protein component (htert) of human telomerase.12 Patient and control skin fibroblasts were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS).

Enzyme measurements The activities of the OXPHOS enzyme complexes in skeletal muscle and fibroblasts were measured according to described protocols.13,14 COX and citrate synthase activities were measured in fibroblast cell extracts as described elsewhere.15

Sequence analysis Total RNA was extracted from cultured skin fibroblast of the patient by use of RNAzol (Campro Scientific). The TSFM cDNA (GenBank accession number NM_005726) was amplified by superscript II reverse transcriptase/Toq DNA polymerase (Life Technologies) in four overlapping fragments (primer sequences available on request). After treatment with ExoI (Promega) and SAP (Promega), the PCR fragments were used as templates in sequencing reactions with the chemistry of BigDye1.1 (Applied Biosystems) according to manufacturer's protocol. The labeled fragments were separated on a 3130xl Genetic Analyzer (Applied Biosystems). Sequence data were evaluated with Sequencher 4.2 sequence-

91 Chapter 4 analysis software (Gene Codes) by comparison with the reference sequence of TSFM. Sequence analysis of the last part of exon 7 of the TSFM gene was performed on genomic DNA by use of the primers 5'-CTTGGGCAGCCATGTGG-3'(forward) and 5'-ACCCATGCATTCTCGGTCTG-3'(reverse).

Blue-native PAGE and immunoblotting Blue-native PAGE was used for separation of the OXPHOS complexes on 6%-15% polyacrylamide-gradient gels.16 Mitoplasts, which were prepared from fibroblasts by treatment with 0.8 mg of digitonin per mg of protein, were solubilized with 1% lauryl maltoside, and 20 |jg of the solubilized protein was used for electrophoresis.17 Assembly of the OXPHOS complexes was established by immunoblot analysis with the use of monoclonal antibodies against subunits of complexes II-V (Molecular Probes) and a polyclonal antibody against the ND1 subunit of Complex I (a kind gift of A. Lombes, Paris). For immunoblotting, fibroblasts were solubilized with 1.5% lauryl maltoside in PBS, and 20 |jg of protein was separated by TRIS-Glycine SDS-PAGE gel. After transfer to a nitrocellulose membrane, individual proteins were detected using monoclonal antibodies against subunits of complexes I-V (Molecular Probes), porin (Sigma), MnSOD (Stressgen), and polyconal antibodies directed against ND1, EFTu (a kind gift of N. Takeuchi, Tokyo), EFTu/Ts (a kind gift of L. Spremulli, Chapel Hill, NC), and EFG1.10

Pulse labeling of mitochondrial translation products In vitro labeling of mitochondrial translation was performed as described elsewhere.18 In brief, cells were labeled for 60 min at 37°C in methionine-free DMEM with 10% dialyzed fetal bovine serum, containing 200 ^Ci/ml of [35S] methionine and 100 |jg/ml of emetine, followed by a 10-min incubation in regular DMEM with 10% fetal bovine serum. Total cellular protein (50 |jg) was resuspended in loading buffer containing 93 mM Tris-HCl (pH 6.7), 7.5% glycerol, 1% SDS, 0.25 mg bromophenol blue per ml, and 3% mercaptoethanol, was sonicated for 3-8 s, and was run on 12%-20% polyacrylamide-gradient gels. The gels were scanned on a Storm Phosphorimager (Molecular Dynamics) and analyzed with ImageQuant software. Under the conditions of the experiment, all signals were in the linear range. cDNA constructs, virus production, and infection Retroviral vectors containing the cDNA sequence of TUFM (GenBank accession number NM_003321) and TSFM were created with the Gateway cloning system (Invitrogen) as described elsewhere.10 The PCR constructs were cloned into a Gateway-modified retroviral-expression vector (pBABE-puro). Retroviral constructs were transiently transfected into Phoenix packaging cell line by use

92 Mitochondrial elongation factor Ts mutation of the HBS/Ca3(PO4)2 method (Helper Dependent Protocol Web site). Patient and control fibroblasts were infected 48 h later by exposure to virus-containing medium in the presence of 4 |jg/ml of polybrene, as described elsewhere.12

Computational modeling The crystal structure of the bovine EFTu/EFTs complex has been described elsewhere.19 On the basis of this model, the structure of wild-type and mutant EFTs protein were determined with the YASARA-WHAT IF twin set software.20 The R333W mutation was predicted using the method of De Fillippis et al.21 Molecular figures were drawn with the POV-Ray module in YASARA. Further modeling details are available at the TS Modelling Web site.

R e s u l t s Skeletal muscle and fibroblasts from patient 1 showed decreased enzyme activities of complexes I, III, and IV of the respiratory chain (table 1). The activities of complexes I, III, and IV in the skeletal muscle of patient 2 were similarly reduced

Figure 1. Pulse labeling of mitochondrial translation products in patient fibroblasts. Fibroblasts from the two patients (P1 and P2) and two controls (C1 and C2) were labeled with [35S]methionine in the presence of emetine, a cytosolic translation inhibitor, as outlined in the "Material and Methods" section. Fibroblasts from patient 1 were also analyzed after transduction with retroviral vectors overexpressing the mitochondrial translation factors EFTu or EFTs. The mitochondrial translation products are indicated on the left of the autoradiogram. COI-COIII = subunits of cytochrome c oxidase; cytb = cytochrome b subunit; ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 = subunits of NADH CoQ oxidoreductase; ATP6 and ATP8 = subunits of ATP synthase. The numbers at the bottom of the gel indicate the total incorporation of [35S]methionine relative to the corresponding control.

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Table 1. OXPHOS enzyme activities in skeletal muscle and fibroblasts of patient 1 Activities (mU/U citrate synthase) in Skeletal muscle Fibroblasts Complex Patient Reference range Patient Reference range I 0 70 - 250 53 100 - 310 III 753 2,200 - 6,610 1,250 1,320 - 2,610 II + III 228 300 - 970 163 110 - 470 IV 186 810 - 3,120 397 680 - 1,190 PDH 60 34 - 122 ND ND Note.-Samples were 600 g of supernatant of skeletal muscle or mitochondrial enriched fraction from fibroblasts. ND = not determined; PDH = pyruvate dehydrogenase complex. to 30%, 14%, and 14% of the control means, respectively, whereas complex II was unaffected (123% of the control mean). In fibroblast mitochondria from patient 2, complexes I, IV, II+III, and II activities were 44%, 25%, 77%, and 107% of the control mean, respectively. Pulse labeling of the mitochondrial translation products in fibroblasts from both patients revealed a generalized mitochondrial protein-synthesis defect; however, different polypeptides were variably affected (fig. 1 and table 2). The overall incorporation of [35S]methionine was 39%-49% of that in controls, and the rate of synthesis of most polypeptides was reduced to 30%-60% of control values. However, the synthesis of the two ATP synthase subunits (ATP6 and ATP8) actually increased relative to control levels in patient 1 and was similar to control levels in patient 2 (table 2). These data suggested that the genetic defect was due to a component of the mitochondrial translation apparatus, and the failure to identify any abnormalities in mtDNA (data not shown) suggested a nuclear gene defect. Two strategies were employed to identify the underlying genetic defect. In patient 1 (and 14 additional patients with a similar combined OXPHOS deficiency), we sequenced the cDNAs for the known mitochondrial translation initiation, elongation, and termination factors. In patient 2, we used the Affymetrix Human Mapping 50K Array Xba240 to genotype 58,495 SNPs to identify regions of homozygosity. The latter analysis identified a long segment of consecutive homozygous SNPs on chromosome 2 spanning 22.1 Mb (213.1-235.2 Mb) and another segment on chromosome 12 spanning 30.79 Mb (26.48-57.27 Mb). With use of the MitoP2 prediction program22 for mitochondrial localization (score >60), two proteins with known function in mitochondrial translation were identified within these regions - the mitochondrial ribosomal protein S35 and mitochondrial translation elongation factor EFTs (encoded by TSFM). Sequence analysis of TSFM revealed the same homozygous C997T mutation in exon 7 in both patients (fig. 2). A BLAST search showed that the C997T mutation

94 Mitochondrial elongation factor Ts mutation was not present in any reported human EST, and the mutation was not found in the other 14 patients tested or in 135 controls. The mutation was heterozygous in the parents in both families and in the two healthy sibs of patient 2 (data not shown). A sib of patient 1 was homozygous wild-type (data not shown). Analysis of microsatellite markers on chromosome 12 demonstrated that the mutation did not arise on a common haplotype in the two patients (fig. 2D). Human TSFM appears to be alternatively spliced, since exon 5 was not present in any of the patient or control cDNAs analyzed. Exon 5 is present in a single EST sequence in the human database, derived from bone marrow of a patient with acute myelogenous leukaemia. The mutation predicts an Arg333Trp substitution in the C-terminal domain of EFTs, and sequence alignment of this protein from several species shows that the mutated amino acid is highly conserved from human to bacteria (fig. 2B). This Arg residue makes favorable hydrophobic and electrostatic interactions within the helical part of subdomain C (fig. 3). Mutational analysis using the crystal structure of the bovine EFTu^EFTs complex shows that it is not possible to introduce a Trp at this position without serious clashes between the Trp side chain and the backbone atoms in the immediate surroundings. The close-up of the region of Ts that harbors the mutation shows three preferred rotamers of Trp, representing

Table 2. Synthesis of individual mitochondrial polypeptides in fibroblasts from patients P1 and P2 after pulse labeling with [35S]methionine Incorporation of [35S]methionine (% of control) + EFT Polypeptide P1 P2 P1 + EFTu P1 s ND5 22 18 59 49 COI 34 32 59 61 ND4 42 33 65 52 ND2/cytb 61 51 56 43 ND1 56 55 58 46 COIII/COII 42 34 56 60 ATP6 134 64 69 31 ND6 59 21 60 72 ND3 103 68 112 91 ND4L 29 30 43 48 ATP8 244 113 94 22 Note.- The results are presented as a percentage of the average incorporation of [35S]methionine in two relevant controls. Mitochondrial translation was also analyzed in patient 1 after transduction with retroviral constructs overexpressing EFTu or EFTs. COI-COIII = subunits of cytochrome c oxidase; cytb = cytochrome b subunit; ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 = subunits of NADH CoQ oxidoreductase.

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Figure 2. Sequence analysis of TSFM. A, DNA sequence chromatogram from a control and patient 1 (homozygous C997T) and a heterozygous chorionic villus sample. B, Domain structure of EFTs (N-terminal in black, subdomain N in white, and subdomain C in gray) showing the predicted Arg333Trp substitution in subdomain C. C, Protein sequence alignment for Homo sapiens, Bos taurus, Caenorhabditis eiegans, and E. Coli. The alignment from human to bacteria shows that the mutated Arg333Trp is highly conserved among different species. D, Analysis of microsatellite markers spanning the TSFM locus on chromosome 12. The markers are arbitrarily assigned to chromosomes in both patients. three different energy minima (fig. 3, lower panel). The rotamer that is predicted to be the most favorable in the absence of a surrounding bumps into the helical domain immediately beneath it, and the other two rotamers that normally are energetically favorable bump into other local residues, as indicated in the legend of figure 3. The mutation is predicted to disrupt the interaction between the long helix and two strands in subdomain C, moving the domain that holds the long helix away from domain III of EFTu19 and preventing it from contributing to EFTu^EFTs binding (fig. 3). To directly test whether the lack of a functional EFTs protein was the cause of the defective mitochondrial translation, we transduced patient fibroblasts with a retroviral vector expressing the wild-type TSFM cDNA. Since EFTs and EFTu form

96 Mitochondrial elongation factor Ts mutation a dimer in human cells, we also tested whether overexpression of wild-type EFTu could suppress the translation defect. Overexpression of either of these constructs (two-to-threefold for EFTu and three-to-fourfold for EFTs; fig. 4) had a dominant negative effect on mitochondrial translation in control cells. In independent experiments with different control lines, global mitochondrial translation was 61% ± 18% (five independent transductions in two control lines) of control in cells overexpressing EFTu and 74% ± 14% of control in cells overexpressing EFTs (four independent transductions in two control lines). In the results shown in figure 1, global mitochondrial translation is 68% and 75% of the control in cells overexpressing EFTu and EFTs, respectively. A relatively modest increase in global mitochondrial protein synthesis was observed in patient cells overexpressing EFTs

Figure 3. Computational modeling based on the crystal structure of the bovine EFTu^EFTs complex. The top panel shows the structure of the Tu^Ts dimer. Tu is on the left side of the model, and Ts on the right. The wild-type Arg 333 residue (depicted in orange) is superimposed on three Trp rotamers (depicted in yellow). The lower panel is a close-up of the region containing the mutation, which shows that the Arg residue fits well in front of the C-terminal side of the helix beneath it. The most favorable Trp rotamer bumps into this same helix. The second- and third-best local minima for the Trp rotamer bump into Glu 644 and Phe 637, respectively. The second rotamer also bumps into its own backbone.

97 Chapter 4 compared with the appropriate control; however, there was a significant increase in the rate of synthesis of several polypeptides of complex I and complex IV, and the rate of synthesis of ATP6 and ATP8 was significantly reduced relative to that observed in untransduced patient fibroblasts (table 2 and fig. 1). The results were generally similar in patient cells overexpressing EFTu. Thus, overexpression of either translation elongation factor could partially suppress the translation defect in the patient cells. To investigate the mechanism of suppression of the translation defect by EFTu, we analyzed the steady-state levels of the translation elongation factors by immunoblot analysis. EFTs and EFTu in patient fibroblasts were reduced to 25% and 40% of control levels, respectively (fig. 4). Overexpression of EFTu partially rescued EFTs levels, and EFTu levels were completely rescued by overexpression of EFTs. Overexpression of EFTs also slightly reduced EFTu levels in control cells. The levels of another mitochondrial translation elongation factor, EFG1, were unaltered in patient cells and were not changed in any of the cells overexpressing EFTs or EFTu.

Figure 4. Immunoblot analysis of fibroblasts from patient 1. Immunoblot analysis of the steady-state levels of three translation elongation factors (EFG1, EFTu, and EFTs) and eight OXPHOS system subunits (COI-49kDa, COI-39 kDa, COI-ND1, COII-70 kDa, COIII-Corel, COIV-COXI, COIV-COXIV, and COV-a) in control and patient fibroblasts before and after transduction with retroviral constructs expressing the mitochondrial translation elongation factors EFTu or EFTs. Porin and manganese superoxide dismutase (MnSOD) were used as loading controls.

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Figure 5. Blue-native PAGE analysis of the assembly of the OXPHOS complexes in patient fibroblasts. Fibroblasts from patient 1 and two controls (C1 and C2) were analyzed by blue-native PAGE before and after transduction with retroviral constructs expressing the mitochondrial translation elongation factors EFTu or EFTs. The gels were immunoblotted with antibodies directed against specific individual subunits to assess the amount of each of the fully assembled complexes.

To investigate whether the increased mitochondrial protein synthesis in patient cells overexpressing EFTu or EFTs resulted in increased OXPHOS function, we examined the assembly of the OXPHOS complexes by blue-native PAGE analysis (fig. 5). Patient fibroblasts showed reduced levels of fully assembled complexes I, IV, and V but normal levels of complex II and III, consistent with the deficiencies demonstrated by enzyme activity assays (table 1). The assembly of all three affected complexes was restored to control levels (in comparison with control fibroblasts overexpressing the same construct) by overexpression of either elongation factor (fig. 5). Functional complementation of the biochemical defect in patient cells overexpressing the elongation factors was also demonstrated by immunoblot analysis, which showed similar steady-state levels of both nuclear- and mitochondrial-encoded subunits of the OXPHOS complexes in patient cells and controls (fig. 4). Interestingly, overexpression of both factors led to slightly higher steady-state levels of some complex I subunits (39 kDa, 49 kDa, and ND1) in the patient cell line compared with controls and a markedly decreased expression of MnSOD, the mitochondrial superoxide dismutase.

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D i s c u s s i o n This study firmly establishes mutations in the mitochondrial translation elongation factor EFTs as the cause of disease in two unrelated patients with mitochondrial dysfunction due to a combined deficiency of OXPHOS enzymes. Several pieces of evidence support this conclusion. First, pulse labeling of the mitochondrial translation products in patient fibroblasts identified a global translation defect in both patients, leading to a failure to assemble adequate amounts of three of the OXPHOS complexes containing subunits encoded in mtDNA. This phenotype could be rescued by retroviral expression of the wild-type cDNAs for either EFTs or EFTu. Second, a homozygous missense mutation, predicting an amino acid substitution at an evolutionarily conserved site, was identified in TSFM in both patients. Molecular modeling predicts that this mutation would disrupt the Tu^Ts dimerization interface. Finally, the steady-state levels of mutant EFTs and wild type EFTu were reduced in patient fibroblasts, and this phenotype was rescued by overexpression of either EFTu or EFTs. Much of our knowledge of the mechanism of mitochondrial translation in mammals comes from studies of prokaryotic organisms, which share most of the same initiation, elongation, and termination factors.4 EFTs is a guanine nucleotide exchange factor that binds to the EFTu^guanosine diphosphate (GDP) complex, promoting the release of GDP and the formation of a stable EFTu^EFTs heterodimer. Guanosine triphosphate (GTP) then promotes dissociation of EFTs from this complex, regenerating EFTu^GTP, which can then bind another aminoacyl tRNA. This ternary complex associates with the ribosomal A site, and, when correct codon-anticodon recognition is established, GTP is hydrolyzed, releasing EFTu^GDP, and the cycle repeats itself. Interestingly, not all organisms require EFTs. For instance, the budding yeast Saccharomyces cerevisiae contains only EFTu, which can function both as a guanosine triphosphatase and its own guanine nucleotide exchange factor.23 Human TSFM maps to chromosome 12q13-q14, has an ORF of 1,040 bp, and comprises seven exons, although it appears that exon 5 is spliced out of the coding sequence in the human protein. EFTs from Escherichia coli is organized into four recognizable modules: N-terminus, core domain (consisting of subdomains N and C), coiled-coil domain, and C-terminal domain.24 The crystal structure of the bovine mitochondrial elongation EFTu^EFTs complex has been determined to a 0.22-nm resolution,19 and it appears to be broadly similar to that in E. coli, with a notable exception of the virtual absence of the coiled-coil and C-terminal domains in the mammalian protein.19 Human EFTu is 95% identical25 and human EFTs is 91% identical26 to the bovine sequence, and this permitted us to model the effects of the homozygous Arg333Trp mutation onto the bovine sequence

100 Mitochondrial elongation factor Ts mutation without altering the backbone structure. Molecular modeling predicts that the mutation will alter the local structure of subdomain C, abolishing most of the important interactions of this portion of EFTs with domain III of EFTu (fig. 3). This is likely to lead to the destabilization of the EFTu^EFTs complex and an increased turnover of its components, explaining the decreased steady-state levels of both factors we observed in the patient cells (fig. 4). Expression of the wild-type version of either factor would stabilize the complex, thus increasing the total EFTu^EFTs pool. This can explain why overexpression of EFTu acts as a suppressor of the translation defect in patient cells. However, the observation that overexpression of either EFTu or EFTs in control cells acts as a dominant negative - decreasing the rate of mitochondrial protein synthesis by ~35%-40% and ~20%-25%, respectively10 (fig. 1) - shows that the relative ratio of these elongation factors is a critical determinant of mitochondrial translation efficiency. Indeed, we observed that overexpression of EFTs slightly reduced the levels of EFTu in control fibroblasts, again suggesting that the relative expression of both factors is finely tuned. Consistent with this interpretation, co-overexpression of both EFTu and EFTs partially rescued the dominant negative effect of EFTu in control fibroblasts.10 It is not clear why the synthesis of the different mtDNA-encoded polypeptides is variably affected as a result of the TSFM mutation, but a similar phenomenon has been observed in fibroblasts from patients with mutations in the elongation factor EFG1.9,10 The most striking difference in this study, and in patients with EFG1 mutations, is in the synthesis of the two complex V subunits (ATP6 and ATP8), for which the rate of synthesis is similar to the control or even increased in the presence of the mutant elongation factor. The most parsimonious interpretation of this observation is that the mRNA coding for these polypeptides, a bicistronic mRNA with overlapping reading frames, can be translated more efficiently than can mRNAs coding for the other polypeptides, most of which (except ND4/4L) are translated from monocistronic mRNAs. Whether this difference reflects an increased efficiency of translation of the ATP6 and ATP8 mRNA per se, or simply reduced competition from other transcripts for a limited number of ribosomes, remains uncertain, since the factors that regulate the efficiency of translation initiation and elongation in mitochondria are largely unknown. The Arg333Trp TSFM mutation in patient 1 led to a fatal mitochondrial encephalomyopathy, with muscle weakness, hypotonia, rhabdomyolysis, and epilepsy as the most prominent clinical signs and symptoms. In striking contrast, the identical mutation in patient 2 was associated with a hypertrophic cardiomyopathy, but the results of neurological examination and CT scan were normal. Although tissue specificity in the clinical phenotype is common in mitochondrial disease, as far as we are aware, this is the first example of a

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nuclear-encoded mitochondrial accessory factor in which the identical mutation is associated with such a remarkable difference in clinical presentation. In addition, in the patients with EFG1 mutations, cardiac function was normal, and the only apparent OXPHOS defect in this tissue was a modest reduction in the assembly of complex IV.9,10 Moreover, none of the other study patients with combined OXPHOS enzyme deficiencies presented with early-onset cardiomyopathy as the major clinical symptom. This suggests the presence of genetic modifiers, but these await identification. Combined enzyme deficiencies of the OXPHOS system are a frequent cause of mitochondrial disorders. The traditional genetic diagnostic approach in affected patients often includes the study of mitochondrial tRNAs and, more recently, sequencing of the entire mitochondrial genome. This study suggests that it will be useful to include a genetic screen of nuclear-encoded translation elongation factors in the diagnostic program for these patients or to perform SNP genotyping in all patients originating from consanguineous families.

A cknowledgements We thank the support staff of the Nijmegen Centre for Mitochondrial Disorders for biochemical analysis of the patient skeletal muscle and fibroblast OXPHOS- system enzyme activities. J.S. and B.v.d.H. are currently supported by grants from the Radboud University Medical Centre Nijmegen, the Prinses Beatrix Fonds, and the European Union (EUMITOCOMBAT [LSHM-CT-2004-503116] and MitoCircle). This research was supported by the Canadian Institutes of Health Research (CIHR) (to E.A.S.), by the Israel Science Foundation (grant 1354-2005 [to O.E. and A.Saada]), and the Israeli Ministry of Science and Technology (grant 3-904 [to O.E.]). G.V. acknowledges BioSapiens, which is funded by the European Commission within its FP6 Programme, under the thematic area "Life sciences, genomics and biotechnology for health," contract LSHG-CT-2003-503265. E.A.S. is an international scholar of the Howard Hughes Medical Institute and a senior scientist of the CIHR.

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w e b r e s o u r c e s Accession numbers and URLs for data presented herein are as follows:

BLAST, http://www.ncbi.nlm.nih.gov/BLAST/ GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for TSFM [accession number NM_005726] and TUFM [accession number NM_003321]) Helper Dependent Protocol, http://www.stanford.edu/group/nolan/protocols/ pro_helper_dep.html Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/ Omim/ (for TSFM) POV-Ray, http://www.povray.org/ TS Modelling, http://swift.cmbi.ru.nl/gv/service/ts/ YASARA, http://www.yasara.org/

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R e f e r e n c e s 1. Thorburn DR: Mitochondrial disorders: prevalence, myths and advances. J Inherit Metab Dis 2004; 27: 349-362. 2. Smeitink J, van den Heuvel L, DiMauro S: The genetics and pathology of oxidative phosphorylation. Nat Rev Genet 2001; 2: 342-352. 3. Jacobs HT: Disorders of mitochondrial protein synthesis. Hum Mol Genet 12 Spec No 2003; 2: R293-R301. 4. Spremulli LL, Coursey A, Navratil T, Hunter SE: Initiation and elongation factors in mammalian mitochondrial protein biosynthesis. Prog Nucleic Acid Res Mol Biol 2004; 77: 211-261. 5. Zhang Y, Spremulli LL: Identification and cloning of human mitochondrial translational release factor 1 and the ribosome recycling factor. Biochim Biophys Acta 1998; 1443: 245-250. 6. O'Brien TW: Evolution of a protein-rich mitochondrial ribosome: implications for human genetic disease. Gene 2002; 286: 73-79. 7. DiMauro S, Schon EA: Mitochondrial respiratory-chain diseases. N Engl J Med 2003; 348: 2656-2668. 8. Taylor RW, Turnbull DM: Mitochondrial DNA mutations in human disease. Nat Rev Genet 2005; 6: 389-402. 9. Coenen MJ, Antonicka H, Ugalde C, et al: Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency. N Engl J Med 2004; 351: 2080-2086. 10. Antonicka H, Sasarman F, Kennaway NG, Shoubridge EA: The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1. Hum Mol Genet 2006; 15: 1835-1846. 11. Miller C, Saada A, Shaul N, et al: Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation. Ann Neurol 2004; 56: 734-738. 12. Lochmuller H, Johns T, Shoubridge EA: Expression of the E6 and E7 genes of human papillomavirus (HPV16) extends the life span of human myoblasts. Exp Cell Res 1999; 248: 186-193. 13. Smeitink J, Sengers R, Trijbels F, van den Heuvel L: Human NADH:ubiquinone oxidoreductase. J Bioenerg Biomembr 2001; 33: 259-266. 14. Janssen AJ, Smeitink JA, van den Heuvel LP: Some practical aspects of providing a diagnostic service for respiratory chain defects. Ann Clin Biochem 2003; 40: 3-8. 15. Zhu Z, Yao J, Johns T, et al: SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome. Nat Genet 1998; 20: 337-343.

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16. Schagger H, von Jagow G: Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 1991; 199: 223-231. 17. Klement P, Nijtmans LG, Van den Bogert C, Houstek J: Analysis of oxidative phosphorylation complexes in cultured human fibroblasts and amniocytes by blue-native-electrophoresis using mitoplasts isolated with the help of digitonin. Anal Biochem 1995; 231: 218-224. 18. Boulet L, Karpati G, Shoubridge EA: Distribution and threshold expression of the tRNA(Lys) mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF). Am J Hum Genet 1992; 51: 1187-1200. 19. Jeppesen MG, Navratil T, Spremulli LL, Nyborg J: Crystal structure of the bovine mitochondrial elongation factor Tu.Ts complex. J Biol Chem 2005; 280: 5071-5081. 20. Vriend G: WHAT IF: a molecular modeling and drug design program. J Mol Graph 1990; 8: 52-56, 29. 21. De Filippis V, Sander C, Vriend G: Predicting local structural changes that result from point mutations. Protein Eng 1994; 7: 1203-1208. 22. Andreoli C, Prokisch H, Hortnagel K, et al: MitoP2, an integrated database on mitochondrial proteins in yeast and man. Nucleic Acids Res 2004; 32: D459-D462. 23. Chiron S, Suleau A, Bonnefoy N: Mitochondrial translation elongation factor tu is essential in fission yeast and depends on an exchange factor conserved in humans but not in budding yeast. Genetics 2005; 169: 1891-1901. 24. Kawashima T, Berthet-Colominas C, Wulff M, Cusack S, Leberman R: The structure of the Escherichia coli EF-Tu.EF-Ts complex at 2.5 A resolution. Nature 1996; 379: 511-518. 25. Woriax VL, Burkhart W, Spremulli LL: Cloning, sequence analysis and expression of mammalian mitochondrial protein synthesis elongation factor Tu. Biochim Biophys Acta 1995; 1264: 347-356. 26. Xin H, Woriax V, Burkhart W, Spremulli LL: Cloning and expression of mitochondrial translational elongation factor Ts from bovine and human liver. J Biol Chem 1995; 270: 17243-17249.

105 Life is not always fair. Sometimes you can get a splinter even sliding down a rainbow. Terri Guillemets C h apter 5 Mutation in subdomain G' of mitochondrial elongation factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle

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Paulien Smits, Hana Antonicka, Peter M. van Hasselt, Woranontee Weraarpachai, Wolfram Haller, Marieke Schreurs, Hanka Venselaar, Richard J.T. Rodenburg, Jan A.M. Smeitink and Lambert P. van den Heuvel

European Journal of Human Genetics 2011; 19(3): 275-279 Chapter 5

A b s t r a c t The mitochondrial translation system is responsible for the synthesis of 13 proteins required for oxidative phosphorylation (OXPHOS), the major energy- generating process of our cells. Mitochondrial translation is controlled by various nuclear encoded proteins. In 27 patients with combined OXPHOS deficiencies, in whom complex II (the only complex that is entirely encoded by the nuclear DNA) showed normal activities and mutations in the mitochondrial genome as well as polymerase gamma were excluded, we screened all mitochondrial translation factors for mutations. Here we report a mutation in mitochondrial elongation factor G1 (GFM1) in a patient affected by severe, rapidly progressive mitochondrial encephalopathy. This mutation is predicted to result in an Chapter Arg250Trp substitution in subdomain G' of the elongation factor G1 protein and is presumed to hamper ribosome-dependent GTP hydrolysis. Strikingly, the 5 decrease in enzyme activities of complex I, III and IV detected in patient fibroblasts was not found in muscle tissue. The OXPHOS system defects and the impairment in mitochondrial translation in fibroblasts were rescued by overexpressing wild type GFM1, establishing the GFM1 defect as the cause of the fatal mitochondrial disease. Furthermore, this study evinces the importance of a thorough diagnostic biochemical analysis of both muscle tissue and fibroblasts in patients suspected to suffer from a mitochondrial disorder, as enzyme deficiencies can be selectively expressed.

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I ntroduction Mitochondria harbor a translation system distinct from the one in the cell's cytoplasm. This system is essential for the functioning of the oxidative phosphorylation (OXPHOS) system, and thus for energy production, through the synthesis of 13 proteins of OXPHOS complexes I, III, IV and V. The remaining OXPHOS system subunits, as well as all other mitochondrial proteins, including those involved in mitochondrial translation, are encoded by nuclear DNA. Defects in the mitochondrial translation machinery, due to either mitochondrial (mt) or nuclear (n) DNA mutations, can lead to the dysfunction of multiple mtDNA-reliant OXPHOS complexes. Mitochondrial disorders caused by OXPHOS deficiencies are often devastating diseases that are estimated to occur in 1:5000 live births.1 Sequencing of all mitochondrial translation factors (MTIF2, MTIF3, TSFM, Chapter TUFM, GFM1, GFM2, MTRF1, MTRF1L and MRRF) in a cohort of 27 patients with combined OXPHOS enzyme deficits in fibroblasts and/or muscle tissue, in whom 5 complex II showed normal activities and mutations in mtDNA and polymerase gamma were excluded, revealed a novel mutation in elongation factor G1 (gene: GFM1, protein: EFG1) in one patient. In this report, we demonstrate that the combined OXPHOS deficiency and mitochondrial protein synthesis defect detected in patient fibroblasts are the result of the GFM1 mutation, ultimately leading to mitochondrial encephalopathy inducing rapid neurological deterioration, therapy-resistant epilepsy and death at 2 years of age.

M a t e r i a l s a n d m e t h o d s Case report The female patient was born at term as the second of a dizygotic twin after an uneventful gestation and birth. She was small for gestational age, with a birth weight of 2330 g at 40 weeks of gestation. Parents were consanguineous (second cousins). Two days after birth she was briefly admitted to the hospital because of feeding problems and reduced consciousness that were attributed to an infection. Cultures of urine and blood, however, remained negative. In the following months, she developed very slowly compared with her healthy twin-sister. In addition, she was severely hypotonic, made poor eye contact and continued to have significant feeding problems. Seizures were noted from the age of 8 weeks onwards. Examination at the age of 3 months showed a borderline microcephalic girl (head circumference was -2.5 SD) with wandering eye movements (suggesting delayed visual maturation) and without dysmorphic features. Axial hypotonia was accompanied by hypertonia of the extremities with brisk tendon reflexes. Liver function tests, including bilirubin levels, were normal, except for a repeatedly low alanine aminotransferase (ALAT range 3-15 U/l, N:

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10-50 U/l; ASAT range 24-68 U/l, N: 20-65 U/l). Blood ammonia was normal. Biochemical examination revealed elevated lactate levels in blood (4.9 mmol/l, N: <2.1mmol/l) and cerebrospinal fluid (CSF) (2.5 mmol/l, N: <2.1 mmol/l) as well as an increased plasma lactate:pyruvate ratio (22, N: 12-18). Amino acids in plasma and CSF were normal. Urine organic acids showed increased excretion of lactate (454 mmol/mol creatinine, N: < 156 mmol/mol creatinine), but no other abnormalities. Ophthalmological and cardiological investigations were normal. Neuroimaging revealed a small frontal cortex, a thin corpus callosum and delayed myelination. Multifocal epileptic discharges were found on electroencephalogram (EEG): a disturbed background pattern with multifocal spikes and waves, no clear hypsarrythmia; seizures consisting of 1-3 s of background suppressions with high

Chapter frequency discharge, predominantly over occipital areas, resulting in nystagmus; and an occasional tonic component, resembling infantile spasm. A muscle biopsy 5 was performed on suspicion of a mitochondrial disease. Histological examination of the skeletal muscle showed normal mitochondrial morphology without ragged- red fibers. On follow-up she deteriorated. She lost the ability to suck within the first year of life. Microaspirations persisted after introducing tube feeding and induced recurrent pneumonia. In addition, she developed hepatomegaly (4 cm below the right costal margin at the age of 8 months). The epilepsy progressed, the EEG evolving into a Lennox-Gastaut syndrome, and was resistant to various antiepileptic drugs (vigabatrin, nitrazepam and ACTH), whereas a ketogenic diet had only a modest favorable effect. She died at the age of 2 years of respiratory insufficiency secondary to pneumonia. The parents did not consent to perform a postmortem.

Cell culture Human skin fibroblasts were cultured in M199 medium (Gibco, Breda, The Netherlands) supplemented with 10% fetal calf serum and antibiotics. For complementation experiments, primary skin fibroblasts were immortalized with a retroviral vector expressing the E7 gene of type 16 human papilloma virus and another retroviral vector expressing the protein component (htert) of human telomerase.2 Patient and control skin fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum.

Enzyme measurements The activities of the OXPHOS enzyme complexes were measured in skin fibroblasts and muscle tissue as described previously.34 The pyruvate oxidation and ATP production rates were determined in muscle tissue.5

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Sequence analysis Total RNA was isolated from cultured skin fibroblasts using RNA-Bee (Tel- Test, Friendswood, TX, USA); complementary DNA (cDNA) was prepared using superscript II reverse transcriptase (Invitrogen, Breda, The Netherlands). Sequence analysis of the open reading frame of GFM1 (GenBank accession number NM_024996) was performed in four overlapping fragments (primer sequences are available on request). The presence of the mutation was confirmed on genomic DNA, isolated from patient fibroblasts and from blood from control subjects and from the patient's parents using salt extraction,6 with primers 5'-CATTACTCTTATTTGCCATTTG-3' (forward) and 5'-GGGTCCTCAATAACTTTTC-3' (reverse). Both strands were used for direct sequencing on a 3130xl Genetic

Analyzer, using the BigDye terminator v1.1 cycle sequencing kit according to Chapter the manufacturer's protocol (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). Sequence data were analyzed with Sequencher 4.8 software (Gene 5 Codes, Ann Arbor, MI, USA) by comparison with the reference sequence of GFM1.

Computational modeling The structures of the wild-type and mutant EFG1 proteins were based on the crystal structure of Thermus thermophilus EFG.7 Modeling, energy minimization and analysis were performed using the YASARA (YASARA Biosciences GmbH, Vienna, Austria) and WHAT IF Twinset software (WHAT IF Foundation, Nijmegen, The Netherlands).8 The human EFG1 and T. thermophilus EFG proteins share 45% sequence identity; the first 43 EFG1 residues could not be modeled. Molecular figures were drawn with the POV-Ray module in YASARA. cDNA constructs and virus production and infection A retroviral vector containing the cDNA sequence of GFM1 was created with the Gateway cloning system (Invitrogen, Carlsbad, CA, USA). The GFM1 cDNA was amplified from I.M.A.G.E. clone No. 5574223 (ATCC) using specific primers and this PCR construct was subsequently cloned into a Gateway-modified retroviral expression vector, pBABE. The fidelity of the cDNA clone was confirmed by automated DNA sequencing. The retroviral construct was transiently transfected into a Phoenix packaging cell line using the HBS/Ca3(PO4)2 method (http://www. stanford.edu/group/nolan/protocols/pro_helper_dep.html). Patient and control fibroblasts were infected 48 h later by exposure to virus-containing medium in the presence of 4 ^g/ml of polybrene, as described.2

Blue-native and SDS-PAGE analysis Blue-native PAGE was used for separation of the OXPHOS complexes on 6-15% polyacrylamide gradient gels.9 Mitoplasts, which were prepared from fibroblasts by

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treatment with 0.8 mg of digitonin/mg of protein, were solubilized with 1% lauryl maltoside, and 20 |jg of the solubilized protein was used for electrophoresis.10 Assembly of the OXPHOS complexes was investigated by immunoblot analysis using monoclonal antibodies against subunits of complexes II-V (Molecular Probes/Invitrogen, Carlsbad, CA, USA) and a polyclonal anti-ND1 antibody for complex I detection (a kind gift of A. Lombes, Paris). One-dimensional 10% SDS-PAGE analysis was performed as described previously.11 A polyclonal antibody directed against EFG112 and a monoclonal antibody against the 70 kDa subunit of complex II (Molecular Probes/Invitrogen, Leiden, The Netherlands) were used for detection.

Chapter Pulse labeling of mitochondrial translation products 35S labeling of mitochondrial translation was performed as described elsewhere.13 5 In short, cells were labeled for 60 min at 37 °C in methionine-free DMEM with 200 |jCi/ml of a [35S]-methionine and -cysteine mixture (Perkin Elmer, Woodbridge, ON, Canada) and 100 |jg/ml emetine, and subsequently chased for 10 min in regular DMEM. Total cellular protein (50 |jg) was resuspended in loading buffer containing 93 mM Tris-HCl (pH 6.7), 7.5% glycerol, 1% SDS, 0.25 mg bromophenol blue per ml and 3% mercaptoethanol, sonicated for 3-8 s and run on 15-20% polyacrylamide gradient gels.

R e s u l t s Patient fibroblasts showed decreased enzyme activities of OXPHOS complexes I, III and IV, whereas complex II activity was normal (Table 1). Strikingly, skeletal muscle biochemistry revealed normal enzyme activities for OXPHOS complexes I and IV, whereas complex III was only moderately decreased compared with the lowest reference range value. The capacity of the mitochondrial energy generating

Table 1. OXPHOS enzyme activities in skeletal muscle and skin fibroblasts Activities (mU/U citrate synthase) in Skeletal muscle Skin fibroblasts Complex Patient Reference range Patient Reference range I 79 70 - 250 34 100 - 310 II 97 67 - 177 595 520 - 845 III 2151 2200 - 6610 1038 1320 - 2610 II+III 319 300 - 970 122 110 - 470 IV 1103 810 - 3120 168 680 - 1190

Abbreviation: OXPHOS, oxidative phosphorylation. Samples were 600 g of supernatant of skeletal muscle or mitochondrial enriched fraction from fibroblasts. Decreased activities as compared with controls are in bold.

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Table 2. Pyruvate oxidation and ATP production rates in skeletal muscle Substrate Patient Reference range [1-14C]pyruvate + malate 1.53 a 3.61 - 7.48 a [1-14C]pyruvate+carnitine 2.07 a 2.84 - 8.24 a ATP + CrP from oxidation of pyruvate 13 b 42 - 81 b Abbreviations: ATP, adenosine triphosphate; CrP, phosphocreatine. a nmol CO2/hr mU citrate synthase b nmol ATP/hr mU citrate synthase system, however, was clearly reduced in muscle tissue, indicated by impairments in pyruvate oxidation and ATP production rates (Table 2). After excluding mutations in mtDNA and polymerase gamma (POLG and POLG2), the nine known mitochondrial translation factors (MTIF2, MTIF3, TSFM, Chapter TUFM, GFM1, GFM2, MTRF1, MTRF1L and MRRF) were sequenced. This revealed a homozygous C to T transition at position 748 of the coding sequence of GFM1 5 (Fig. 1A). The mutation was not present in any reported human EST or in 100 control subjects and was found heterozygous in both parents (Figure 1a). This mutation is predicted to result in an Arg250Trp substitution in helix A of the G' subdomain, which is part of domain I of the EFG1 protein (Figure 1b). Sequence alignment of EFG1 orthologs in several species shows that this amino acid is highly conserved from humans to bacteria (Figure 1c). Immunoblot analysis revealed a severe reduction in the steady-state level of EFG1 in patient fibroblasts (Figure 1d). Figure 2 depicts how the Arg residue is positioned on the periphery of the protein and forms a hydrogen bond with Glu 153; this bond is lost when the Trp residue is introduced. Furthermore, a positive charge, which could be important for interactions between helix A and other proteins, is lost because of the mutation. In addition, the large and aromatic Trp side chain could interfere with these protein interactions. To investigate whether the lack of functional EFG1 protein was responsible for the combined OXPHOS system defect, we transduced patient fibroblasts with a retroviral vector expressing the wild-type GFM1 cDNA. Patient fibroblasts displayed markedly reduced levels of fully assembled OXPHOS complexes (Figure 3b), corresponding to the deficiencies detected using enzyme activity assays (Table 1), as well as a clear impairment in mitochondrial protein synthesis (Figure 3a). These phenotypes were rescued by overexpressing wild-type GFM1 (Figures 3a and b). The mitochondrial translation rate increased from 51 to 92% of control levels and the activity of complex IV was restored from 20 to 88% of the activity in control fibroblasts.

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Chapter 5

Figure 1. Mutational analysis of GFM1. (a) DNA sequence chromatogram from the patient (homozygous c.748C>T) and both parents (heterozygous). (b) EFG1 protein structure with the functional domains showing the predicted Arg250Trp substitution in the G' subdomain of domain I. (c) Protein sequence alignment from humans to bacteria. The alignment shows that the mutated Arg250Trp is highly conserved among different species. (d) Immunoblot analysis in patient (P) and control (C) fibroblasts reveals the absence of the EFG1 protein. The 70 kDa subunit of complex II (CII) was used as a loading control.

Figure 2. Computational modeling based on the crystal structure of the T. thermophilus EFG protein. The left panel shows the structure of the entire human EFG1 protein and the right panel is a close-up of the outlined region containing the mutation. The wild-type Arg 250 residue is depicted in green and the mutant Trp is depicted in red (bound GDP is depicted in yellow). The right panel shows that the Trp residue fits in the structure; however, the hydrogen bond that Arg forms with Glu 153 is lost.

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Chapter 5

Figure 3. Rescue of the mitochondrial translation defect and combined OXPHOS deficiency in patient fibroblasts by expression of GFM1. Control (C) and patient (P) fibroblasts were transduced with a retroviral vector expressing the wild-type GFM1 cDNA and analyzed by a mitochondrial translation assay (a) and BN-PAGE (b). The mitochondrial translation products are indicated on the left: seven subunits of complex I (ND), one subunit of complex III (cyt b), three subunits of complex IV (COX) and two subunits of complex V (ATP). Co I - Co V = complex I to V.

D i s c u s s i o n A homozygous mutation in subdomain G' of translation elongation factor G1 (Arg250Trp) led to a combined OXPHOS deficiency and a clinical picture consistent with severe, rapidly progressive mitochondrial encephalopathy in our patient. EFG1 is a GTPase that catalyzes the translocation step of mitochondrial protein synthesis, during which the deacylated tRNA moves from the ribosomal peptidyl (P) to the exit (E) site, the peptidyl-tRNA moves from the acceptor (A) to the P site and the mRNA is advanced by one codon, preparing for a new elongation cycle.14 EFG1 consists of five domains; GTP-binding domain I (or G) contains a large insert, subdomain G', which is indirectly required for translocation. The Arg250Trp mutation replaces a highly conserved arginine residue by tryptophan in helix A of subdomain G', which possibly disturbs the protein structure because of the loss of a stabilizing hydrogen bond and might hamper interaction of helix A with other

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proteins. The G' domain of bacterial EF-G has been demonstrated to interact with ribosomal protein L7/L12 (L7 and L12 refer to the W-acetylated and unacetylated forms of the same protein).15,16 Multiple copies of L7/L12, together with protein L10, make up a peripheral stalk of the large ribosomal subunit; the end of the stalk contains the C-terminal domain (CTD) of L7/L12.17 The CTD of L7/L12 promotes recruitment of EF-G to the ribosome, greatly stimulates its GTP hydrolysis activity and controls the release of inorganic phosphate.1718 Research by Nechifor and Wilson indicated that helix A of EF-G domain G' is directly involved in ribosome- activated GTP hydrolysis and thereby exerts indirect effects on translocation.19 Mutating the conserved residue 224 or 228 in helix A of Escherichia coli EF-G (corresponding to amino acids 255 and 259 in human EFG1), thus disrupting

Chapter electrostatic interactions with L7/L12, resulted in severe defects in GTP hydrolysis in the presence of the ribosome (without effects on basal GTPase activity) and 5 smaller defects in ribosomal translocation. Hence, the Arg250Trp mutation in EFG1 reported in the current study is presumed to hamper ribosome-dependent GTP hydrolysis, causing impairments in mitochondrial translation and oxidative phosphorylation and consequently fatal mitochondrial encephalopathy. Previously, three other EFG1 defects have been described. The first patient harbored a homozygous mutation in the GTP-binding domain I (Asn174Ser),20 and subsequent reports revealed compound heterozygous missense and nonsense mutations affecting other domains of EFG1: Ser321Pro (on the boundary between domains I and II)+Leu607X (predicted to remove domain V)12 and Met496Arg (in domain III)+Arg47X (predicted to eliminate basically all domains).21 Mitochondrial translation was impaired in a similar manner in all patients: a severe overall decrease in the rate of mitochondrial translation, with the expression of subunits ND5, ND6, COX I, II and III generally being the lowest. ND3 expression, on the contrary, was often increased compared with control levels. The previously reported patients all presented with OXPHOS enzyme deficiencies in muscle and fibroblasts, in contrast to our patient, in whom decreased enzyme activities were detected merely in fibroblasts. Remarkably, the muscular symptoms were relatively mild in all cases. Lowered respiratory chain activities in fibroblasts with normal values in muscle tissue are an uncommon finding in mitochondrial disorders, whereas the opposite is often observed. It should be noted that the capacity of the mitochondrial energy-generating system was affected substantially in muscle tissue of our patient. The sensitivity of the assays used to determine this capacity, such as pyruvate oxidation, is much higher than that of the standard enzyme activity measurements.22 However, the mechanism underlying the (near) normal enzyme activities in muscle is currently unclear. Adaptive processes (eg, compensatory changes in the levels of other translation factors), which can differ quantitatively and qualitatively between tissues and patients, are likely involved.

116 Mitochondrial elongation factor G1 mutation

Two of the previous reports on EFG1 defects described a very severe phenotype characterized by intrauterine growth retardation, microcephaly, hypertonicity and profound liver dysfunction, resulting in liver failure and death within the first weeks or months.12,20 The clinical symptomatology in our patient is reminiscent of the somewhat milder phenotype described by Valente et al,21 which was characterized by feeding problems, axial hypotonia, increased limb muscle tone and infantile-onset epilepsy. Notably, liver dysfunction was absent, as in our patient. Whereas ß-hydroxybutyrate in blood and urine were normal in our patient, Valente et al reported increased levels. The biochemical findings appear to correlate with disease severity: our patient showed lower blood lactate levels (4.9 versus 17 mmol/l) and lactate:pyruvate ratios (22 versus 38) compared with the first reported EFG1 patient,20 suggesting a more modest disturbance of Chapter mitochondrial functioning. The patient investigated here was part of a cohort of 27 patients with combined 5 OXPHOS deficiencies. Sequence analysis of all mitochondrial translation factors additionally revealed a homozygous mutation in elongation factor Ts (TSFM) in one patient: Arg333Trp, which has been reported before in two unrelated patients.23 All three patients presented with extensive lactic acidosis and muscular hypotonia within 3 days after birth, displayed combined complex I, III and IV deficiencies in fibroblasts as well as muscle tissue, and died in the first few months of life. W hereas the mutation in TSFM was associated with either mitochondrial encephalomyopathy or hypertrophic cardiomyopathy in the previously published cases, our patient was affected by both. In addition, hepatomegaly was observed. The current study evinces the importance of a thorough diagnostic biochemical analysis of muscle tissue and fibroblasts in patients suspected to suffer from a mitochondrial disorder, as OXPHOS defects do not always manifest evidently in both tissues. Future research should elucidate the compensatory and regulatory mechanisms, at the mitochondrial, cellular and tissue levels, responsible for the differences in phenotypic presentation of mitochondrial disorders due to GFM1 and other mitochondrial translation factor mutations.

A cknowledgements This work was supported by the European Union's Sixth Framework Program, contract number LSHMCT-2004-005260 (MITOCIRCLE). We thank Benigna Geurts (Nijmegen Center for Mitochondrial Disorders) for performing sequence analysis of GFM1 on control samples.

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R e f e r e n c e s 1. Schaefer AM, Taylor RW, Turnbull DM, Chinnery PF: The epidemiology of mitochondrial disorders--past, present and future. Biochim Biophys Acta 2004; 1659: 115-120. 2. Lochmuller H, Johns T, Shoubridge EA: Expression of the E6 and E7 genes of human papillomavirus (HPV16) extends the life span of human myoblasts. Exp Cell Res 1999; 248: 186-193. 3. Janssen AJ, Smeitink JA, van den Heuvel LP: Some practical aspects of providing a diagnostic service for respiratory chain defects. Ann Clin Biochem 2003; 40: 3-8. 4. Smeitink J, Sengers R, Trijbels F, van den Heuvel L: Human NADH:ubiquinone oxidoreductase. J Bioenerg Biomembr 2001; 33: 259-266. Chapter 5. Janssen AJ, Trijbels FJ, Sengers RC et al: Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to 5 human pathology. Clin Chem 2006; 52: 860-871. 6. Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215. 7. Hansson S, Singh R, Gudkov AT, Liljas A, Logan DT: Structural insights into fusidic acid resistance and sensitivity in EF-G. J Mol Biol 2005; 348: 939-949. 8. Krieger E, Koraimann G, Vriend G: Increasing the precision of comparative models with YASARA NOVA--a self-parameterizing force field. Proteins 2002; 47: 393-402. 9. Schagger H, von Jagow G: Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 1991; 199: 223-231. 10. Klement P, Nijtmans LG, van den Bogert C, Houstek J: Analysis of oxidative phosphorylation complexes in cultured human fibroblasts and amniocytes by blue- native-electrophoresis using mitoplasts isolated with the help of digitonin. Anal Biochem 1995; 231: 218-224. 11. Ugalde C, Vogel R, Huijbens R, van den Heuvel B, Smeitink J, Nijtmans L: Human mitochondrial complex I assembles through the combination of evolutionary conserved modules: a framework to interpret complex I deficiencies. Hum Mol Genet 2004; 13: 2461-2472. 12. Antonicka H, Sasarman F, Kennaway NG, Shoubridge EA: The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1. Hum Mol Genet 2006; 15: 1835-1846. 13. Leary SC, Sasarman F: Oxidative phosphorylation: synthesis of mitochondrially encoded proteins and assembly of individual structural subunits into functional holoenzyme complexes. Methods Mol Biol 2009; 554: 143-162.

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14. Wintermeyer W, Peske F, Beringer M, Gromadski KB, Savelsbergh A, Rodnina MV: Mechanisms of elongation on the ribosome: dynamics of a macromolecular machine. Biochem Soc Trans 2004; 32: 733-737. 15. Gao YG, Selmer M, Dunham CM, Weixlbaumer A, Kelley AC, Ramakrishnan V: The structure of the ribosome with elongation factor G trapped in the posttranslocational state. Science 2009; 326: 694-699. 16. Nechifor R, Wilson KS: Crosslinking of translation factor EF-G to proteins of the bacterial ribosome before and after translocation. J Mol Biol 2007; 368: 1412-1425. 17. Diaconu M, Kothe U, Schlunzen F et al: Structural basis for the function of the ribosomal L7/12 stalk in factor binding and GTPase activation. Cell 2005; 121: 991 1004. Chapter 18. Savelsbergh A, Mohr D, Kothe U, Wintermeyer W, Rodnina MV: Control of phosphate release from elongation factor G by ribosomal protein L7/12. EMBO J 2005; 24: 5 4316-4323. 19. Nechifor R, Murataliev M, Wilson KS: Functional interactions between the G' subdomain of bacterial translation factor EF-G and ribosomal protein L7/L12. J Biol Chem 2007; 282: 36998-37005. 20. Coenen MJ, Antonicka H, Ugalde C et al: Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency. N Engl J Med 2004; 351: 2080-2086. 21. Valente L, Tiranti V, Marsano RM et al: Infantile encephalopathy and defective mitochondrial DNA translation in patients with mutations of mitochondrial elongation factors EFG1 and EFTu. Am J Hum Genet 2007; 80: 44-58. 22. Janssen AJ, Schuelke M, Smeitink JA et al: Muscle 3243A-->G mutation load and capacity of the mitochondrial energy-generating system. Ann Neurol 2008; 63: 473-481. 23. Smeitink JA, Elpeleg O, Antonicka H et al: Distinct clinical phenotypes associated with a mutation in the mitochondrial translation elongation factor EFTs. Am J Hum Genet 2006; 79: 869-877.

119 The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and there would be no music. Lewis Thomas C h apter 6 Reconstructing the evolution of the mitochondrial ribosomal proteome

Paulien Smits, Jan A.M. Smeitink, Lambert P. van den Heuvel, Martijn A. Huynen and Thijs J.G. Ettema

Nucleic Acids Research 2007; 35(14): 4686-4703 Chapter 6

A b s t r a c t For production of proteins that are encoded by the mitochondrial genome, mitochondria rely on their own mitochondrial translation system, with the mitoribosome as its central component. Using extensive homology searches, we have reconstructed the evolutionary history of the mitoribosomal proteome that is encoded by a diverse subset of eukaryotic genomes, revealing an ancestral ribosome of alpha-proteobacterial descent that more than doubled its protein content in most eukaryotic lineages. We observe large variations in the protein content of mitoribosomes between different eukaryotes, with mammalian mitoribosomes sharing only 74 and 43% of its proteins with yeast and Leishmania mitoribosomes, respectively. We detected many previously unidentified mitochondrial ribosomal proteins (MRPs) and found that several have increased in size compared to their bacterial ancestral counterparts by addition of functional domains. Several new MRPs have originated via duplication of existing MRPs as well as by recruitment from outside of the mitoribosomal Chapter proteome. Using sensitive profile-profile homology searches we found hitherto undetected homology between bacterial and eukaryotic ribosomal proteins, as 6 well as between fungal and mammalian ribosomal proteins, detecting two novel human MRPs. These newly detected MRPs constitute, along with evolutionary conserved MRPs, excellent new screening targets for human patients with unresolved mitochondrial oxidative phosphorylation disorders.

122 Evolution of mitochondrial ribosomal proteome

I ntroduction Mitochondria are primary ATP-producing organelles which originated from an ancestral alpha-proteobacterial endosymbiont.12 The oxidative phosphorylation (OXPHOS) pathway, consisting of the mitochondrial respiratory chain and the ATP synthase complex, is the final biochemical pathway in energy conversion.3 However, mitochondria have been found to perform pivotal roles in many other metabolic, regulatory and developmental processes. During the evolution of the mitochondria, most genes of the ancestral endosymbiont have been transferred from the mitochondrial to the nuclear genome. The genes that reside on the mitochondrial DNA (mtDNA), e.g. 13 OXPHOS-related genes in human, are translated into their respective protein products by the mitochondrial translation machinery, which resembles the prokaryotic translation system,4 comprising a mitochondrial ribosome (mitoribosome) and several translation factors. In mammals, all proteins that constitute the mitochondrial translation machinery are encoded by the nuclear genome. Also the mitochondrial genomes of fungi Chapter generally encode only very few mitochondrial ribosomal proteins (MRPs). In contrast, the mitochondrial genome of many protozoa and plants encode numerous MRPs. Most notably, the 'primitive' mitochondrial genome of the 6 freshwater protist Reclinomonas americana was found to contain as many as 27 MRP-encoding genes.5 Mitoribosomes have undergone major remodeling during their evolution. For instance, it has been found that, despite the fact that their rRNA content is only half the content of bacterial ribosomes,6 mitoribosomes generally exceed the bacterial ribosomes both in molecular mass as well as in physical dimensions.6-8 Mass spectrometry studies of the bovine and yeast mitoribosomes, which have served as model systems during the past years, revealed that mammalian mitoribosomes comprise about 80 MRPs.9-13 In yeast, about 70 MRPs have been identified thus far, although protein-protein interaction data and mutational analysis suggest that this number might be substantially higher.14-16 Interestingly, a recent proteomics survey of mitochondrial ribosome-related complexes in the kinetoplast-mitochondria of Leishmania tarentolae revealed an additional protein complex which was found to be specifically associated with the small subunit (SSU), but not with the large subunit (LSU) of the mitoribosome.17 The biological role of the additional SSU-associated complex, which mostly consists of proteins unique to kinetoplastida, remains unknown. Apparently, mitoribosomes have expanded their protein content in the course of evolution by acquiring numerous extra 'supernumerary' MRPs. Currently information regarding the functions of these supernumerary MRPs is rather limited. In addition, loss of MRPs that are normally part of the 'bacterial core

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ribosome' is also observed. Recently, several attempts have been made to identify the MRPs in other eukaryotes, such as Neurospora crassa,18 Arabidopsis thaliana19 and L. tarentolae,20 however, a comprehensive study of the evolution of the mitoribosomal proteome remains to be established. Such a study bears relevance for the potential identification of genes that cause mitochondrial dysfunctions in human patients. Since mitochondria perform many fundamental functions, mitochondrial dysfunction results in a wide variety of multisystemic diseases, predominantly affecting tissues with high metabolic energy rates.21 These disorders are mostly caused by the dysfunction of one or more enzyme complexes of the OXPHOS system and several mutations have been identified in mtDNA22 as well as in nuclear DNA23. Not only mutations in structural OXPHOS genes, but also mutations in genes involved in the mitochondrial transcription or translation, or in the assembly of OXPHOS complexes can result in OXPHOS disease. Although the vast majority of components of the mitochondrial translation system are nuclear encoded, thus Chapter far most mutations associated with mitochondrial translation defects have been reported in mtDNA-encoded tRNAs and rRNAs.22,24 Recently, mutations in four 6 different nuclear gene products involved in mitochondrial protein synthesis have been reported in patients with mitochondrial disease: elongation factors EFG1,25 EFTs26 and EFTu,27 and small ribosomal subunit protein MRPS16.28 Additional information regarding which MRPs could be implicated in disease could be obtained by studying the evolutionary conservation of the MRPs. The aim of the present study is 2-fold, from an evolutionary and a disease point of view: (i) gain insight into the evolution of the mitoribosome and its protein content in various eukaryotic species; (ii) prioritize MRPs as candidates for their involvement in mitochondrial disease. We have performed a comprehensive comparative genomics analysis of the MRPs in 18 eukaryotic species from which both the complete nuclear and the organellar genome sequences were available. Representatives are included from different phylogenetic groups: six metazoa (Homo sapiens, Mus musculus, Tetraodon nigroviridis, Drosophila melanogaster, Anopheles gambiae and Caenorhabditis elegans), two fungi (Saccharomyces cerevisiae and N. crassa), one microsporidian (Encephalitozoon cuniculi), one mycetozoan (Dictystelium discoideum), one plant (A. thaliana), one alga (Chlamydomonas reinhardtii), one apicomplexa (Plasmodium falciparum), one ciliophora (Tetrahymena thermophila), two kinetoplastida (Trypanosoma brucei and Leishmania major), one diplomonad (Giardia lamblia) and the mitochondrial genome of the freshwater protist Reclinomonas americana. In addition, we included a balanced, phylogenetically diverse subset of completely sequenced prokaryotic genomes in our survey.

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Our analysis retrieved multiple previously unidentified MRPs in several species, two of which constitute potential novel human MRPs. Furthermore, the current study established orthology relationships between seven MRPs reported to be fungi-specific and ribosomal proteins from other eukaryotes. The establishment of these homology relationships enabled us to trace the origins of some of the supernumerary MRPs and to predict their molecular functions. In addition, we have investigated all MRPs on the presence of additional, newly acquired protein domains that might point at mitochondria-specific adaptations of the mitoribosome, and we have reconstructed the evolutionary history of the mitoribosomal proteome in terms of gains and losses of the MRPs along the different eukaryotic lineages. The newly detected mitochondrial ribosomal genes constitute, in addition to the set of evolutionary conserved MRPs, excellent new screening targets for human patients with unresolved mitochondrial oxidative phosphorylation disorders.

M a t e r i a l s a n d m e t h o d s Chapter

Use of genomic data Genome data (both proteomes and genomes) of the eukaryotes described in 6 this study were obtained from the Integr8 database (http://www.ebi.ac.uk/ integr8/, Release 41) at EBI,29 except for the genomes of Tetrahymena thermophila (version 2 obtained from TIGR, ftp://ftp.tigr.org/pub/data/Eukaryotic_Projects/t_ thermophila/), Trypanosoma brucei (version 4 obtained from Sanger, ftp:// ftp.sanger.ac.uk/pub/databases/T.brucei_sequences/), N. crassa (version 7.0 obtained from Broad institute at MIT, http://www.broad.mit.edu/annotation/ genome/neurospora/Downloads.html) and Chlamydomonas reinhardtii (version 3.0 obtained from JGI, ftp://ftp.jgi-psf.org/pub/JGI_data/Chlamy/v3.0/). For most eukaryotic species, the organelle genomes were included in the gene set per species, otherwise those were downloaded separately from GOBASE30 (http:// megasun.bch.umontreal.ca/ogmp/projects/other/mt_list.html). In addition to the eukaryotic genomes mentioned above, we have also included a balanced, phylogenetically diverse subset of completely sequenced prokaryotic genomes. We have explicitly chosen not to include the complete set of completely sequenced prokaryotic genomes available, since the large number of prokaryotic proteins might cause an imbalance in the PSI-BLAST analysis described below. Sequence data (proteomes and genomes) of the selected prokaryotic genomes were all downloaded from the Integr8 database at EBI, and included: Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae (gamma-proteobacteria), Bacillus subtilis, Clostridium acetobutylicum (firmicutes), Borrellia burgdorferi (spirochetes), Agrobacterium tumefaciens WashU, Brucella suis, Rickettsia prowazeckii,

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Caulobacter crescentus, Rhizobium meliloti, Rhodopseudomonas palustris (alpha- proteobacteria), Nostoc sp. PCC 7920, Synechocystis sp. PCC 6803, Prochlorococcus marinus SS120 (cyanobacteria), Deinococcus radiodurans (Deinococcus-Thermus group), Aquifex aeoliticus (aquificae), Sulfolobus solfataricus (crenarchaea), Pyrococcus furiosus (euryarchaea).

Detection of MRPs in eukaryotic genomes For each of the experimentally verified proteins of the Leishmania,1120 fungal14-16 and mammalian6 mitochondrial ribosomal proteins we have performed iterative PSI-BLAST searches31 against a locally assembled protein database comprised of the proteomes of the organisms indicated above. For each of these searches, an E-value inclusion threshold of 0.005 was applied, and the proteins that were retrieved after five iterations were aligned using MUSCLE.32 For those cases where a candidate MRP, from a species from which the query MRP was still missing, appeared in the PSI-BLAST hit list, but with an Chapter E-value between 0.005 and 10, a reciprocal search was performed. If this search successfully retrieved a bona fide MRP within five iterations (E-value inclusion 6 threshold of 0.005), it was added to the initial set of homologous proteins, and a new alignment was created. Whenever a given MRP was still not found in the predicted proteome of a eukaryotic organism, a hidden Markov model (HMM) of the respective protein was created from the above-mentioned alignments using the HMMER program.33 Subsequently, the HMM was used to screen the genomic sequences at the DNA level. The latter screen served as a final control for missing MRPs that represent unpredicted genes in the genome annotation process. To delineate orthologous groups, neighbor-joining trees were derived from these alignments using Kimura distances.34 A bootstrap analysis using 1000 samples was conducted for each phylogenetic tree. The selection of the orthologous group for each MRP was subsequently done by manually examining all trees. Assignment of orthologous relationships among the eukaryotic sequences was based on the species-phylogeny for eukaryotes3536 and the presence of these sequences within a monophyletic branch of the tree that contained known MRPs. Homologies between different families of MRPs, if not already found during the PSI-BLAST searches described above, were detected using the hhsearch algorithm,37 by comparing HMM profiles constructed from aligned MRPs with each other, using an E-value cut-off of 1e-4.

Phylogenetic analyses Maximum-likelihood (ML) trees were created using PHYML (version 2.2.4)38 based on alignments of the above-mentioned orthologous groups. Prior to the analyses, global and pairwise gaps were removed from the alignments. The appropriate

126 Evolution of mitochondrial ribosomal proteome model of protein evolution was selected using MODELSELECTOR.39 Bootstrap support values were derived from 100 replicates.

Detection of newly evolved MRP domains All MRPs homologous to bacterial core RPs that were identified in the current research were examined for additional, potentially newly evolved domains. Using HMMER,33 each MRP was analyzed with an HMM profile that was built from an alignment of its ancestral bacterial counterpart. These parts of the MRPs that were not covered by the respective HMM profile (minimum length 30 amino acids), were searched against the PFAM40 and CDD41 databases using HMMER33 and RPS-BLAST41 respectively. Hits below an E-value threshold of 0.01 were regarded significant.

R e s u l t s Detection of MRPs in eukaryotic genomes Chapter We have employed a systematic analysis of nuclear and mitochondrial genomes in order to identify the mitoribosomal proteome content encoded in a wide variety of eukaryotic species. To avoid confusion regarding different nomenclatures that are 6 used for ribosomal proteins, we have adopted the nomenclature for human MRPs, approved by the Human Genome Nomenclature Committee (http://www.gene. ucl.ac.uk/nomenclature/). All proteins are designated MRPSxx or MRPLxx, where S and L stand for small and large subunit, respectively, and xx is the number of the corresponding bacterial ribosomal protein. The MRPs lacking bacterial orthologs (supernumerary proteins) are assigned higher numbers, starting at MRPS22 and MRPL37 for the small and large subunit, respectively. In yeast, unfortunately, the nomenclature is as variable as the methods employed for their characterization. Therefore, in order to prevent further confusion about proteins with the same name in human and yeast that are not part of the same orthologous group, we have indicated all human MRPs in capital letters throughout the manuscript (e.g. MRPS28), whereas for the yeast proteins we only capitalized the first letter (e.g. Mrps28, which corresponds to MRPS15 in human, also see Table 1 and Supplementary Data). Starting from a curated list of MRPs that have been identified in various studies, we first searched for homologs using several search algorithms (see Materials and Methods). Subsequently, the list of homologs was subjected to a phylogenetic analysis in order to define orthologous groups of MRPs. Not only does our analysis distinguish MRPs from their cytosolic counterparts, it also enabled for differentiation between 'true' MRPs and homologous ribosomal proteins of other organelles, such as the chloroplast (in plants) or apicoplast (in the apicomplexa P falciparum). We realize that this approach has its limitations,

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Table 1. Phylogenetic distribution of SSU mitochondrial ribosomal proteins and their bacterial counterparts

Chapter 6

(continued)

128 Evolution of mitochondrial ribosomal proteome

continued

Pet123 Rsm26 Mrp10 * Rsm28 Mrp8 Mhr1 Lm F03.0220 Lm F03.0630 Lm F15.0080 Lm F15.0410 Lm F15.0780 Lm F15.1400 Lm F16.1230 Lm F22.0830 Lm F22.0990 Lm F22.1220 Lm F24.0830 Lm F25.2160 Lm F26.2270 Chapter Lm F27.0180 Lm F27.0630 Lm F27.1895 6 Lm F28.2180 Lm F29.0430 Lm F30.0650 Lm F30.0740 Lm F30.3220 Lm F30.3530 Lm F32.0650 Lm F33.0010 Lm F33.2510 Lm F35.2230 Lm F35.3490 Lm F35.3940 Lm F36.4120

The first and second column contain, if1 applicable, the MRP identifiers according to the human and yeast nomenclature, respectively. MRPs and bacterial RPsthat were identified on the predicted proteome are indicated in yellow, MRPs that were not found in thie predicted proteome, but for which the unpredicted gene couldbe identified on the genomic DNA are indicated in b b er MRPs that have been experimental^ verified as bemg associatedwith the mitoribosome by means of large-scale proteomics mteraction srudies or small-scale functional s tu d e are indicated in green (exeept for the human MRPs, for wlhich mostly the bovine counterparts were experimentally verified). MRPs that are encoded by the mitochondrial genome are indicated in red. A bracket (#) denotes a peculiar case of a split MRPS3 gene in D. discoideum, which is encoded by iTs mitochondrial genome. The 5' and 3' ends are fused to genes of1 unknown funrtion (ORF4c5 and ORF1740, respectively)/9-80 It is unknown f the split MRPS3 genes results in a functional protein. An asterisk (*) denotes cases where a given MRP was not identified most probably as a result of incompleteness of the genome sequence, i.e. the MRP was not found in the predicted proteome, nor was it found by analyzing the genomic DNA, but could be identified in a closely related species. For example, MRPS28 and MRP10 were not detected in T. nigroviridis, but could be found in Danio rerio. See Supplementary Data for protein identifiers. Species abbreviations: Hsa: Homo sapiens; Mmu: Mus musculus; Tni: Tetraodon nigroviridis; Dme:

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Drosophila melanogaster; Aga: Anopheles gambiae; Cel: Caenorhabditis elegans; Sce: Saccharamyces cerevisiae; Ncr: Neurospora crassa; Ecu: Encephalitozoon cuniculi; Ddi: Dictyostelium discoideum; Ath: Arabidopsis thaliana; Cre: Chlamydomonas reinhardtii; Pfa: Plasmodium falciparum; Tth: Tetrahymena thermophila; Ram (mt): Reclinomonas americana (mitochondrial genome); Tbr: Trypanosoma brucei; Lma: Leishmania major; Gla: Giardia lamblia; Eco: Escherichia coli; Bsu: Brucella suis; Rpr: Rickettsia prowazekii.

as it does not account for potential retargeting events that have been shown to occur, such as in A. thaliana, where a chloroplast-type S13 protein encoded by a diverged duplicated nuclear gene has been demonstrated to be targeted to the mitochondrion.42 In order to account for such cases, we included such an experimentally verified protein in our list, whenever our analysis did not suggest a candidate MRP. Altogether, our survey resulted in the identification of highly variable numbers of MRPs across the genomes of the investigated eukaryotic species, several of which had not been identified as MRPs previously (see Table 1, Table Chapter 2 and Supplementary Data). The total number of identified MRPs ranged from 81 in most metazoan species, to 80 in yeast, to 63 in plants, down to a mere 39 6 MRPs in the apicom plexan P. falciparum. We recognize the fact that the latter numbers are probably an underestimate of the actual numbers of MRPs in these species, as there are no experimental studies to identify potential lineage specific supernumerary MRPs. Moreover, since protein sequences of these species have been found to evolve relatively fast,43 it is at least feasible that we failed to identify some MRPs that have evolved beyond the detection limits of our search algorithms. Apart from mapping MRPs that are encoded by eukaryotic genomes, we were able to find hitherto undetected homology between experimentally verified supernumerary MRPs and bacterial ribosomal proteins that were presumed to have been lost from the primitive eukaryotic mitoribosome. Using sensitive profile profile searches for detection of distant homology between protein families, we connected the MRPS24 orthologous group to the bacterial RPS3 ribosomal protein family (E-value 2.2e-5) and similarly, we connected the MRPL47 orthologous group to the bacterial RPL29 ribosomal protein family (E-value 8.6e-7) (see Figure 1). The complementary phylogenetic distribution patterns of the respective genes are in support of this suggestion. For example, MRPL47 is present in all eukaryotes analyzed (except for E. cuniculi and G. lamblia), but not in bacteria. The opposite phylogenetic pattern is observed for bacterial ribosomal protein RPL29, for which no homolog was identified in any eukaryotic genome. Apparently, these MRPs have diverged almost beyond homology detection limits during the course of evolution (see Figure 1), possibly as an adaptation to specific mitoribosomal functionality. Given the detected homology between the above-mentioned MRPs and bacterial

130 Evolution of mitochondrial ribosomal proteome ribosomal proteins, we have placed the MRPs in the orthologous group of their respective bacterial counterparts (see Tables 1 and 2). We also encountered some cases where we could not detect an expected MRP ortholog in some species, which might be caused by incompleteness of the respective genomes. For example, despite the fact that we could identify orthologs of MRPL50, MRPS28 and Mrp10 in all metazoa analyzed in this survey, as well as in the Danio rerio genome (data not shown), we were unable to detect these MRPs in the genom e of T. nigroviridis. Likewise, MRPL56 was found in all metazoa, including A. gambiae, but not in D. melanogaster (see Table 2). In addition, in some instances our analysis proposed different proteins than have been reported in earlier reports. The discrepancies between our study and that of others are outlined in the Supplementary Data. Not unexpectedly, MRPs were not detected in the genomes of the microsporidian E. cuniculi and the diplomonad G. lamblia. These organisms do not contain full-fledged mitochondria, but instead they contain mitosomes (E. cuniculi) or mitochondrial remnants (G. lamblia), which typically lack organellar Chapter DNA. Consequently, these organisms have no need for mitochondrial translation machineries, explaining the observed loss of MRP-encoding genes. 6

Identification of novel (human) MRPs Our analysis expanded orthologous groups that were previously reported to contain fungi-specific MRPs to include other eukaryotic proteins, resulting in the identification of two potentially novel human MRPs, namely the human orthologs of yeast SSU proteins Rsm22 and Mrp10, extending the total number of human MRPs to 81. The detection of human orthologs of yeast MRP proteins Rsm22 and Mrp10 is of particular interest, as defects within these genes might be linked to mitochondrial dysfunctions in human. Interestingly, extensive biochemical data is available for the yeast gene products, enabling to speculate about their respective molecular functions. Yeast Rsm22 is homologous to an rRNA methyltransferase and has been shown to be part of the SSU of the yeast mitochondrial ribosome.44 The association of Rsm22 with the SSU has also been confirmed in a large-scale affinity-capture mass spectrometry study.45 We have detected an ortholog of Rsm22 in most eukaryotes, as well as in some prokaryotes. Interestingly, the STRING database46 shows that the gene encoding the Rsm22 ortholog in some prokaryotes is clustered on the genome with genes that encode ribosomal proteins (not shown), suggesting that the Rsm22 orthologs in these species are also associated with the ribosome. In Schizosaccharomyces pombe, the genes encoding Rsm22 and Cox11 are fused. Cox11 is an assembly factor of the OXPHOS enzyme complex cytochrome c oxidase and in S. cerevisiae, Cox11 has been shown to associate

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Table 2. Phylogenetic distribution of LSU mitochondrial ribosomal proteins and their bacterial counterparts

1 z LU Q < Ö CL i— CC 1- —i O LU CD CC L1 MrpM 12 Rml2 # L3 Mrpl9 L4 Yml6 E L5 Mrpl7 L6 Mrpl6 L9 Mrpl50 L10 MrpM 1 L11 Mrpl19 L12 Mnp1 1 L13 Mrpl23 L14 Mrpl38/Mrpl34 L15 Mrpl 10/Mrpl18 L16 Mrpl16 = Chapter L17 Mrpl8 L18 L19 Imq1 6 L20 L21 Mrpl49 L22 Mrpl22 L23 Mrpl41/Mrp20 L24 Mrpl40 L25 L27 Mrpl2/Mrp7 L28 Mrpl24/Mrpl14 L29/L47 Mrpl4 L30 Mrpl33 i L31 Mrpl36 L32 Mrpl32 L33 Mrpl39 L34 Ydr115w L35 L36 Ypl183w-a L37 L38 Mrpl35 L39 L40 Mrpl28 L41 Mrpl27 L42 L43 Mrpl51 I L44 Mrpl3 L45 Mba1 L46 Mrpl17/Mrpl30 L48 = (continued)

132 Evolution of mitochondrial ribosomal proteome

co ntinued

L49 Img2 L50 * L51 L52 L53 Mrpl44 L54 Mrpl37 1=z - L55 L56 ! * = ■ Mrpl13 - Mrpl15 Mrpl20 Mrpl25 Mrpl31 11 For explanation of used MRP protein names, color coding and species abbreviations, see comments below Table 1. Subunits that are indicated to be experimentally characterized for L. major were in fact identified to be part of the mitoribosome of the closely related species L tarentolae.17 A bracket (#) denotes a speciai case ot rhe A. thaliana MRPL2, of which the N-terminal half is encoded by the mitochondrial genome and the C-terminal half is nuclear encoded. See 77upplementary Data for protein identifiers. An asterisk (*) denotes cases where a given MRP was not identified as a result of Chapter incompleteness of tine genome sequence, i.e. the MRP was not found in the predicted pfoteome, nor was if found by analyzing the genomic DNA, but covld be identified in a closely related species. For example, MRPL56 was not detected in D. melanogaster, but was found in the genomes of D. willistoni 6 and in A. gambiae. Originally, MRPS32 was identified as a protein in the small subunit9 and MRPL42 as a protein in the large subunit (MRPL31 in reference89). However, the protein sequences of these proteins were found to be identical. Later, MRPS32 was omitted since its identification was probably caused by contamination in the preparation of small subunits, and thus was in fact MRPL42 (Thomas O'Brien, personal communication). with the mitoribosome and is postulated to function in co-translational insertion of copper ions into Coxl, a subunit of cytochrome c oxidase.47 Finally, Rsm22- deficient yeast strains exhibit a growth defect on non-fermentable (respiratory) carbon sources, indicative of mitochondrial respiratory dysfunction.44 All these lines of evidence suggest that Rsm22 is a protein of the mitoribosomal small subunit that is essential for proper mitochondrial function. As Rsm22 is a predicted rRNA methyltransferase, it might be involved in methylation of mitochondrial rRNA, and as such play a role in the regulation of protein translation efficiency. Yeast MrplO was shown to be a component of the SSU of mitoribosomes as well and disruption of the gene resulted in a mitochondrial translation defect and a tendency to accumulate deletions in mtDNA.48 Jin et al observed low sequence similarity to yeast Mrpl37, a constituent oPthe LSU.49 Howevee, we identified yeast Mrfrl37 as an ortholog of human MRPL54 ared did not detect any significant sequence homology between MrplO and Mrpl37. Nevertheless, we did find MrplO orthologs in many eukaryotes, all of which were found to contain a CHCH domain (Coiled coil 1-Helix 1-Coiled coil 2-Helix 2, PFAM domain 06747). Within each helix of this CHCH domain, two invariant cysteine residues are present in

133 Chapter 6

Chapter Figure 1. Distant homology between mitochondrial and bacterial ribosomal protein families detected 6 in this study. Multiple sequence alignments of MRPL47 and RPL29 proteins (A) and of MRPS24 and RPS3 proteins (B). Amino acid residues are shaded according to the following physicochemical properties: t: tiny (blue font on yellow shading); m: amphoteric (red font on green shading); h: hydrophobic (white font on black shading); o: positive (red font on blue shading); p: polar (black font on green shading); s: small (green font on yellow shading); a: aliphatic (red font on gray shading); r aromatic (blue font on gray shading); c: charged (white font on blue shading). Sequences are annotated by the organism name and Swiss-Prot protein identifier and the boundaries of the segment that is used in the alignment are indicated. A 90% consensus sequence is depicted below the alignments using the same classification, with invariant residues being indicated in capitals.

a C-X9-C motif. Besides in Mrp10, CHCH domains have also been detected in Cox19 and in the respiratory complex I (NADH-ubiquinone oxidoreductase) 19 kDa subunit (NDUFA8).50 In yeast, Cox19 is present in the cytoplasm as well as in the inter membrane space of the mitochondria, where it is suggested to play a posttranslational role in the assembly of cytochrome c oxidase through copper transport to mitochondria. It has been proposed that the conserved cysteine residues of the CHCH motif in Cox19 mediate as ligands for the copper ions.51,52 Most likely, yeast Mrp10 and their orthologs in other eukaryotes, including human, constitute an essential component of the mitochondrial translation machinery and we anticipate that the CHCH domains that are present in this orthologous group, as well as in complex I subunit NDUFA8, are involved in trafficking of metal ions. We also detected a potential third novel human MRP, Ppel, however this case is less clear-cut than these described above. Ppel is a protein, which has been shown to be part of the SSU of the yeast mitoribosome.53 We detected Ppel orthologs in all eukaryotes analyzed, except for P falciparum and T. thermophila

134 Evolution of mitochondrial ribosomal proteome

(and the mtDNA lacking eukaryotes). However, the localization of Ppel in the mitochondrion is questionable, as the report by Kitakawa et al is the only indication for this.53 Moreover, the yeast and human Ppel have previously been reported to function as protein phosphatase methylesterases of Protein phosphatase 2A,5455 which are mainly localized in the nucleus and cytoplasm.56 Additionally, yeast Ppel mutant strains do not exhibit any growth defects,55 while mutations affecting synthesis or function of respiratory chain components generally result in growth impairment on a non-fermentable carbon source. Thus currently there is, besides the reported association with SSU of the mitoribosome in yeast, no clear indication that Ppel is implicated in mitochondrial translation. Therefore, additional research is needed to corroborate the cellular localization and function of yeast Ppel and its orthologs in other eukaryotes.

The evolution of the mitochondrial ribosomal proteome along eukaryotic lin eag es The protein composition of mitoribosomes is variable, not only in terms of the Chapter number of MRPs, but also in terms of the specific MRPs contained (see Tables l and 2). By mapping the distribution of the orthologous groups of MRPs onto 6 a reconstructed tree of the eukaryotes35 while always considering the most parsimonious scenario, we obtained an overview of the history of gains and losses of MRPs during the divergence of eukaryotes (see Figure 2). Mitoribosomes originated from the bacterial ribosome present in the alpha-proteobacterial ancestor of mitochondria, consisting of a microbial core of 54 ribosomal proteins in total. Already in the earliest stage of eukaryotic evolution, prior to the major divergence of the fungal, plant and metazoan lineages, several supernumerary proteins were recruited to the mitoribosome, while only one bacterial core protein was lost at this stage (RPS20, see Figure 2), resulting in a primitive eukaryotic mitoribosome of 68 MRPs. Subsequent to the gain of this primary set of l5 proteins, the mitoribosome diversified along the various eukaryotic lineages and a number of lineage-specific gain and loss events can be observed. Another gain of MRPs occurred at the level of the metazoa, prior to the bifurcation of nematodes and eumetazoa. At this stage, l4 proteins were recruited to the metazoan mitoribosome, whereas seven bacterial core proteins were lost. Major protein gains at these two time points have also been observed in the evolutionary history of complex I.57 Additionally, l l fungi-specific MRPs were recruited after the animal-fungi radiation. In the lineage towards the kinetoplastida, a major surge of 29 proteins is observed. These proteins have been shown to comprise a protein complex that is associated with the SSU of kinetoplastid-mitoribosomes.:L7 The role of this additional protein complex and its association with the SSU is still unclear. Apart from this major gain in the kinetoplastida, we observe loss of several

l3 5 Chapter 6

D. melanogaster (81) M. musculus (81) H. sapiens (81) Mrps35, Mrp51, Mrp13, Mrp1 T. nigroviridis (80) \ / Pet123, Rsm26, Mhr1 Mrpl 15, Mrpl20, Mrpl25, Mrpl31 A. gambiae (81)

C. elegans (77) S1, S3, S4, S10 S. cerevisiae (80) Z 5 S21, S33, S35, S36 C. reinhardtii (47) S18(2X), S22 Rsm22, MrplO S26-S28, S30 L5, L9, L18, L25 E. cuniculi (0) L37, L39, L42, L48 L28, L32, L41 y A. thaliana (63) L50, L51, L55 ■fsTI D. discoide um (59)

P. falciparum (39)

T. thermophila (45)

Chapter R. americana - mt (27)

G. lamblia (0) 29 SSU associateci kinetoplastida L. major (66) 6 specific MRPs Mammals T. brucei (66) ■ Nematodes

■ Amoebozoa Bacterial core (54)

A VE group

Figure 2. Reconstruction of the evolutionary history of the mitochondrial proteome across the different eukaryotic lineages. (A) Incoming and outgoing arrows indicate the gain and losses of the MRPs that are indicated in the box. Genes encoding proteins of the bacterial core are shown in bold. The scenario of gene gain and loss was adjusted according to the MRP content encoded by the mitochondrial genomes of rice and R. americana5 (dashed line, position in tree inferred from reference89). Dotted lines indicate lineages with highly reduced mitochondria in which all MRP-encoding genes have been lost. Species for which the mitoribosomal proteome has been experimentally investigated are highlighted in a gray shaded box. Numbers following the species names denote the total number of MRPs identified in that species. Note that several MRPs have been lost independently in different lineages. For example, MRPSl is lost independently in C. reinhardtii, A. thaliana (MRPSl is encoded on the mitochondrial genomes of Oryza sativa and R. americana) and in the kinetoplastida and alveolata lineages. (b) Evolutionary trajectory towards the human mitochondrial ribosomal proteome. Main gene gain events can be observed at major branching points of the tree, such as in the ancestral eukaryote and at the origin of the metazoa, after which the mitoribosomal proteome is maintained at a constant level. AVE: alveolates, viridiplantae and excavates branch.

l3 6 Evolution of mitochondrial ribosomal proteome proteins in the AVE (alveolates, viridiplantae and excavates) lineage, predominantly consisting of bacterial core proteins, but also including supernumerary MRPs in some lineages. Possibly, some of these proteins remained undetected due to sequence divergence. The fact that the AVE lineages are dominated by loss events of MRPs is likely due to the lack of experimental data of mitoribosomal content in these species. Compared to the 54 ribosomal proteins present in the alpha-proteobacterial ancestor, 8 l MRPs in human represent a substantial gain in complexity (see Figure 2B). However, the observed increase in complexity following the endosymbiotic event is not uncommon, as it has also been reported for electron transport chain complexes.57 It has been proposed that the recruited supernumerary proteins are mainly involved in the assembly and stabilization of the complexes.5758 The mammalian supernumerary MRPs are shown to be mostly localized to the peripheral regions of the mitoribosome.59-61 Since all proteins synthesized by metazoan mitoribosomes are inserted into the inner mitochondrial membrane, at least some of the metazoa-specific supernumerary MRPs are presumed to be Chapter associated with positioning the mitoribosome during co-translational insertion of nascent polypeptides into the membrane. Interestingly, a protein that has been 6 proposed to be essential for co-translational insertion in yeast, M bal (MRPL45, see 'Origins from outside of the mitoribosomal proteome - MRP recruitment'), was already part of the primitive eukaryotic mitoribosome and has been recruited from the alpha-proteobacterial ancestor. Similar to the evolution of complex I, we observe the recruitment of proteins of bacterial origin to the mitoribosome (MRPL45 and Rsm22), however, this number is considerably lower (only two, compared to six in complex I), especially considering that in the evolution of the mitoribosome more proteins were added compared to complex I (37 and 32 in the human mitoribosome and complex I, respectively). tracing the origins of supernumerary MRPs As outlined above, the protein content of the mitoribosome has significantly increased in most species during the evolution of the mitochondria. Our analysis indicates that in several stages of the mitoribosomal evolution various extra proteins, the supernumerary MRPs, have been added to the mitoribosomal proteome. Some of these supernumerary MRPs were found to be present in all eukaryotes that were investigated, but not in bacteria, such as MRPS29 (see Table 1). Clearly, these MRPs have been recruited in an early stage of the evolution of the eukaryotes, before the formation of major eukaryotic kingdoms. In contrast, other MRPs have originated relatively recently, such as MRPL37, which is specific for the metazoa. However, it is not only interesting to know 'when' certain MRPs were added to the mitoribosome, it is also enticing to trace their origins. We found

137 Chapter 6

table 3. Paralogous relationships involving MRPs MRP Mitochondrial [cytosolic] Other ribosome homolog SSU

S10 L48, [S20] S18 S18A, S18B, S18C S26 Several coiled coil containing proteins, including subunit 10 theta of the eukaryotic translation initiation factor 3 S27 TPR/PPR repeat proteins/salt-inducible proteins (Arabidopsis) S29 DAP3 S30 L37 PDCD9 M rpl Rsm26 (yeast) Superoxide dismutase Petl23 Several coiled coil containing proteins Chapter Mrpl0 COX19, NDUFA8 Rsm28 Kinases 6 LmjF30.3530 Rhodanese, inactive domain LmjF25.2l60 Ubiquitin LmjF32.0650 AKAP7

LSU

L2 [L8] NUZM (fungi-specific OXPHOS complex I subunit) L38 Phosphatidylethanolamine-binding protein 1 (PEBP-1) and Flowering Locus T L39 Threonyl- and alanyl-tRNA synthetase L43 S25 Respiratory Complex I subunit B8 L44 Mrpll5, Mrpl3 (yeast) Ribonuclease III and other ribonuclease_3 domain containing proteins L45 Tim44 L46 mRNA decapping enzyme 2 (DCP2) and Nudix hydrolase (NUDT) L56 Beta-lactamase Mrpl20 Mesenchymal stem cell protein

Names of MRPs are indicated in the first column and supernumerary MRPs and proteins from outside the ribosome are indicated in the second and third column, respectively. In addition, if a homologous cytosolic ribosomal protein is present, it is indicated between square brackets in the second column.

138 Evolution of mitochondrial ribosomal proteome that several of the supernumerary MRPs are homologous to other proteins, some of which are also part of the mitoribosome (see Table 3). The latter finding implies that gene duplication events have played a substantial role in the expansion of the mitoribosomal proteome (see Table 3). Below we will discuss some of the most protruding cases.

Chapter 6

Figure 3. Maximum-likelihood tree of the MRPS18 orthologous group. The metazoa contain three copies of this family (MRPS18A, MRPS18B and MRPS18C) as a result of two consecutive duplication events at the root of the metazoan radiation. Proteins are indicated by a species name and a protein identifier. Bootstrap values for partitions that are supported with values above 50% (out of 100 replicates) are displayed. ______l3 9 Chapter 6

Origins from within the mitoribosomal proteome - MRP duplication Some supernumerary MRPs are homologous to MRPs that are part of the bacterial core of the mitoribosome. These proteins have seemingly been duplicated from within the mitoribosomal proteome. One of the most straightforward examples is the emergence of three variants of MRPS18 in the metazoan lineage resulting from gene duplication events (see Figure 3). In contrast with earlier reports,9 we identified three MRPS18 variants in C. elegans and we found that all three metazoan variants emerged from the same ancestral metazoan sequence, which is unlikely to be derived from chloroplast S18, as was reported by Cavdar Koc and co-workers9 (see Figure 3). All three C. elegans MRPS18 variants seem to be essential in embryonal development, as targeted disruption of these genes result in an embryonic lethal phenotype.62 Independent from the observed MRPS18 duplications in metazoa, it is interesting to note that most sequenced actinobacterial genomes encode two variants of the S18 gene, probably the result of a gene duplication event in these species. Currently, experimental evidence Chapter regarding the functions of the different MRPS18 versions in metazoa is lacking, but it is believed that each mitoribosome contains only one copy of MRPS18, 6 giving rise to a heterogeneous population of mitoribosomes.9 Another example can be found in the duplication of MRPS10 in the lineage of the metazoa, which gave rise to MRPL48 (see Table 3 and Figure 4). This duplication event constitutes one of the cases where the duplicate gene product has become part of the other mitoribosomal subunit, in this case from the SSU (MRPS10) to the LSU (MRPL48). Likewise, a duplication event at the base of the metazoan radiation seems to have given rise to the supernumerary MRPs MRPS30 and MRPL37, both of which are only present in metazoa (See Figure 5). Interestingly, MRPS30 is also known as programmed cell death protein 9 (PDCD9 or p52).63,64 Another supernumerary protein, MRPS29, is also implicated in apoptosis, as it has been shown to be identical to death-associated protein 3 (DAP3).63,65 Given the dual roles of these MRPs, the processes of mitochondrial translation and apoptosis are apparently closely coupled.66

Origins from outside of the mitoribosomal proteome - MRP recruitment We found that some supernumerary MRPs have probably been recruited to the mitoribosome, as they display significant homology to other mitochondrial proteins, some of which are also implicated to play a role in mitochondrial translation (see Table 3). Of course, it cannot always be resolved if an MRP has been part of the mitoribosome prior to the duplication event. For some instances, we can use the phylogenetic distribution patterns of the copies in order to point out which was first. One of such examples, where a pre-existing protein has been recruited to the mammalian mitoribosome, is the acquisition of MRPL39, which is

140 Evolution of mitochondrial ribosomal proteome

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Figure 4. Maximum-likelihood tree showing the origin of supernumerary MRPL48 as a result of a duplication event at the base of the metazoan radiation within the MRPS10 orthologous group. All depicted proteins were connected using PSI-BLAST (see Materials and Methods for details). For instance, a search started with human MRPS10 retrieved both the human MRPL48 (Q96GC5) and the cytosolic SSU ribosomal protein RPS20 (RS20_HUMAN) in the second iteration with E-values of le-9 and 0.001, respectively. Proteins are indicated by a species name and a protein identifier. Bootstrap values for partitions that are supported with values above 50% (out of 100 replicates) are displayed. homologous to the W-terminal domain of threonyl-tRNA synthetases and which has a universal phylogenetic distribution. This homology has been noticed before and it is suggested that MRPL39 can bind tRNA in a similar manner as the E. coli threonyl-tRNA synthetase.67 Conceivably, the W-terminal tRNA-binding domain of a mitochondrial threonyl-tRNA synthetase was recruited to the mitoribosome in order to compensate for the loss of bacterial ribosomal proteins that are involved in tRNA binding, such as RPS13 and RPL5 (see Tables 1 and 2).

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The most striking example of MRP recruitment is that of MRPL45 (See Figure 6). First we found that MRPL45 and yeast protein Mba1 belong to the same orthologous group, which in addition comprises proteins from all metazoa, the plant A. thaliana, the alga C. reinhardtii as well as from the mycetozoan D. discoideum. Mba1 is a mitochondrial protein associated with the matrix face of the inner membrane, which associates with the highly conserved inner membrane protein Oxa1. Mba1 and Oxa1 orchestrate the insertion of both mitochondrial- and nuclear-encoded proteins from the mitochondrial matrix into the inner membrane.68,69 Moreover, we detected homology between MRPL45 (including Mba1) and Tim44 (see Figure 6), a peripheral mitochondrial inner membrane protein that is an essential component of the import motor PAM of the TIM23 complex, which is implicated in the translocation of proteins from the inter membrane space to the mitochondrial matrix (for reviews see references7071). The apparent sequence homology, as well as functional homology (i.e. protein translocation), suggests that MRPL45 and Tim44 share a common alpha- Chapter proteobacterial origin. Many alpha-proteobacteria contain two distinct types of 6 Tim44/MRPL45-like genes (see Figure 6).

Figure 5. Maximum-likelihood tree showing that metazoan-specific supernumerary MRPs MRPS30 and MRPL37 originated as a result of a duplication event at the base of the metazoan radiation. The depicted proteins were connected using PSI-BLAST (see Materials and Methods for details). For instance, a search started with human MRPS30 retrieves human MRPL37 within the first iteration with an E-value of 0.001. All proteins are indicated by a species name and a protein identifier. Bootstrap values for partitions that are supported with values above 50% (out of 100 replicates) are displayed.

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Figure 6. Maximum-likelihood tree indicating common ancestry of supernumerary mitoribosomal protein MRPL45 and Tim44, an essential component of the TIM23/PAM complex, which mediates the translocation of nuclear-encoded proteins across the mitochondrial inner membrane. The tree contains alpha-proteobacterial sequences from which mitochondrial MRPL45 and Tim44 have evolved. Note that there are two paralogous Tim44-like families within the alpha-proteobacteria, resulting from a duplication within that lineage. The depicted proteins were connected using PSI-BLAST as outlined in the Materials and Methods. For instance, a search started with human MRPL45 retrieves the Rhizobium meliloti Type I Tim44-like protein (Q92TE6) in the second iteration with an E-value of 1e-7. The yeast Mba1 and human Tim44 proteins were both retrieved in the third iteration (with E-values of 2e-13 and 2e-10, respectively). Proteins are indicated by a species name and a protein identifier. Bootstrap values for partitions that are supported with values above 50% (out of 100 replicates) are displayed.

A close examination of MRPL45 and Tim44 proteins reveals that the /V-terminal region of Tim44 is absent in MRPL45 (not shown). This region is supposed to be located in the mitochondrial matrix where it interacts with mitochondrial Hsp70 chaperone, which is also a component of the PAM complex.72 Apart from its association with Oxa1, MRPL45 has been shown to be a constituent of the mitoribosomal LSU in multiple studies.10,73 Moreover, it has been shown that in

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yeast, transcription of MBA1 is tightly co-regulated with that of genes encoding MRPs,74 implying a functional link between the respective gene products. The anticipated association of the mitoribosome and membrane-bound constituents of the mitochondrial protein insertion machinery would explain the fact that up to 50% of the bovine mitoribosomes are found to associate with the inner membrane fraction of mitochondria.75 Taking all this in consideration, we postulate that MRPL45 functions as a bridging factor between the mitoribosome and Oxa1, and as such constitutes an essential component for successful co- translational insertion of proteins into the inner membrane of mitochondria in eukaryotes. Interestingly, the MRPL45 orthologous group, which also includes the bacterial Tim44-like proteins, shows a similar phylogenetic distribution pattern as the orthologous group of Cox11, an assembly factor of cytochrome oxidase, which is involved in the co-translational insertion of copper ions into the nascent Cox1 protein.47 The observed pattern of co-presence and absence of these genes across a phylogenetically diverse set of species indicates that their gene products Chapter might be involved in the same process.76 As Cox11 has been shown to associate transiently with the mitoribosome as well, it is feasible that MRPL45 and Cox11 6 act together in order to functionally insert Cox1 into the inner mitochondrial membrane.47 Other examples of MRPs that have been recruited to the mitoribosome are the supernumerary proteins MRPL43 and MRPS25 (see Table 3), which are homologous to each other as well as to the B8 subunit (NI8M) of respiratory complex I (NADH- ubiquinone oxidoreductase) of the OXPHOS system.57,77 Presumably, MRPL43 and the B8 subunit of complex I originated from a single gene that was present in the primitive eukaryote, as they are present in a phylogenetically wide range of organisms. Most likely, another duplication of MRPL43 then gave rise to MRPS25, which is only found in metazoa and fungi. More examples of MRP recruitment include yeast MRPs Mrp1 and Rsm26, which are both part of the iron/manganese superoxide dismutase family,44 and MRPL44, which is homologous to a wide range of double-stranded RNA-binding proteins (see Table 3).

Detection of orthologs of MRPs previously reported to be fungi-specific Several comprehensive experimental studies on the yeast mitochondrial translation machinery have significantly extended its mitoribosomal proteome.14-16 However, for a large number of MRPs that have been characterized in yeast thus far, no significant or conclusive sequence similarity has been reported to ribosomal proteins from other sources. In the current study, we were able to link seven of these seemingly fungi-specific MRPs to known ribosomal proteins in other species, as will be outlined below.

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Apart from the homology between yeast Mba1 and MRPL45, which has already been discussed above, we identified Mrp49 as the ortholog of MRPS25. It is, however, surprising that the orthologs of MRPS25 found in S. cerevisiae as well as in N. crassa are reported to be located in the large instead of the small subunit of the mitoribosome,1878 in contrast with the location of bovine MRPS25.59 It is possible that the subunit localization of the MRPS25 orthologs is different between metazoa and fungi; however, further experimental evidence is needed in order to clarify this. We also connected Rsm27, a yeast protein of the small subunit,44 to the MRPS33 protein family. In addition, we detected MRPS33 orthologs in all metazoa as well as D. discoideum and A. thaliana. Moreover, we found that yeast MRPs Mrpl28,53 Mrpl44,79 Mrpl5053 and Mrpl4053 are in fact the orthologs of MRPL40, MRPL53, MRPL9 and MRPL24, respectively. Both MRPL24 and Mrpl40 were found to contain a KOW motif (PFAM domain pfam00467), which is present in a variety of ribosomal proteins as well as in the bacterial transcription elongation factor NusG.80 The homology between Mrpl50 and bacterial RPL9 has been noted Chapter before,14 but it was dismissed as being insignificant, since the sequence similarity was not conserved throughout the full length of Mrpl50. In our analysis, however, 6 we did detect significant homology between Mrpl50 and the MRPL9 family (including bacterial RPL9). Our finding is supported by the fact that Mrpl50 and MRPL9 representatives reside within the same protein superfamily (SSF55658, 'L9 N-domain-like').81 Finally, we were able to confirm the distant homology between Var1 and Rps5, two mtDNA-encoded homologs of bacterial RPS3 in fungi, as was previously reported by Lang and colleagues.82 Var1 and Rps5, which are the only mtDNA- encoded MRPs in yeast and N. crassa, respectively, have both been identified as essential components of the SSU of the mitoribosome, showing similar phenotypes in mutant strains.82 Despite the fact that RPS3 has been shown to play an essential role in bacterial ribosome assembly, and that Var1 and Rps5 are both essential for assembly of the mitochondrial SSU in fungi, we were unable to detect candidate mitochondrial RPS3 homologs in metazoa. In the latter organisms, this essential role in ribosome assembly could be performed by MRPS24, which displays distant homology to RPS3 (see Figure 1). Moreover, we could detect mtDNA- encoded RPS3 hom ologs in T. thermophila and D. discoideum,8283 the latter of which constitutes a peculiar case where the RPS3 encoding gene is split and both the 5' and 3' parts are fused to an ORF of unknown function (ORF425 and ORF1740, respectively). In any case, the fact that genes encoding RPS3 homologs are present on several mitochondrial genomes implies an alpha-proteobacterial origin of this protein.

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Figure 7. Domain architectures of MRPs to which additional functional domains have been added. Protein identifiers are depicted as in Table 1 or 2 and species distribution is indicated. Proteins are drawn approximately according to scale. Domain abbreviations: (1) Pkinase (pfam00069): Protein kinase catalytic domain. (2) DUF298 (pfam03556): Domain of unknown function, containing a basic helix-loop-helix leucine zipper motif. (3) Collagen (pfam01391): Collagen triple helix repeat. (4) VAR1 (pfam05316): This domain is specific for some fungal mitochondrial ribosomal S3 proteins and is often present in two copies (also see text). (5) AAA (pfam00004): ATPase family associated with various cellular activities. Proteins containing AAA domains often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. (6) RRM (pfam00076): RNA-binding domain, found in a variety of RNA-binding proteins (also see text). (7) Cu (pfam02298): Copper-binding domain found in various proteins (also see text). (8) In plants, the N-terminus (S3_N, pfam00417) and C-terminus (S3_C, pfam00189) of MRPS3 are separated as a result of an insertion of a protein domain of unknown function (pfam B domain PB012879). (9) COG5373: Predicted membrane protein present in prokaryotes. (10) CBS (pfam00571) and PB1 (pfam00564) domains: CBS domains are small intracellular modules, which have a regulatory role in making proteins sensitive to adenosyl carrying ligands. PB1 is present in many eukaryotic cytoplasmic signaling proteins. (11) COG0021: Transketolase (1-deoxyxylulose-5-phosphate synthase).

Adaptive evolution of core MRP s Compared to bacterial ribosomes, mammalian mitoribosomes contain scarcely half as much rRNA and over twice as much protein, due to the presence of enlarged bacterial core proteins and supernumerary proteins.6 Previously, it has been hypothesized that these proteins might compensate for the loss of rRNA segments in the mitoribosome.684 Recently however, it was shown that many

146 Evolution of mitochondrial ribosomal proteome supernumerary MRPs occupy new quaternary positions in both the large and small subunits of the mitoribosome,61 inconsistent with this hypothesis. We compared the sequences of all core MRPs with their bacterial counterparts in order to explore the possibility that core MRPs contain additional domains that perform extra, mitochondria-specific functions. Indeed, we observe that MRPs are significantly larger (on average almost twice as large) than their bacterial ancestral sequences (see Supplementary Data, Figure S8). In some cases, we could identify functional domains within these newly evolved regions, which we expect to be linked to ribosome assembly or function (see Figure 7). For instance, we identified a RNA recognition motif (RRM_1, pfam00076) and a copper-binding domain (Cu_bind_like, pfam02298) in the N-terminal regions of A. thaliana MRPS19 and MRPL22, respectively (see Figure 7). The RRM motif that is present in MRPS1985 is found in a wide variety of RNA-binding proteins, which are implicated in a wide variety of cellular processes. Here, the motif is most likely involved in increasing the binding affinity to and/or stabilization of the tertiary structure of rRNA. The role of the copper-binding domain in MRPL22 is less clear. It might be Chapter involved in the co-translational insertion of copper ions into proteins that require copper for activity, in a similar way as has been observed for the cytochrome c 6 oxidase assembly factor in yeast, Cox1147 (also see above). The functions of other functional domains that are fused to MRPs are still obscure, such as a bacterial- type membrane protein (COG5373) to MRPL27 in N. crassa (see Figure 7).

D i s c u s s i o n

In the present study we have mapped the evolution of the mitochondrial ribosomal proteome using a comparative genomics approach that combined the most recent experimental data and computational techniques. The results of our study hint at a complex evolutionary scenario in which an ancestral ribosome of alpha-proteobacterial descent doubled its amount of protein in most eukaryotic lineages by elongation of its proteins and by recruiting new proteins of diverse origins. We observe that the mitoribosome has a complex history in terms of MRP gene gain and loss events during evolution. A major protein gain of the mitoribosome in eukaryotic evolution occurred prior to the divergence of the main eukaryotic domains, resulting in a primitive eukaryotic mitoribosome of 68 MRPs. Subsequently, the mitoribosome diversified along the different eukaryotic lineages with a continuous recruitment of new proteins, including a large gain after the bifurcation of the animal-fungi clade in each lineage. Another notable surge has taken place in the kinetoplastida lineage, where 29 new proteins were found to form a new, distinct protein complex with unknown function, which associates with the SSU of the Leishmania mitoribosomes.17

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In addition to the gain of MRPs, we also observe that numerous MRPs have been lost during the course of evolution, some of which have occurred independently in different lineages, as guided from ribosomal protein content that is encoded by the 'ancestral' mitochondrial genome of R. americana.5 The extensive loss of genes encoding core MRPs in some lineages contrasts with the evolutionary scenario that is observed for another mitochondrial protein complex with an alpha-proteobacterial ancestry, respiratory complex I (NADH:Ubiquinone oxidoreductase). During the evolution of this protein complex, which has approximately tripled its size in most eukaryotes, hardly any gene loss was observed, except for these lineages in which the complete respiratory complex was lost.57 Moreover, the bacterial core of complex I has been restricted from gene loss events in all eukaryotic genomes analyzed thus far, which contrasts with the scenario of the mitoribosome. What is the underlying reason for the observed differences in the evolutionary trajectories? Evidently, some of the core ribosomal proteins are not as essential as the core subunits of complex I for Chapter proper functioning of the protein complex, and are therefore expendable. This is the case for RPS20 and RPL25, which are absent from most mitoribosomes, 6 for example. RPS20-deficient E. coli strains were found to be viable, but showed an increased misreading ability of nonsense codons;86 RPL25-defective and wild type E. coli ribosomes were found to translate at the same rate in vitro, albeit that the mutant type was less efficient.87 In contrast, some MRPs that are missing in most eukaryotic species, such as MRPS1 and MRPL5, are indispensable for translation in E. coli and result in lethal phenotypes when mutated.8788 Moreover, MRPL5-deficient yeast strains display a respiratory-deficient phenotype, probably caused by a defect in the mitochondrial translation machinery.15 Apparently, the evolution of the mitoribosomal proteome involved several drastic events that altered the absolute necessity of some ribosomal proteins. In addition, the present study was performed in order to prioritize MRPs for their potential involvement in mitochondrial respiratory diseases. Based on the fact that essential genes appear more evolutionary conserved than non-essential genes, the bacterial core ribosomal proteins and translation factors that are conserved in most eukaryotes constitute excellent screening targets for human mitochondrial disorders. Mutations in non-essential MRPs could be risk factors for these diseases. Thus far, mutations in patients with mitochondrial respiratory disorders have been reported for the gene encoding small ribosomal subunit protein MRPS16.28 But what about the supernumerary MRPs? In general, they are phylogenetically less conserved than the bacterial core proteins; however, their recruitment bears functional relevance, as is indicated by knock-out studies in yeast and C. elegans. As such, the two novel human MRPs, orthologs of yeast Rsm22 and Mrp10, are good candidates for unresolved mitochondrial disorders,

148 Evolution of mitochondrial ribosomal proteome since they show a wide phylogenetic distribution, have not been investigated before and yeast mutants show clear respiratory deficient phenotypes. Translation-related mitochondrial disorders are not solely caused by mutations in mitoribosomal genes, but also by mutations in genes encoding factors that functionally interact with the mitoribosome, such as mitochondrial translation elongation factors EFG125 and EFTs.26 In addition to the newly discovered human MRPs, these interactors constitute excellent screening targets for human patients with unresolved mitochondrial OXPHOS system disorders. Altogether, our study reveals that the mitoribosomal proteome has, after initially having been acquired via the alpha-proteobacterial endosymbiont, been subjected to a complex evolution that involved numerous gain and loss events, resulting in a large variety in mitoribosomal protein content between different eukaryotic lineages. Potentially, the increased amount of proteins that seem to be associated with all modern eukaryotic mitoribosomes might reflect specific adaptations to mitochondria-specific functions, or, it might even reflect the increased complexity of eukaryotes themselves. Finally, we expect that additional Chapter experimental studies of mitoribosomal proteomes, such as the one that was recently performed on Leishmania mitoribosomes, will reveal an even more 6 dramatic picture of the evolutionary trajectory of this protein complex. It will be a major challenge to gain more insight in the functional roles of the newly acquired MRPs.

A cknowledgements

This work was supported by a Rubicon grant from The Netherlands Organization for scientific research (NWO) and by the European Union's 6th Framework Program, contract number LSHM-CT-2004-503116 (EUMITOCOMBAT) and contract number LSHM-CT-2004-005260 (MITOCIRCLE). We thank Siv Andersson (Evolutionary Biology Center, Uppsala University, Sweden) for reading the manuscript and for helpful suggestions prior to submission. Funding to pay the Open Access publication charges for this article was provided by the European Union's 6th framework program EUMITOCOMBAT.

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59. Cavdar Koc E, Burkhart W, Blackburn K, Moseley A, Koc H, Spremulli LL: A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins. J Biol Chem 2000; 275: 32585-32591. 60. Mears JA, Sharma MR, Gutell RR et al: A structural model for the large subunit of the mammalian mitochondrial ribosome. J Mol Biol 2006; 358: 193-212. 61. Sharma MR, Koc EC, Datta PP, Booth TM, Spremulli LL, Agrawal RK: Structure of the mammalian mitochondrial ribosome reveals an expanded functional role for its component proteins. Cell 2003; 115: 97-108. 62. Sonnichsen B, Koski LB, Walsh A et al: Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 2005; 434: 462-469. 63. Cavdar Koc E, Ranasinghe A, Burkhart W et al: A new face on apoptosis: death- associated protein 3 and PDCD9 are mitochondrial ribosomal proteins. FEBS Lett 2001; 492: 166-170. 64. Sun L, Liu Y, Fremont M et al: A novel 52 kDa protein induces apoptosis and concurrently Chapter activates c-Jun N-terminal kinase 1 (JNK1) in mouse C3H10T1/2 fibroblasts. Gene 1998; 208: 157-166. 6 65. Kissil JL, Cohen O, Raveh T, Kimchi A: Structure-function analysis of an evolutionary conserved protein, DAP3, which mediates TNF-alpha- and Fas-induced cell death. EMBO J 1999; 18: 353-362. 66. O'Brien TW, O'Brien BJ, Norman RA: Nuclear MRP genes and mitochondrial disease. Gene 2005; 354: 147-151. 67. Spirina O, Bykhovskaya Y, Kajava AV et al: Heart-specific splice-variant of a human mitochondrial ribosomal protein (mRNA processing; tissue specific splicing). Gene 2000; 261: 229-234. 68. Herrmann JM, Neupert W: Protein insertion into the inner membrane of mitochondria. IUBMB Life 2003; 55: 219-225. 69. Preuss M, Leonhard K, Hell K, Stuart RA, Neupert W, Herrmann JM: Mba1, a novel component of the mitochondrial protein export machinery of the yeast Saccharomyces cerevisiae. J Cell Biol 2001; 153: 1085-1096. 70. Dolezal P, Likic V, Tachezy J, Lithgow T: Evolution of the molecular machines for protein import into mitochondria. Science 2006; 313: 314-318. 71. Wiedemann N, Frazier AE, Pfanner N: The protein import machinery of mitochondria. J Biol Chem 2004; 279: 14473-14476. 72. Pavlov PF, Glaser E: Probing the membrane topology of a subunit of the mitochondrial protein translocase, Tim44, with biotin maleimide. Biochem Biophys Res Commun 2002; 293: 321-326.

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73. Ott M, Prestele M, Bauerschmitt H, Funes S, Bonnefoy N, Herrmann JM: Mba1, a membrane-associated ribosome receptor in mitochondria. EMBO J 2006; 25: 1603 1610. 74. Hughes TR, Marton MJ, Jones AR et al: Functional discovery via a compendium of expression profiles. Cell 2000; 102: 109-126. 75. Liu M, Spremulli L: Interaction of mammalian mitochondrial ribosomes with the inner membrane. J Biol Chem 2000; 275: 29400-29406. 76. Marcotte EM, Pellegrini M, Thompson MJ, Yeates TO, Eisenberg D: A combined algorithm for genome-wide prediction of protein function. Nature 1999; 402: 83-86. 77. Ton C, Hwang DM, Dempsey AA, Liew CC: Identification and primary structure of five human NADH-ubiquinone oxidoreductase subunits. Biochem Biophys Res Commun 1997; 241: 589-594. 78. Fearon K, Mason TL: Structure and function of MRP20 and MRP49, the nuclear genes for two proteins of the 54 S subunit of the yeast mitochondrial ribosome. J Biol Chem 1992; 267: 5162-5170. Chapter 79. Matsushita Y, Kitakawa M, Isono K: Cloning and analysis of the nuclear genes for two mitochondrial ribosomal proteins in yeast. Mol Gen Genet 1989; 219: 119-124. 6 80. Kyrpides NC, Woese CR and Ouzounis CA: KOW: a novel motif linking a bacterial transcription factor with ribosomal proteins. Trends Biochem Sci 1996; 21: 425-426. 81. Madera M, Vogel C, Kummerfeld SK, Chothia C, Gough J: The SUPERFAMILY database in 2004: additions and improvements. Nucleic Acids Res 2004; 32: D235-239. 82. Bullerwell CE, Burger G, Lang BF: A novel motif for identifying rps3 homologs in fungal mitochondrial genomes. Trends Biochem Sci 2000; 25: 363-365. 83. Iwamoto M, Pi M, Kurihara M, Morio T, Tanaka Y: A ribosomal protein gene cluster is encoded in the mitochondrial DNA of Dictyostelium discoideum: UGA termination codons and similarity of gene order to Acanthamoeba castellanii. Curr Genet 1998; 33: 304-310. 84. Suzuki T, Terasaki M, Takemoto-Hori C et al: Structural compensation for the deficit of rRNA with proteins in the mammalian mitochondrial ribosome. Systematic analysis of protein components of the large ribosomal subunit from mammalian mitochondria. J Biol Chem 2001; 276: 21724-21736. 85. Sanchez H, Fester T, Kloska S, Schroder W, Schuster W: Transfer of rps19 to the nucleus involves the gain of an RNP-binding motif which may functionally replace RPS13 in Arabidopsis mitochondria. EMBO J 1996; 15: 2138-2149. 86. Ryden-Aulin M, Shaoping Z, Kylsten P, Isaksson LA: Ribosome activity and modification of 16S RNA are influenced by deletion of ribosomal protein S20. Mol Microbiol 1993; 7: 983-992.

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87. Korepanov AP, Gongadze GM, Garber MB, Court DL, Bubunenko MG: Importance of the 5 S rRNA-binding ribosomal proteins for cell viability and translation in Escherichia coli. J Mol Biol 2006; 366: 1199-1208. 88. Sorensen MA, Fricke J, Pedersen S: Ribosomal protein S1 is required for translation of most, if not all, natural mRNAs in Escherichia coli in vivo. J Mol Biol 1998; 280: 561 569. 89. Simpson AG, Inagaki Y, Roger AJ: Comprehensive multigene phylogenies of excavate protists reveal the evolutionary positions of "primitive" eukaryotes. Mol Biol Evol, 2006; 23: 615-625.

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156 Evolution of mitochondrial ribosomal proteome

S upplementary d a t a Supplementary text Discrepancies between our study and other reports This section describes the differences between the current analysis and some comparative analyses performed by others.1-4 In some cases we failed to detect MRPs where these were proposed, and in other cases our proposed candidate did not correspond to the candidate proposed. In either such case, we have performed bidirectional homology searches and a phylogenetic analysis in order to verify the proposed identity. Koc et al. reported orthologs for MRPS36 and MRPL52 for C. elegans (CAA90335 and CAA21620, respectively).2,3 We, however, performed extensive homology searches but failed to connect the proposed C. elegans candidate proteins to the respective MRPs. We therefore conclude that the genes encoding MRPS36 and MRPL52 are missing in C. elegans. In addition, we were unable to detect significant sequence homology between N. crassa protein NCU05552 and yeast Mrpl13, which contrasts with results from Gan et al,5 Chapter who detected sequence homology between these proteins. However, given the limited sequence similarity (less than 15% over the full length of the proteins) and given the fact that we were unable to connect these proteins in bidirectional homology searches, 6 it is conceivable that NCU05552 and Mrpl13 are unrelated and thus constitute different components of the LSU. Further experiments are needed to corroborate this however. Additionally, several differences were observed between the results of the current study and a study performed by Bonen and Calixte, who compiled data for the bacterial-origin MRPs from A. thaliana and Oryza sativa.1 In our study, we failed to detect mitochondrial candidates for ribosomal proteins S1, S20, L10, L31 and L35, suggesting that these genes have been lost. Bonen and Calixte also report these MRPs to be missing, but they propose that proteins from a non-alpha-proteobacterial origin might have substituted for these MRPs, as has been experimentally verified for MRPS8 (divergent copy of cytosolic-origin S8)6 and MRPS13 (divergent copy of chloroplast- origin S13).6,7 Alternatively, they propose that nuclear-encoded chloroplast-type homologs might be dually targeted to the chloroplast as well as to the mitochondrion.1 We confirmed a chloroplast origin for the proposed S1, S20, L10, L21, L31, L34 and L35 candidates (not shown). However, they have not been included in our dataset since evidence of mitochondrial targeting of these proteins is still lacking. Bonen and Calixte also proposed a protein of chloroplast origin for mitochondrial S21 in plants, but we detected a more likely candidate of bacterial origin (See Table 1 and Supplementary Table 4). Moreover, we were able to detect proteins with significant sequence similarity to bacterial RPL25 encoded by the nuclear genomes of A. thaliana, O. sativa, as well as D. discoideum, indicating that the genes encoding these proteins have not been lost in the early stage of plant evolution as is suggested by Bonen and Calixte.1

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Supplementary data MS-Excel files containing supplementary information of the orthologous groups of MRPs used in this study can be downloaded from the NAR website. For small subunit: 'Supplementary Table 4 (SSU).xls' For large subunit: 'Supplementary Table 5 (LSU).xls'

ssu

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S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S21 Ribosomal protein LSU g 400 I RP 350 i MRP

300

I 250

! 200 <

150 i

100

L1 L2 L3 L4 L5 L6 L9 L10 L11 L12 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L24 L27 L28 L30 L31 L32 L33 L34 L35 L36 Ribosomal protein I Supplementary Figure S8. Mitochondrial ribosomal proteins of the small (A) and large subunit (B) have increased in size compared to their bacterial counterparts. The average lengths of MRPs are indicated with purple bars and their bacterial counterparts (RP) with orange bars. The lengths of the respective ribosomal proteins as depicted on the Y-axis was calculated by averaging the length of all the proteins that were present in the same orthologous group as defined by the COG database (RPs) or as defined in this study (MRPs). Only those ribosomal proteins are depicted that have a mitochondrial counterpart.

158 Evolution of mitochondrial ribosomal proteome

R e f e r e n c e s i n supplementary information 1. Bonen L, Calixte S: Comparative analysis of bacterial-origin genes for plant mitochondrial ribosomal proteins. Mol Biol Evol 2006; 23: 701-712. 2. Cavdar Koc E, Burkhart W, Blackburn K, Moseley A, Spremulli LL: The small subunit of the mammalian mitochondrial ribosome. Identification of the full complement of ribosomal proteins present. J Biol Chem 2001 ; 276: 19363-19374. 3. Cavdar Koc EC, Burkhart W, Blackburn K et al: The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present. J Biol Chem 2001; 276: 43958-43969. 4. Gan X, Kitakawa M, Yoshino K, Oshiro N, Yonezawa K, Isono K: Tag-mediated isolation of yeast mitochondrial ribosome and mass spectrometric identification of its new components. Eur J Biochem 2002; 269: 5203-5214. 5. Gan X, Arita K, Isono S, Kitakawa M et al: Identification and comparative analysis of the large subunit mitochondrial ribosomal proteins of Neurospora crassa. FEMS Chapter Microbiol Lett 2006; 254: 157-164. 6. Adams KL, Daley DO, Whelan J, Palmer JD: Genes for two mitochondrial ribosomal 6 proteins in flowering plants are derived from their chloroplast or cytosolic counterparts. Plant Cell 2002; 14: 931-943. 7. Mollier P, Hoffmann B, Debast C, Small I: The gene encoding Arabidopsis thaliana mitochondrial ribosomal protein S13 is a recent duplication of the gene encoding plastid S13. Curr Genet 2002; 40: 405-409.

159 I am not discouraged, because every wrong attempt discarded is another step forward. Thomas Edison C h apter 7 Sequence variants in four candidate genes (NIPSNAP1, GBAS, CHCHD1 and METT11D1) in patients with combined oxidative phosphorylation system deficiencies

Paulien Smits, Richard J.T. Rodenburg, JanY A.M. Smeitink and Lambert P. van den Heuvel

Journal of Inherited Metabolic Disease 2009; January 24th, Short Report #149 Chapter 7

A b s t r a c t The oxidative phosphorylation (OXPHOS) system, comprising five enzyme complexes, is located in the inner membrane of mitochondria and is the final biochemical pathway in oxidative ATP production. Defects in this energy- generating system can cause a wide range of clinical symptoms; these diseases are often progressive and multisystemic. Numerous genes have been implicated in OXPHOS deficiencies and many mutations have been described. However, in a substantial number of patients with decreased enzyme activities of two or more OXPHOS complexes, no mutations in the mitochondrial DNA or in nuclear genes known to be involved in these disorders have been found. In this study, four nuclear candidate genes - NIPSNAP1, GBAS, CHCHD1 and METT11D1 - were screened for mutations in 22 patients with a combined enzymatic deficiency of primarily the OXPHOS complexes I, III and IV to determine whether a mutation in one of these genes could explain the mitochondrial disorder. For each variant not yet reported as a polymorphism, 100 control samples were screened for the presence of the variant. This way we identified 14 new polymorphisms and 2 presumably non-pathogenic mutations. No mutations were found that could explain the mitochondrial disorder in the patients investigated in this study. Chapter Therefore, the genetic defect in these patients must be located in other nuclear genes involved in mtDNA maintenance, transcription or translation, in import, processing or degradation of nuclear encoded mitochondrial proteins, or in assembly of the OXPHOS system.

162 Genes for combined OXPHOS deficiencies?

I ntroduction In this study, four nuclear candidate genes for combined deficiencies of the oxidative phosphorylation (OXPHOS) system were screened for mutations in a group of 22 patients: nipsnap homolog 1 (C. elegans) (NIPSNAP1), glioblastoma amplified sequence (GBAS), coiled-coil-helix-coiled-coil-helix domain containing 1 (CHCHD1), and methyltransferase 11 domain containing 1 (METT11D1). The oxidative phosphorylation system is located in the inner membrane of mitochondria and is the primary source of ATP in all aerobic organism s.1 In case of a defect in the energy-generating system, we speak of a mitochondrial disorder. Mitochondrial disorders are a group of genetically and clinically heterogeneous diseases that are often progressive and multisystemic. They are among the most common inherited human diseases with an estimated incidence of 1:5000 living births.2 Currently, therapy is mainly focused on symptom relief and only in up to 25% of the cases diagnosed at our centre can the underlying genetic defect of the mitochondrial disorder be found. Accordingly, in many families prenatal diagnosis is not possible, although in selected cases prenatal diagnosis on the basis of biochemical measurements can be considered.3 The human OXPHOS system is composed of the four enzyme complexes (complexes I-IV) that make up the mitochondrial respiratory chain, and the ATP synthase Chapter complex (complex V), which uses the energy generated by electron transport along the respiratory chain to produce ATP. All the complexes contain nuclear (n) as well as mitochondrial (mt) DNA-encoded protein subunits with the exception of complex II, which is entirely encoded by the nuclear genome. Consequently, a reduced enzyme activity of one or more of the complexes can be caused by mtDNA or nDNA mutations. Approximately 30% of the OXPHOS deficiencies in fresh muscle biopsies investigated at our centre consist of combined enzymatic deficiencies.4 Numerous mutations have been described in mtDNA as well as in nDNA.5 However, in a substantial number of patients with combined OXPHOS system deficiencies, despite extensive investigations, no mutations in the mtDNA or in nuclear genes known to be involved in these disorders have been found. Thus, research into new candidate genes is warranted. The four nuclear candidate genes investigated in this study were selected on the basis of their potential role in the pathogenesis of combined OXPHOS deficiencies. The function of NIPSNAP1 and GBAS (also known as NIPSNAP2) is am biguous. Both genes belong to an evolutionary well-conserved family consisting of four human NIPSNAP genes, divided into two subfamilies: (a) NIPSNAP1 and GBAS, and (b) NIPSNAP3A and NIPSNAP3B (also known as NIPSNAP3 and NIPSNAP4, respectively).6 NIPSNAP1 has a strong sequence similarity to the central portion of a hypothetical Caenorhabditis elegans protein, which is located between a

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4-nitrophenylphosphatase (NIP) domain and non-neuronal SNAP25-like protein.7 Chromosomal region 7p12, which contains GBAS, is amplified in approximately 40% of glioblastomas.8 The transcripts of both genes are expressed ubiquitously at variable levels, with NIPSNAP1 m RNA being most abundant in liver, brain and kidney7,9 and GBAS mRNA in brain, skeletal muscle and heart.7,8 These tissues have high energy demands and have been shown to be often affected in mitochondrial diseases. All NIPSNAP proteins have been reported to play a role in vesicular transport, mainly on the basis of homology data and the location of the C. elegans NIPSNAP gene in an operon with the SNAP25-like gene;6,7,10,11 the SNAP25-like protein is implicated in synaptic vesicle docking and fusion. However, functional studies have not confirmed this hypothesis yet, at least not for NIPSNAP1 and GBAS. More recently, NIPSNAP1 was shown to be located in the mitochondrion, in the inner membrane.12,13 Additionally, NIPSNAP1 was found to be associated with mtDNA, possibly functioning as a mitochondrial nucleoid protein in mtDNA maintenance and segregation.14 Since mtDNA defects can cause mitochondrial disorders, NIPSNAP1 and its close homolog GBAS are candidate genes for these diseases and for combined OXPHOS deficiencies in particular. For instance, defects in the mtDNA maintenance protein POLG (polymerase gamma), the only mtDNA polymerase and also a constituent of mitochondrial nucleoids, are Chapter associated with a variety of mitochondrial disorders.15,16 The other two candidate genes, CHCHD1 and METT11D1, were shown to be orthologues of yeast genes coding for mitochondrial ribosomal small subunit proteins MRP10 and RSM22, respectively.17 Two human mitochondrial ribosomal proteins (MRPs) of the small subunit, MRPS16 and MRPS22, have already been shown to be implicated in combined OXPHOS diseases.18,19 The two newly identified human MRPs are good screening targets for unresolved combined OXPHOS disorders because they show a wide phylogenetic distribution, they have not been investigated before, and yeast mutants show clear respiratory-deficient phenotypes. We performed sequence analysis on the DNA level for the four candidate genes in a group of 22 patients with a combined enzymatic deficiency of primarily the OXPHOS complexes I, III and IV in muscle and/or fibroblasts to determine whether a mutation in one of these genes could explain the mitochondrial disorder. This revealed 14 new polymorphisms and 2 probably non-pathogenic mutations. Consequently, the genetic defect in this group of patients must be located in other nuclear genes involved in processes such as mtDNA maintenance, transcription or translation.

164 Genes for combined OXPHOS deficiencies?

M a t e r i a l s a n d m e t h o d s Sub jects The patient group consisted of 22 patients diagnosed at our centre with a combined OXPHOS deficiency of primarily the complexes I, III and IV; the enzyme activities were measured in skeletal muscle tissue and/or cultured skin fibroblasts (see Table 1). In all patients, mtDNA mutations were excluded by sequencing the entire mitochondrial genome. Additionally, POLG and all known mitochondrial translation factors (MTIF2, MTIF3, TSFM, TUFM, GFM1, GFM2, MTRF1, MTRF1L and MRRF) and multiple MRPs (MRPS12, MRPS14, MRPS16, MRPL11, MRPL12 and MRPL45) were systematically screened; no convincing pathogenic sequence variants were found. The control material for our standard control group was derived from 100 healthy persons who had been referred to our centre for reasons not related to mitochondrial dysfunction. The two ethnically matched control groups each consisted of 100 inhabitants of The Netherlands who either had a Dutch or a Turkish background.

Cell culture and biochemical measurements Human skin fibroblasts were cultured in M199 medium (Gibco, Breda, The Netherlands) supplemented with 10% fetal calf serum and antibiotics. The Chapter activities of the OXPHOS enzyme complexes were measured in skin fibroblasts and muscle tissue as described previously.20,21

Sequence analysis DNA was isolated from cultured skin fibroblasts (patients) or blood (controls) using salt extraction.22 The entire coding sequences including the intron-exon boundaries were screened for mutations. The sequence of the oligonucleotide primers and the PCR conditions used for amplification can be obtained upon request. Both strands were used for direct sequencing on a 3130xl Genetic Analyzer, using the BigDye terminator v1.1 cycle sequencing kit according to the manufacturer's protocol (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). Sequence data were analysed with Sequencher 4.8 software (Gene Codes, Ann Arbor, MI, USA) by comparison with the reference sequences. Genbank accession numbers: AC005529.7 (NIPSNAP1), AC092579.3 (GBAS), AC022400.9 (CHCHD1), AF321003.1 (METT11D1).

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R e s u l t s Direct sequencing of the four genes NIPSNAP1, GBAS, CHCHD1 and METT11D1 in 22 patients with a combined OXPHOS deficiency revealed many known polymorphisms (data not shown) but no pathogenic mutations. However, 14 new polymorphisms and 2 mutations, presumably non-pathogenic, were identified by screening 100 controls for the genetic variants found in the patients (see Table 2). The numbering of the variants is based on the following coding DNA reference sequences: NM_003634.1 (NIPSNAP1), NM_001483.1 (GBAS), NM_203298.1 (CHCHD1), and NM_001029991.1 (METT11D1). We classified a sequence variation as a new polymorphism when detected in at least 1 % and as a mutation when found in less than 1 % of the control population. Four variants were found in less than 1% of our standard control group and therefore we also screened 100 ethnically matched controls for these variations. Both GBAS c.93-51T>C and METT11D1 c.517G>T (p.Ala173Ser) were present in a Turkish control group with an allele frequency of 0.02 and were consequently regarded as new polymorphisms. The two other variants were considered to be non-pathogenic mutations. In NIPSNAP1, a heterozygous mutation was identified in patient 21, a transversion of a G to a C located in the 3' UTR (untranslated region), 170 nucleotides after the stop codon (c.*170G>C). This mutation was also found to be heterozygous in one Turkish control. In METT11D1, patient 20 harboured a heterozygous G-to-A transition located 21 nucleotides before exon 6 (c.529-21G>A). The mutation was not detected in a Dutch control population. Investigation on the cDNA level revealed no effect on splicing. We also screened 100 controls for the presence of the following 11 known polymorphisms found in the four genes we investigated: rs34114704, rs9625833, rs1058646, rs35864418, rs7098766, rs11538172, rs35402434, rs34207344, rs2771348, rs35308618, and rs2771350. None of these polymorphisms was found in our patient or control group. Additionally, we detected two discrepancies between Genbank reference sequences and patient sequences. The NIPSNAP1 reference sequence (NM_003634.1) contains CG on position c.468_469, whereas all patients and the Ensembl reference sequence (ENST00000216121) have GC on this position. The GBAS reference sequence (NM_001483.1) contains a G on position c.*1082; however, all patients and the Ensembl reference sequence (ENST00000322090) show a T at this location. BLAST searches revealed that both sequence variations deposited in Genbank are present in only one of the available human expressed sequence tags.

166 Genes for combined OXPHOS deficiencies?

Table 1. OXPHOS enzyme activities of all patients Patient Tissue CI CII CIII SCC CIV 1 Muscle 19 175 22 110 22 Fibroblast 53 95 66 210 20 2 Muscle 63 73 69 123 69 Fibroblast 144 121 ND 159 127 3 Muscle 0 ND ND 159 44 Fibroblast 17 151 94 192 30 4 Muscle 80 118 68 91 96 Fibroblast 110 129 78 118 65 5 Muscle 21 ND 27 50 25 Fibroblast 182 ND 153 144 125 6 Muscle 80 163 71 105 143 Fibroblast 75 ND 85 133 66 7 Fibroblast 43 123 76 113 14 8 Muscle 38 ND ND 249 54 Fibroblast 35 ND 87-136a 173 30 9 Muscle 80 67 51 64 95 Fibroblast 117 ND 127 111 136 10 Fibroblast 64 87 71 51 39 11 Muscle 54 91 78 ND 51 Fibroblast 27 93 65 ND 23 12 Muscle 31 222 25 77 22 Fibroblast 164 ND 142 100 121 13 Muscle 0 184 18 120 13 Chapter Fibroblast 166 153 126 189 137 14 Fibroblast 47 102 59 147 41 15 Muscle 60 224 69 96 23 Fibroblast 144 ND 66 110 64 16 Muscle 24 160 55 152 61 Fibroblast 169 ND 146 175 124 17 Muscle 6 ND 73 94 19 Fibroblast 231 ND 183 144 107 18 Muscle 17 109 56 113 54 Fibroblast 120 246 241 329 146 19 Muscle 116 158 78 101 157 Fibroblast 53 193 65 177 53 20 Muscle 26 178 109 142 78 Fibroblast 31 254 52 85 13 21 Muscle 4 ND 82 97 21 Fibroblast 119 ND 155 200 104 22 Muscle 9 130 35 102 26 Fibroblast 144 ND 144 194 148 CI—IV, complexes I-IV; SCC, succinate cytochrome c oxidoreductase; ND, not determined a Complex III activity was decreased in one fibroblast sample and normal in another sample. All enzyme activities are expressed as percentage of the lowest control reference value. Lowered activities are shown in bold type. In total 10 patients had lowered enzyme activities in muscle tissue, but normal values in fibroblasts. In 10 other patients decreased enzyme activities were measured in muscle tissue as well as fibroblasts (in multiple cases not all three of the complexes I, III and IV were affected or measured in both tissues). For 3 patients muscle tissue was not available and therefore the enzyme activities were only measured in fibroblasts.

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table 2. New sequence variants found in our patient group Gene Sequence variant Submitter SNP ID Allele frequency Patients Controls NIPSNAP1 c. -157C>T ss99308674 0.05 0.04 c. -123G>T ss99308675 0.02 0.03 c. -105G>A ss99308676 0.02 0.02 c.790+11C>G ss99308677 0.02 0.05 c.*170G>C - 0.02 0/0.005b GBAS c.93-51T>C ss107796293 0.02 0.005/0.02b c.444 + 17A>G ss99308678 0.11 0.23 c.444 + 197G>A ss99308679 0.11 0.23 c.585 + 130_585 + 131insATATATGTAT ss99308680 0.80 0.75 c.585 + 152_585 + 153delAC ss99308681 0.05 0.02 c.586-41delT a - 0.50 0.50 CHCHD1 c.-6-40delT ss99308682 0.05 0.03 METT11D1 c.76-37_76-36insGT ss99308683 0.02 0.02 c.364 + 55G>A ss99308684 0.02 0.03 c.517G>T ss107796294 0.02 0/0.02b c.529-21G>A - 0.02 0/0b Chapter c.997-32T>C ss99308685 0.02 0.02

a This is probably not a SNP but a sequence artefact due to a T-stretch of 10 nucleotides. b These sequence variants were screened in our standard control group and in ethnically matched control groups (Dutch for METT11D1 c.529-21G>A, Turkish for the 3 other variants); the first number refers to the allele frequency in the standard control group and the second number to the frequency in the ethnically matched group.

D i s c u s s i o n In this study we screened four nuclear genes, NIPSNAP1, GBAS, CHCHD1 and METT11D1, which were selected based on their potential role in the pathogenesis of combined OXPHOS deficiencies, for the presence of mutations in a group of 22 patients with primarily an OXPHOS complex I, III and IV deficiency. Sequence analysis revealed no mutations that could explain the mitochondrial disorder of the patients. However, many sequence variations were detected, including 14 new polymorphisms and 2 mutations. Both mutations are considered to be non- pathogenic on the basis of the location in the sequence (in non-coding regions), the presence in a control subject (applies only to the NIPSNAP1 mutation), the absence of an effect on splicing (only tested for the METT11D1 mutation), and the fact that the mutations were both heterozygous. The mutation in the 3' UTR of NIPSNAP1 could have an effect on the stability of the mRNA or the efficiency of binding of the mRNA to the ribosomes; this has not been investigated.

168 Genes for combined OXPHOS deficiencies?

Nevertheless, we do not expect this single nucleotide change to be the cause of the mitochondrial disorder. Additionally, 11 sequence variations reported in the NCBI SNP database were not observed in either our patient group or the control group. For most of these variations, population frequency data were lacking or data showed the absence of the sequence variant (rs9625833, rs2771348, and rs2771350), and two SNPs (rs34207344 and rs35308618) were found with an allele frequency of only 0.013. Since the variations are probably all rare polymorphisms, it is not surprising that these SNPs were not detected in our study. Furthermore, we found two discrepancies between the NIPSNAP1 and GBAS Genbank reference sequences and the patient sequences. Because the Genbank variations were only present in one of the available expressed sequence tags, we suspect that these variations are rare polymorphisms, mutations, or errors in the Genbank sequences. A possible explanation for the absence of disease-causing mutations in our patient group in the four candidate genes we screened is that the roles hypothesized for the proteins in mtDNA maintenance (NIPSNAP1 and GBAS) and mitochondrial translation (CHCHD1 and METT11D1) could be incorrect. Functional studies with human CHCHD1 and METT11D1 have not been performed so far. NIPSNAP1 has Chapter been shown to be associated with mtDNA14 but its potential role as nucleoid protein has not been investigated further; moreover, the function of GBAS is less certain, since it was not found in association with mtDNA. Additionally, NIPSNAP1 was recently reported to function as a negative modulator of the selective Ca2+ channel TRPV6 located in the kidney.9 Although the mechanism of this inhibition is not clear, vesicular trafficking processes appear not to be involved, even though other studies have reported the role of multiple NIPSNAP proteins in vesicular transport.6,7,10,11 Thus the function of the NIPSNAP proteins remains elusive and further research is warranted to clarify this. Moreover, there are of course many more candidate genes for combined OXPHOS complex I, III, and IV deficiencies: not only all genes involved in m tDNA maintenance, transcription and translation, in import, processing and degradation of nuclear-encoded mitochondrial proteins, but also genes involved in assembly of the OXPHOS system, including assembly of the complexes themselves and of the supercomplexes (containing, for instance, complex I, III and IV). All these processes are extremely complex and tightly regulated, which makes the identification of the remaining genes causing combined OXPHOS disorders a real challenge. Linkage analysis and homozygosity mapping are powerful tools for the identification of candidate genes and localization of the genetic defects, as has been shown for combined OXPHOS defects as well.23-26 Because we only have samples from single patients, we could not put these techniques to use. A method that has been shown

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to be useful on single patients is microcell-medicated chromosome transfer;27 however, this is a very time-consuming technique. Hopefully, it will be possible to apply whole-genome sequencing to our patient cohort in the near future. Much data will be generated with this technique; therefore, bioinformatic tools will have to be developed and optimized to make whole genome sequencing a feasible and relatively fast method - compared with screening candidate genes one by one - to find the genetic defects in patients with unresolved combined OXPHOS deficiencies.

A cknowledgements

This work was supported by the European Union's 6th Framework Program, contract number LSHMCT-2004-005260 (MITOCIRCLE).

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170 Genes for combined OXPHOS deficiencies?

R e f e r e n c e s 1. Saraste M: Oxidative phosphorylation at the fin de siecle. Science 1999; 283: 1488-1493. 2. Schaefer AM, Taylor RW, Turnbull DM, Chinnery PF: The epidemiology of mitochondrial disorders--past, present and future. Biochim Biophys Acta 2004; 1659: 115-120. 3. Niers L, van den Heuvel L, Trijbels F, Sengers R, Smeitink J: Prerequisites and strategies for prenatal diagnosis of respiratory chain deficiency in chorionic villi. J Inherit Metab Dis 2003; 26: 647-658. 4. Loeffen JL, Smeitink JA, Trijbels JM, et al: Isolated complex I deficiency in children: clinical, biochemical and genetic aspects. Hum Mutat 2000; 15: 123-134. 5. Zeviani M, Carelli V: Mitochondrial disorders. Curr Opin Neurol 2007; 20: 564-571. 6. Buechler C, Bodzioch M, Bared SM, et al: Expression pattern and raft association of NIPSNAP3 and NIPSNAP4, highly homologous proteins encoded by genes in close proximity to the ATP-binding cassette transporter A1. Genomics 2004; 83: 1116-1124. 7. Seroussi E, Pan HQ, Kedra D, Roe BA, Dumanski JP: Characterization of the human NIPSNAP1 gene from 22q12: a member of a novel gene family. Gene 1998; 212: 13-20. Chapter 8. Wang XY, Smith DI, Liu W, James CD: GBAS, a novel gene encoding a protein with tyrosine phosphorylation sites and a transmembrane domain, is co-amplified with EGFR. Genomics 1998; 49: 448-451. 9. Schoeber JP, Topala CN, Lee KP, et al: Identification of Nipsnap1 as a novel auxiliary protein inhibiting TRPV6 activity. Pflugers Arch 2008; 457: 91-101. 10. Lee AH, Zareei MP, Daefler S: Identification of a NIPSNAP homologue as host cell target for Salmonella virulence protein SpiC. Cell Microbiol 2002; 4: 739-750. 11. Satoh K, Takeuchi M, Oda Y, et al: Identification of activity-regulated proteins in the postsynaptic density fraction. Genes Cells 2002; 7: 187-197. 12. Da Cruz CS, Xenarios I, Langridge J, Vilbois F, Parone PA, Martinou JC: Proteomic analysis of the mouse liver mitochondrial inner membrane. J Biol Chem 2003; 278: 41566-41571. 13. Mootha VK, Bunkenborg J, Olsen JV, et al: Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria. Cell 2003; 115: 629-640. 14. He J, Mao CC, Reyes A, et al: The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization. J Cell Biol 2007; 176: 141-146. 15. Chen XJ, Butow RA: The organization and inheritance of the mitochondrial genome. Nat Rev Genet 2005; 6: 815-825.

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16. Hudson G, Chinnery PF: Mitochondrial DNA polymerase-gamma and human disease. Hum Mol Genet 2006; 15 Spec No 2: R244-R252. 17. Smits P, Smeitink JA, van den Heuvel LP, Huynen MA, Ettema TJ: Reconstructing the evolution of the mitochondrial ribosomal proteome. Nucleic Acids Res 2007; 35: 4686-4703. 18. Miller C, Saada A, Shaul N, et al: Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation. Ann Neurol 2004; 56: 734-738. 19. Saada A, Shaag A, Arnon S, et al: Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation. J Med Genet 2007; 44: 784-786. 20. Janssen AJ, Smeitink JA, van den Heuvel LP: Some practical aspects of providing a diagnostic service for respiratory chain defects. Ann Clin Biochem 2003; 40: 3-8. 21. Smeitink J, Sengers R, Trijbels F, van den Heuvel L: Human NADH:ubiquinone oxidoreductase. J Bioenerg Biomembr 2001; 33: 259-266. 22. Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215. 23. Bykhovskaya Y, Casas K, Mengesha E, Inbal A, Fischel-Ghodsian N: Missense mutation in pseudouridine synthase 1 (PUS1) causes mitochondrial myopathy and sideroblastic Chapter anemia (MLASA). Am J Hum Genet 2004; 74: 1303-1308. 24. Edvardson S, Shaag A, Kolesnikova O, et al: Deleterious mutation in the mitochondrial arginyl-transfer RNA synthetase gene is associated with pontocerebellar hypoplasia. Am J Hum Genet 2007; 81: 857-862. 25. Kaukonen J, Juselius JK, Tiranti V, et al: Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science 2000; 289: 782-785. 26. Smeitink JA, Elpeleg O, Antonicka H, et al: Distinct clinical phenotypes associated with a mutation in the mitochondrial translation elongation factor EFTs. Am J Hum Genet 2006; 79: 869-877. 27. Coenen MJ, Antonicka H, Ugalde C, et al: Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency. N Engl J Med 2004; 351: 2080-2086.

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173 From the bitterness of disease man learns the sweetness of health. Catalan proverb C h apter 8 Mutation in mitochondrial ribosomal protein MRPS22 leads to Cornelia de Lange- like phenotype, brain abnormalities and hypertrophic cardiomyopathy

Paulien Smits, Ann Saada, Saskia B. Wortmann, Angelien J. Heister, Maaike Brink, Rolph Pfundt, Chaya Miller, Dorothea Haas, Ralph Hantschmann, Richard J.T. Rodenburg, Jan A.M. Smeitink and Lambert P. van den Heuvel

European Journal of Human Genetics 2011; 19(4): 394-399 Chapter 8

A b s t r a c t The oxidative phosphorylation (OXPHOS) system is under control of both the mitochondrial and the nuclear genomes; 13 subunits are synthesized by the mitochondrial translation machinery. We report a patient with Cornelia de Lange- like dysmorphic features, brain abnormalities and hypertrophic cardiomyopathy, and studied the genetic defect responsible for the combined OXPHOS complex I, III and IV deficiency observed in fibroblasts. The combination of deficiencies suggested a primary defect associated with the synthesis of mitochondrially encoded OXPHOS subunits. Analysis of mitochondrial protein synthesis revealed a marked impairment in mitochondrial translation. Homozygosity mapping and sequence analysis of candidate genes revealed a homozygous mutation in MRPS22, a gene encoding a mitochondrial ribosomal small subunit protein. The mutation predicts a Leu215Pro substitution at an evolutionary conserved site. Mutations in genes implicated in Cornelia de Lange syndrome or copy number variations were not found. Transfection of patient fibroblasts, in which MRPS22 was undetectable, with the wild-type MRPS22 cDNA restored the amount and activity of OXPHOS complex IV as well as the 12S rRNA transcript level to normal values. These findings demonstrate the pathogenicity of the MRPS22 mutation and stress the significance of mutations in nuclear genes, including genes that have no counterparts in lower species like bacteria and yeast, for mitochondrial translation defects.

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176 Mitochondrial ribosomal protein MRPS22 mutation

I ntroduction Mitochondrial disorders are generally caused by dysfunction of the oxidative phosphorylation (OXPHOS) system. The OXPHOS system, comprising five enzyme complexes located in the mitochondrial inner membrane, is responsible for the production of most of the cell's ATP. Synthesis of this energy-generating system is controlled by both the mitochondrial and the nuclear genomes (mtDNA and nDNA). The mtDNA codes for 13 subunits of the OXPHOS complexes I, III, IV and V as well as the tRNAs and rRNAs required for the translation of these transcripts. All other mitochondrial proteins, including over 70 OXPHOS subunits and all proteins of the mitochondrial translation machinery, are encoded by nuclear genes. The majority of mutations associated with combined OXPHOS deficiencies because of impaired mitochondrial translation are located in mtDNA genes;1 however, the list of nDNA mutations is steadily growing with defects reported in mitochondrial translation factors,2-5 mitochondrial ribosomal proteins,6,7 mitochondrial tRNA synthetases8-10 and tRNA-modifying enzymes.11-13 We investigated a group of 33 patients with combined OXPHOS deficiencies (and normal complex II activities) by mutational analysis of the entire mtDNA, polymerase gamma and nuclear genes implicated in mitochondrial translation. In this study, we report a mutation in mitochondrial ribosomal protein MRPS22 detected in one patient and establish its pathogenicity.

M a t e r i a l s a n d m e t h o d s Chapter Case report The male patient was the first child of healthy, first-grade consanguineous Pakistani parents. Fetal ultrasound disclosed microcephaly with dilatation of the third 8 ventricle and a left ventricular hypertrophic cardiomyopathy (HCM). The patient was born by spontaneous vaginal delivery at 35 weeks of gestation with a birth weight of 2610 grams (P50), length 44 cm (P10) and head circumference 29 cm (

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following months, dysphagia and failure to thrive necessitated gastrostomy. The biventricular HCM remained stable over the years with a shortening fraction of 39%. ECG revealed Wolf-Parkinson-White syndrome. At the age of 2 years, he developed tonic seizures and EEG displayed a low background activity with multifocal sharp-slow waves. Seizures and EEG abnormalities disappeared spontaneously. Ophthalmological examination and hearing tests were repeatedly normal. Growth remained poor; growth parameters at the age of 4 years are as follows: weight 8.5 kg (<<right) with sparse but dystonic movements. He is unable to sit freely and communicate, but he reacts to light and sounds.

Cell culture Human skin fibroblasts were cultured in M199 medium (Gibco, Breda, The Netherlands) supplemented with 10% fetal calf serum (FCS) and antibiotics. For complementation experiments, human skin fibroblasts were grown in DMEM (Dulbecco's modified Eagle's medium, Kibbutz Beit Haemek, Israel) with 4.5 g/l of glucose, in the presence of 10% FCS, 50 |jg/ml uridine and 110 |jg/ml pyruvate. Selection of transfected cells was done in glucose-free RPMI (Roswell Park Memorial Institute) supplemented with 50 |j M galactose and 10% dialyzed FCS.14 Viability was assessed by trypan-blue exclusion. Chapter Enzyme measurements 8 The activities of the OXPHOS enzyme complexes were measured in cultured skin fibroblasts as described previously; citrate synthase (CS) served as a control.15,16

Pulse labeling of mitochondrial translation products In vitro labeling of mitochondrial translation was performed as described elsewhere,17 with a few minor adaptations. In short, cells were labeled for 60 min at 37 °C in methionine-free DMEM with 10% dialyzed FCS, 200 ^Ci/ml [35S]methionine (Tran35S- Label, MP Biomedicals, Eindhoven, The Netherlands) and 100 |jg/ml emetine, and subsequently chased for 10 min in regular DMEM with 10% FCS. Total cellular protein (100 ^g) was resuspended and incubated for 10 min in PBS containing 2% lauryl maltoside. Unsolubilized material was removed by centrifugation at 10 000 g for 10 min and loading buffer was added to the supernatant to a final concentration of 100 mM Tris-HCl (pH 6.8), 20% glycerol, 1% SDS, 0.02% Coomassie Blue G-250 and 1% mercaptoethanol. Samples were run on 16% polyacrylamide gels,18 which were subsequently scanned on a FLA5100 (Fujifilm Life Science, Düsseldorf, Germany). Equal protein loading was confirmed by Coomassie blue staining.

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Homozygosity mapping Patient DNA was isolated from cultured skin fibroblasts by salt extraction.19 Whole-genome homozygosity mapping was performed using the GeneChip Human Mapping 10K Array Xba 142 version 2.0 (Affymetrix, High Wycombe, UK) in accordance with the manufacturer's guidelines.20 This array contains 10 204 single neutral polymorphisms (SNPs) with a mean intermarker distance of 258 kb, equivalent to 0.36 cM, and an average SNP heterozygosity of 0.38.

Sequence analysis Genomic DNA (gDNA) was extracted from cultured skin fibroblasts (patient) or blood (parents and controls) by standard procedures.19 Total RNA was isolated from patient fibroblasts using RNA-Bee (Tel-Test, Friendswood, TX, USA); complementary DNA (cDNA) was prepared using superscript II reverse transcriptase (Invitrogen, Breda, The Netherlands). Sequence analysis of MRPS22 (GenBank accession number NM_020191.2) was carried out on cDNA in two overlapping fragments; the parts of the first and last exon not covered, including the intron-exon boundaries, were amplified on gDNA. The presence of the mutation in exon 4 was confirmed on gDNA and subsequently screened for in both parents and 109 Pakistani controls. Sequence analysis of the entire open reading frame including the intron-exon junctions of NIPBL, SMC1A and SMC3 (GenBank accession numbers NM_015384.4, NM_005445.3 and NM_006306.2, respectively) was performed on gDNA. Primer sequences and PCR conditions used for amplification can be obtained upon request. Both strands were used for direct Chapter sequencing on a 3130xl Genetic Analyzer, using the BigDye terminator v1.1 cycle sequencing kit according to the manufacturer's protocol (Applied Biosystems, 8 Nieuwerkerk a/d IJssel, The Netherlands). Sequence data were analyzed with Sequencher 4.8 software (Gene Codes, Ann Arbor, MI, USA) by comparison with the reference sequences.

Affymetrix NspI SNP array hybridization and analysis Copy number variation (CNV) screening by means of microarray analyses was carried out on the Affymetrix GeneChip 250k (NspI) SNP array platform (Affymetrix, Inc., Santa Clara, CA, USA), which contains 25-mer oligonucleotides representing a total of 262 264 SNPs. Hybridizations were performed according to the manufacturer's protocols. Copy numbers were determined using the 2.0 version of the CNAG (Copy Number Analyzer for Affymetrix GeneChip mapping) software package,21 by comparing SNP probe intensities from patient DNA with those of a sex-matched pooled reference DNA sample (DNA from 10 healthy male individuals). The average resolution of this array platform, described by McMullan et al,22 is 150-200 kb.

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Blue-native and SDS-PAGE analysis and in-gel activity assay Blue-native (BN) PAGE was used for separation of the OXPHOS complexes on 5-15% polyacrylamide-gradient gels as described before.23 After electrophoresis of 40 ^g mitochondrial protein from fibroblasts, gels were further processed for complex I in-gel activity assay and western blotting. Assembly of the OXPHOS complexes was investigated using monoclonal antibodies against subunits of complexes I, III, V (Molecular Probes, Leiden, The Netherlands), II and IV (MitoSciences, Eugene, OR, USA). One-dimensional 10% SDS-PAGE analysis was performed as described previously.24 For detection of MRPS22, a polyclonal antibody raised against a GST- MRPS22 (full length) fusion protein (ProteinTech Group, Chicago, IL, USA) was used.

Transfection with wild-type MRPS22 cDNA Wild-type MRPS22 cDNA was cloned into the pLenti6/V5-D-TOPO 6969-bp expression vector by directional TOPO cloning (primers and PCR conditions are available upon request). The recombinant vector was propagated in One Shot Stbl3 Escherichia coli and introduced into 293FT cells after co-transfection with pLP1, pLP2 and pLP/VSVG plasmids using lipofectamine according to the manufacturer's instructions (ViraPower, Invitrogen, Carlsbad, CA, USA). Control and patient cells were infected with the lentiviral construct carrying the wild-type MRPS22, and clones of stably transfected cells were established by blasticin (4 |jg/ Chapter ml) selection and by selection on glucose-free medium.7,25 Patient cells were also transiently transfected with the lentiviral vector carrying an expression control 8 plasmid, pLenti6/V5-GW/lacZ (Invitrogen).

Determination of complex IV levels Protein was determined by Bicinchoninic Acid (BCA) protein assay kit (Sigma- Aldrich, Rehovot, Israel). Amount of complex IV was estimated using the MitoProfile Biogenesis Dipstick assay kit (MitoSciences) according to the manufacturer's instructions. Results were obtained after digital imaging with TINA 2.0 software.

Determination of mitochondrial 12S rRNA and 16S rRNA levels Total mRNA isolated using TRI reagent (Sigma-Aldrich) was reverse transcribed with ImProm-II reverse transcriptase (Promega, Madison, WI, USA). The cDNA was amplified in a PCR reaction to detect the expression of specific nucleic acid sequences using SYBR Green (Applied Biosystems, Roche, NJ, USA). ß-Actin served as an endogenous control and mitochondrial 12S rRNA or 16S rRNA as the target cDNAs (primers are available on request). The levels of all cDNAs were equalized and real-time PCR reactions were performed using each primer set

180 Mitochondrial ribosomal protein MRPS22 mutation separately. The relative expression of the target transcript was calculated with the comparative Ct method (Applied Biosystems User Manual) and analyzed by two tailed Student's t-test.

R e s u l t s Enzyme activities of OXPHOS complexes I, III and IV in patient fibroblasts were reduced to 45, 59 and 36% of the lowest control value, respectively; complex II was not affected (Table 1). BN-PAGE analysis confirmed this combined OXPHOS deficiency: complex I and IV were hardly detectable and complex III showed a slight decrease in the amount of fully assembled complex, whereas the level of complex II was normal (Figure 1). Additionally, complex I activity was again clearly lowered.

Table 1. OXPHOS enzyme activities in fibroblasts Activities (mU/U citrate synthase)ab Complex Patient Reference range I 45 100 - 310 II 606 520 - 845 III 782 1320 - 2610 II+III 186 110 - 470 IV 242 680 - 1190 a Measured in mitochondrial-enriched fractions from fibroblasts. b The activity of citrate synthase was 208 mU/mg protein (reference range 144-257 mU/mg protein). Decreased activities as compared with controls are shown in bold numbers. Chapter

C1 P C2 8

Figure 1. BN-PAGE analysis in patient fibroblasts. Antibodies directed against specific individual subunits were used to assess the amount of each of the fully assembled complexes in the patient (P) and controls (C1 and C2). Complex II was used as a loading control. CI-CIV=complex I-IV; IGA=in-gel activity.

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Figure 2. In vitro labeling of mitochondrial translation products. The patient (P) translation rates of the different subunits are expressed as percentages of the average [35S]methionine incorporation in two controls (C1 and C2). The level of translation of ND5 was obtained from an identical assay, in which this product was clearly visible. COI-COIII=subunits of cytochrome c oxidase (complex IV); cyt b=cytochrome b subunit (of complex III); ND1-6=subunits of NADH CoQ oxidoreductase (complex I); ATP6-8=ATP synthase (complex V) subunits 6 and 8.

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Figure 3. MRPS22 mutation, conservation of the mutated residue, protein structure prediction and protein level in patient cells. (a) DNA sequence analysis of the patient (homozygous c.644T>C) and both parents (heterozygous). (b) Protein sequence alignment from human to worm, which shows that the mutated leucine residue is highly conserved among different species. (c) Prediction of the MRPS22 protein structure, displaying part of the protein with the Leu215Pro substitution located in the middle of a helix. (d) Immunoblot analysis in patient (P) and control (C1 and C2) fibroblasts reveals the (near) absence of the MRPS22 protein. Complex II was used as a loading control.

182 Mitochondrial ribosomal protein MRPS22 mutation

Pulse labeling of mitochondrial protein synthesis products revealed a marked and generalized mitochondrial translation defect (Figure 2): total incorporation of [35S]methionine was 26% of that in controls. The decrease in mitochondrial translation varied between different subunits, with the translation rates of ND5, ND6 and COIII/COII (10-15%) being most severely and of ATP8 and ND4L/ATP6 (60-70%) being the least affected.

C C+MRPS22 P P+MRPS22(1) P+MRPS22(2) P+lacZ

b 160

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C C+MRPS22 P P+MRPS22(1) P+MRPS22(2) P+lacZ Figure 4. Restoration of mitochondrial rRNA levels by MRPS22 transfection. Patient (P) and control (C) fibroblasts were transfected with a viral vector carrying the wild-type MRPS22 cDNA. Stably transfected clones (C + MRPS22, P+MRPS22(1) and P+MRPS22(2)) were isolated by antibiotic selection. Patient cells were also transfected with a control vector (lacZ). 12S rRNA and 16S rRNA levels were quantified relative to ß-actin and presented relative to control values as mean percentages ± SD (a, b) or as the relative ratios of 12S rRNA:16S rRNA ± SD (c) of triplicate determinations. *Significantly different from the control (P<0.05).

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Table 2. Restoration of complex IV activity and amount by MRPS22 transfection Activities (mU/U citrate synthase)0 Amountb Sample Complex IV Complex II Complex IV Control 2230 190 2.49 Control + MRPS22 1780 190 3.35 Patient 890 230 1.26 Patient+MRPS22(1)c 2440 190 2.88 Patient+MRPS22(2)c 2630 250 2.99 Patient+lacZ 650 240 0.40 a Measured in fibroblast homogenates. b Expressed relative to frataxin. c Stably transfected Patient+MRPS22 clones 1 and 2.

After excluding mutations in the mtDNA, polymerase gamma (POLG and POLG2) and all nine known mitochondrial translation factors (MTIF2, MTIF3, TSFM, TUFM, GFM1, GFM2, MTRF1, MTRF1L and MRRF) by direct sequencing, we used homozygosity mapping to guide further investigations. Multiple homozygous regions were found, containing mitochondrial ribosomal proteins, among others. On the basis of the mitochondrial translation deficit in patient fibroblasts and previously reported mutations, we selected MRPS16 (located in the largest homozygous region, spanning 110.9 Mb on chromosome 10) and MRPS22 (located in a region of 16.1 Mb between SNPs rs977683 and rs1112189 on chromosome 3) as plausible candidate genes. Sequence analysis revealed a Chapter homozygous T to C transition at position 644 of the coding sequence of MRPS22, which is predicted to result in the substitution of the highly conserved leucine 8 for proline at position 215 (Figure 3a and b). The mutation was heterozygous in both parents and absent in 109 ethnically matched controls. As MRPS22 has no ortholog in bacteria and no crystal structure exists for modeling, we used the PSIPRED server to predict the secondary structure of the MRPS22 protein.26 The mutation seems to be located in the middle of a helix (Figure 3c) and presumably disturbs it because of the lack of stabilizing hydrogen bond formation by proline and the possible introduction of a kink. The homology-based tools PolyPhen (Polymorphism Phenotyping, http://genetics.bwh.harvard.edu/pph/index.html)27 and SIFT (Sorting Intolerant From Tolerant, http://sift.jcvi.org/)28 were applied to indicate the functional impact of the missense mutation. Both PolyPhen and SIFT predicted the mutation to be deleterious (with a PSIC score difference of 2.243 and a score of 0.00, respectively). Western blotting revealed the (near) absence of MRPS22 in patient fibroblasts (Figure 3d). To investigate whether the MRPS22 deficit causes the OXPHOS system deficiency, we introduced wild-type MRPS22 cDNA into patient cells. The complex IV enzyme activity increased from 40% to 109-118% and its protein level went from

184 Mitochondrial ribosomal protein MRPS22 mutation

51% to 116-120% of the control values (Table 2). Quantification of mitochondrial rRNA levels showed a restoration of 12S and 16S rRNA content from 36 and 77% to 71-109% and 70-120% of control levels (Figure 4a and b), respectively, resulting in normal 12S rRNA/16S rRNA ratios (Figure 4c). Complex IV activity and amount, as well as rRNA content, were decreased after transfection of patient fibroblasts with a control vector. Subsequently, genes known to be implicated in Cornelia de Lange syndrome were screened for mutations to shed light on the genetic origin of the Cornelia de Lange-like dysmorphic features. No mutations were found in the genes NIPBL, SMC1A and SMC3. In addition, analysis of patient DNA on the 250K NspI SNP array revealed a normal male profile, thus excluding any potentially clinically relevant copy number variants.

D i s c u s s i o n This study confirms the importance of mitochondrial ribosomal protein MRPS22 for mitochondrial translation and consequently for the formation and functioning of the OXPHOS system. We described the second mutation in MRPS22 and determined its pathogenic role in the patient's mitochondrial disorder. The combined OXPHOS complex I, III and IV deficiency, impairment of mitochondrial translation and absence of mutations in the mtDNA in our patient suggested that the genetic defect was located in a nuclear gene encoding a constituent of the mitochondrial translation machinery. Sequence analysis of all mitochondrial translation factors followed by homozygosity mapping led to Chapter the identification of a homozygous missense mutation in MRPS22, predicted to substitute a highly conserved leucine for proline (Leu215Pro). In patient 8 fibroblasts, the MRPS22 protein was undetectable and expressing the wild-type MRPS22 cDNA rescued the disease phenotype, evinced by the restoration of OXPHOS complex IV amount and activity, as well as 12S rRNA transcript level, establishing the MRPS22 defect as the cause of the disease. Human MRPS22 is a 360-amino-acid protein that has been found in a limited number of species, having no ortholog in fungi or prokaryotes.29 The exact location of MRPS22 in the three dimensional structure of the ribosome and its exact function are currently unknown. Recently, a MRPS22 defect was shown to strongly hamper assembly of the small ribosomal subunit, whereas assembly of the large subunit and part of the small subunit were only mildly affected.30 This is consistent with both our and previous findings that mutations in mitochondrial ribosomal small subunit proteins cause a marked decrease in the abundance of the 12S rRNA transcript and nearly no reduction in the level of 16S rRNA,67 since rRNAs are readily degraded unless they are incorporated into a ribosomal subunit.31 Thus, although MRPS22 is evolutionary not well conserved, it is indispensable

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for the assembly of the human mitochondrial ribosome and consequently, as we have shown, for effective mitochondrial protein synthesis. Previously, a missense mutation in MRPS22 (Arg170His)7 and a non-sense mutation in MRPS16 (Arg111X),6 which is highly conserved and essential for ribosome assembly,293032 were reported to be lethal. The clinical phenotypes of patients with MRPS16 or MRPS22 mutations bear resemblances. All three mutations are associated with muscle hypotonia, lactic acidemia and edema of the limbs. Furthermore, both MRPS22 defects led to hypertrophic cardiomyopathy. The patient harboring the MRPS22 Arg170His mutation additionally presented with tubulopathy and no brain abnormalities were noted, in contrast to the patient reported in this study. It remains unclear whether the brain abnormalities observed in our patient are primarily due to the genetic defect or developed secondary to intrauterine ischemia; presumably both contributed. The dysmorphic features and brain abnormalities of our patient are similar to those seen in the MRPS16 patient, however, the latter did not develop hypertrophic cardiomyopathy. On the basis of the identification of a MRPS22 mutation and the absence of mutations or copy number variations responsible for the Cornelia de Lang-like symptoms in our patient, we advise to perform echocardiography, metabolic screening (plasma lactate, plasma amino acid analysis and urinary organic acid analysis) and cerebral MRI in patients with dysmorphic features suggestive of Cornelia de Lange syndrome. Notably, in our patient these features were quite prominent at birth but diminished over the years. Chapter The patient reported in this study was part of a cohort of 33 patients with combined OXPHOS deficiencies. In seven other patients from this cohort, the 8 causative genetic defect was found. Mutations were identified in the mitochondrial tRNALys, tRNAArg and tRNATrp genes (four patients),33 POLG (one patient) and translation factors TSFM and GFM1 (two patients), including three mutations that had not been published before. Our findings emphasize that nuclear gene mutations contribute substantially to mitochondrial translation deficits. Vastly exceeding 100 proteins, among which 81 mitochondrial ribosomal proteins,29 are (in)directly involved in the mitochondrial translation process, whereas in merely nine of these proteins mutations have been found up till now. Therefore, many more such defects are expected to be discovered. Progress in high-throughput sequencing technologies will hopefully make the difficult task of selecting the right candidate genes obsolete. In the meantime, supernumerary proteins such as MRPS22, lacking homologs in bacteria and other lower species, should not be disregarded during investigations of mitochondrial disorders as they evidently perform important roles.

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A cknowledgements This work was supported by the European Union's Sixth Framework Program, contract number LSHMCT-2004-005260 (MITOCIRCLE), and in part by the Association Française contre les Myopathies (AFM) and the Israeli Ministry of Health to AS. We thank Ilse Pronk and Benigna Geurts (Nijmegen Center for Mitochondrial Disorders) for their work on the sequence analysis of MRPS22 and Hanka Venselaar (Center for Molecular and Biomolecular Bioinformatics, Nijmegen) for the secondary structure prediction. Maleeha Azam and Dr Raheel Qamar (COMSATS Institute of Information Technology, Islamabad) are acknowledged for providing the Pakistani control samples. We are grateful to Dr Michael Marusich (MitoSciences, OR, USA) for supplying the MitoProfile assay kit. Finally, Dr Avraham Shaag (Hadassah-Hebrew University Medical Center, Jerusalem) is acknowledged for helpful comments regarding the rRNA quantification.

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R e f e r e n c e s 1. MITOMAP: A Human Mitochondrial Genome Database. http://www.mitomap.org, 2009. 2. Antonicka H, Sasarman F, Kennaway NG, Shoubridge EA: The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1. Hum Mol Genet 2006; 15: 1835-1846. 3. Coenen MJ, Antonicka H, Ugalde C et al: Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency. N Engl J Med 2004; 351: 2080 2086. 4. Smeitink JA, Elpeleg O, Antonicka H et al: Distinct clinical phenotypes associated with a mutation in the mitochondrial translation elongation factor EFTs. Am J Hum Genet 2006; 79: 869-877. 5. Valente L, Tiranti V, Marsano RM et al: Infantile encephalopathy and defective mitochondrial DNA translation in patients with mutations of mitochondrial elongation factors EFG1 and EFTu. Am J Hum Genet 2007; 80: 44-58. 6. Miller C, Saada A, Shaul N et al: Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation. Ann Neurol 2004; 56: 734-738. 7. Saada A, Shaag A, Arnon S et al: Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation. J Med Genet 2007; 44: 784-786. 8. Edvardson S, Shaag A, Kolesnikova O et al: Deleterious mutation in the mitochondrial arginyl-transfer RNA synthetase gene is associated with pontocerebellar hypoplasia. Chapter Am J Hum Genet 2007; 81: 857-862. 9. Scheper GC, van der Klok T, van Andel RJ et al: Mitochondrial aspartyl-tRNA synthetase 8 deficiency causes leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation. Nat Genet 2007; 39: 534-539. 10. Isohanni P, Linnankivi T, Buzkova J et al: DARS2 mutations in mitochondrial leukoencephalopathy and multiple sclerosis. J Med Genet 2010; 47: 66-70. 11. Bykhovskaya Y, Casas K, Mengesha E, Inbal A, Fischel-Ghodsian N: Missense mutation in pseudouridine synthase 1 (PUS1) causes mitochondrial myopathy and sideroblastic anemia (MLASA). Am J Hum Genet 2004; 74: 1303-1308. 12. Fernandez-Vizarra E, Berardinelli A, Valente L, Tiranti V, Zeviani M: Nonsense mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by myopathy, lactic acidosis and sideroblastic anaemia (MLASA). J Med Genet 2007; 44: 173-180. 13. Zeharia A, Shaag A, Pappo O et al: Acute infantile liver failure due to mutations in the TRMU gene. Am J Hum Genet 2009; 85: 401-407. 14. Robinson BH: Use of fibroblast and lymphoblast cultures for detection of respiratory chain defects. Methods Enzymol 1996; 264: 454-464.

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15. Janssen AJ, Smeitink JA, van den Heuvel LP: Some practical aspects of providing a diagnostic service for respiratory chain defects. Ann Clin Biochem 2003; 40: 3-8. 16. Smeitink J, Sengers R, Trijbels F, van den Heuvel L: Human NADH:ubiquinone oxidoreductase. J Bioenerg Biomembr 2001; 33: 259-266. 17. Boulet L, Karpati G, Shoubridge EA: Distribution and threshold expression of the tRNA(Lys) mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF). Am J Hum Genet 1992; 51: 1187-1200. 18. Schagger H, von Jagow G: Blue Native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 1991; 199: 223-231. 19. Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215. 20. Matsuzaki H, Loi H, Dong S et al: Parallel genotyping of over 10,000 SNPs using a one-primer assay on a high-density oligonucleotide array. Genome Res 2004; 14: 414 425. 21. Nannya Y, Sanada M, Nakazaki K et al: A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays. Cancer Res 2005; 65: 6071-6079. 22. McMullan DJ, Bonin M, Hehir-Kwa JY et al: Molecular karyotyping of patients with unexplained mental retardation by SNP arrays: a multicenter study. Hum Mutat 2009; 30: 1082-1092. 23. Calvaruso MA, Smeitink J, Nijtmans L: Electrophoresis techniques to investigate defects in oxidative phosphorylation. Methods 2008; 46: 281-287. Chapter 24. Ugalde C, Vogel R, Huijbens R, van den Heuvel B, Smeitink J, Nijtmans L: Human mitochondrial complex I assembles through the combination of evolutionary 8 conserved modules: a framework to interpret complex I deficiencies. Hum Mol Genet 2004; 13: 2461-2472. 25. Saada A, Edvardson S, Rapoport M et al: C6ORF66 is an assembly factor of mitochondrial complex I. Am J Hum Genet 2008; 82: 32-38. 26. Jones DT: Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 1999; 292: 195-202. 27. Ramensky V, Bork P, Sunyaev S: Human non-synonymous SNPs: server and survey. Nucleic Acids Res 2002; 30: 3894-3900. 28. Ng PC, Henikoff S: SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res 2003; 31: 3812-3814. 29. Smits P, Smeitink JA, van den Heuvel LP, Huynen MA, Ettema TJ: Reconstructing the evolution of the mitochondrial ribosomal proteome. Nucleic Acids Res 2007; 35: 4686-4703.

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30. Emdadul HM, Grasso D, Miller C, Spremulli LL, Saada A: The effect of mutated mitochondrial ribosomal proteins S16 and S22 on the assembly of the small and large ribosomal subunits in human mitochondria. Mitochondrion 2008; 8: 254-261. 31. Dennis PP, Young RF: Regulation of ribosomal protein synthesis in Escherichia coli B/r. J Bacteriol 1975; 121: 994-999. 32. Cavdar KE, Burkhart W, Blackburn K, Moseley A, Spremulli LL: The small subunit of the mammalian mitochondrial ribosome. Identification of the full complement of ribosomal proteins present. J Biol Chem 2001; 276: 19363-19374. 33. Smits P, Mattijssen S, Morava E et al: Functional consequences of mitochondrial tRNA Trp and tRNA Arg mutations causing combined OXPHOS defects. Eur J Hum Genet 2010; 18: 324-329.

Chapter 8

190 Mitochondrial ribosomal protein MRPS22 mutation

Chapter 8

191 Most of our so-called reasoning consists in finding arguments for going on believing as we already do. James Harvey Robinson C h apter 9 General discussion and future perspectives

«• Chapter 9

C o m b i n e d o x p h o s deficiencies - h o w t o u n r a v e l t h e i r g e n e t i c ORIGIN? This thesis tackles the difficult task of unraveling the molecular genetic defects underlying combined oxidative phosphorylation (OXPHOS) complex I, III and IV deficiencies. Combined OXPHOS deficiencies are a subset of diseases generally known as mitochondrial disorders, which are frequent and often debilitating diseases that can lead to death early in life. Considering that mitochondrial function depends on thousands of genes, encoded by the mitochondrial (mt) as well as the nuclear (n) genome, and the intricate relationships between all proteins involved, it is hardly surprising that in many cases the genetic cause of the mitochondrial disorder remains unknown. Besides the genetic complexity and heterogeneity among patients, the general lack of a genotype-phenotype correlation also complicates the molecular diagnosis. Especially when multiple complexes of the OXPHOS system are affected, locating the pathogenic mutation is a daunting task. We aimed to identify new nuclear gene defects that cause combined OXPHOS complex I, III and IV deficiencies, with the focus on deficiencies originating from mitochondrial translation defects, to improve diagnostic possibilities, our understanding of underlying disease mechanisms, and hopefully curative therapy in the future.

For this purpose, we assembled a cohort of 33 patients (31 children and two adults) with decreased enzyme activities of complex I, III and IV - and normal complex II activities - in fibroblasts and/or muscle tissue. After mtDNA mutations were excluded, 29 patients remained for mutational analysis of nuclear genes. Genes known to be implicated in combined OXPHOS deficits as well as new candidate genes were screened, leading to a total of 30 genes. This revealed pathogenic Chapter mutations in 'known' nuclear genes (POLG, TSFM, GFM1 and MRPS22) in four patients, including two not previously reported mutations. No clearly deleterious 9 genetic defects were found in the remaining 25 patients. The results of these studies will be discussed in more detail and reflected upon in this chapter.

G e n e t i c d e f e c t s u n d e r l y i n g c o m b i n e d o x p h o s c o m p l e x I, III AND i v deficiencies At present, mutations resulting in mitochondrial disease have been identified in over 100 different mitochondrial and nuclear genes.1 The number of genes implicated in combined OXPHOS deficiencies is much lower but is growing rapidly. The mitochondrial tRNA genes and POLG contain a large proportion of the mutations currently known to cause combined OXPHOS deficits, but in 2004 the first mutation in one of the mitochondrial translation factors was

194 General discussion and future perspectives identified,2 defining a new class of gene defects underlying these disorders. In addition to the mitochondrial rRNA and tRNA genes, there are now 21 nuclear genes - required for nucleotide synthesis or transport, mtDNA replication, or mitochondrial translation - reported to be responsible for combined complex I, III and IV defects.3 Many more disease genes are expected and needed to be found to explain the numerous as yet unsolved cases of combined deficiencies.

Mutational analysis of mitochondrial DNA First of all, we screened the entire mitochondrial genome for mutations in our cohort of 33 patients with combined complex I, III and IV deficiencies. This revealed pathogenic tRNA mutations in four patients. It is possible, however, that heteroplasmic mtDNA mutations were missed because the affected tissue was not investigated. Approximately a quarter of all patients had normal OXPHOS enzyme activities in fibroblasts, and thus the mtDNA mutation load is expected to be low and perhaps undetectable in this tissue. Nevertheless, for all patients mutational analysis was performed in DNA isolated from cultured fibroblasts since the amount of muscle tissue available was often insufficient for DNA extraction. Two patients harbored a mutation in tRNALys (MT-TK), m.8363G>A, which has previously been associated with a variety of clinical manifestations.4 Mitochondrial protein synthesis was slightly decreased in fibroblasts of both patients. Furthermore, an extra band appeared below the ND4 polypeptide, which has also been reported for the common m.8344A>G tRNALys mutation and which has been identified as a truncated COXI polypeptide arisen from premature termination of translation.5 In two other patients, novel tRNATrp (MT-TW) and tRNAArg (MT-TR) mutations were detected. Chapter 3 demonstrates that these mutations affect the conformation and steady-state levels of the tRNAs, causing mitochondrial translation impairment and consequently combined OXPHOS deficiencies. After Chapter publication of this paper, three new mutations in tRNATrp have been reported.6,7 These findings emphasize the rapid progress that is still being made in 9 the identification of mitochondrial tRNA mutations and that such mutations are important contributors to mitochondrial disease. What should be noted, however, is that a considerable proportion of the reported tRNA mutations are not supported by sufficient evidence to be regarded as pathogenic.8,9 On the other hand, the tRNATrp mutation that we clearly demonstrated to be deleterious in our patient was previously listed as a polymorphism. Scoring systems8,9 could be used to determine whether additional studies, such as analysis of the effect on tRNA stability and structure, are required to properly classify a nucleotide change in a mitochondrial tRNA gene.

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Mutational analysis of nuclear DNA Polymerase gamma Subsequently, we performed sequence analysis of POLG and POLG2, the genes coding for the catalytic and accessory subunits of the mtDNA polymerase, respectively. With over 150 deleterious mutations, POLG is a major locus for a wide range of mitochondrial disorders.10 POLG mutations are associated with a variety of OXPHOS enzyme deficits as well as with an entirely normal OXPHOS system; generally, multiple enzymes (sometimes including complex II) are affected in muscle tissue, whereas in fibroblasts often normal enzyme activities are seen.11,12 The most prevalent POLG mutation, Ala467Thr,10 was found compound heterozygous with Gly848Ser in one of our patients, who showed symptoms of Alpers syndrome. In patients with Alpers syndrome, Ala467Thr is the most frequent mutation and Gly848Ser is very common as well.13 This patient showed profound combined OXPHOS complex I, III and IV deficiencies in muscle tissue, in contrast with previous reports on such cases.12,14 Three other patients from our cohort also harbored formerly described mutations in POLG, Asn468Asp (1 patient) and Gly517Val (2 patients), but these mutations were heterozygous without a second mutation being detected. The Asn468Asp variant has been presented as a recessive mutation, yet the pathogenicity of the mutation is unclear: patients were found to be compound heterozygous and healthy family members were found to be carriers, suggesting that the other heterozygous mutation resulted in dominant disease.15,16 In cases where Asn468Asp is the only POLG mutation, as in our patient, it is possible that the disease-causing mutation is actually located in another gene.17 Thus, Asn468Asp may just be a neutral polymorphism or a disease-modifying mutation. Nonetheless, one report showed dominant cosegregation of Asn468Asp.18 A Chapter similar situation applies to the Gly517Val mutation, which was originally published as a dominant mutation19 but is now viewed as an unclassified sequence variant 9 with unknown clinical relevance.1720 Therefore, the Asn468Asp and Gly517Val mutations were considered not to be responsible for the mitochondrial disorder in these three patients. To exclude deletion of one or more POLG exons on one allele, which could be missed by standard sequencing methods and could lead to disease on its own or in combination with a (recessive) mutation on the other allele, multiplex ligation-dependent probe amplification (MLPA) should still be performed for all patients. This is a multiplex PCR technique that can be used to detect the relative copy number of all exons within a gene.21 Furthermore, one possibly pathogenic and not previously reported sequence variant was detected: Pro635Leu. This mutation was heterozygous on both DNA and RNA level in one patient and not found in 97 healthy controls. It is located in the middle of the linker region of POLG, which is involved in binding the accessory

196 General discussion and future perspectives subunit POLG2. The homology-based tools PolyPhen (Polymorphism Phenotyping, http://genetics.bwh.harvard.edu/pph/index.html)22 and SIFT (Sorting Intolerant From Tolerant, http://sift.jcvi.org/)23 were applied to indicate the functional impact of the missense mutation. Both PolyPhen and SIFT predicted the mutation to be deleterious (with a PSIC, Position-Specific Independent Counts, score difference of 2.342 and a score of 0.04, respectively), but the reliability of the predictions is decreased due to the limited number of sequences represented at that position. Additional research, among which family history to assess the possibility of autosomal dominant inheritance, MLPA and mtDNA depletion/deletion screening, is called for to determine the significance of this mutation. Subsequently, the effect of the mutation on POLG functions such as catalytic activity, DNA binding ability and interaction with the accessory subunit could be analyzed.24 Pro635Leu is a neutral sequence variant or a deleterious mutation, either dominant or recessive, with in the latter case either an intronic mutation on the other POLG allele, the absence of (part of) the other allele, or a mutation in a related gene such as the mtDNA helicase, resulting in digenic disease.25 No mutations were found in POLG2 in our patient group. To date, two mutations in POLG2 have been described to cause mitochondrial disease, in particular progressive external ophthalmoplegia (PEO).26,27 The POLG mutation history illustrates the importance of critical evaluation of mutation reports as the mode of inheritance and clinical relevance cannot always be established accurately without analysis of multiple families, and even then the role of certain mutations remains unclear, as is the case for above mentioned Asn468Asp and Gly517Val mutations. Therefore, mutations should be revisited and if necessary reclassified in the light of new findings until they are fully characterized, especially in such a fast moving field with over 150 mutations discovered in just a decade. Much is still to be learned about the currently known Chapter POLG and POLG2 mutations and those awaiting identification. 9 Mitochondrial translation factors Up to date, three out of nine mitochondrial translation factors have been implicated in human mitochondrial disease. The first two mutations in mitochondrial translation factors, GFM1 and TSFM, were detected at our center but in different patient cohorts than the one that is subject of this thesis (TSFM: see chapter 4).2 Subsequently, the first mutation in TUFM and additional mutations in GFM1 were identified.2829 All nine mitochondrial translation factors that have been characterized - MTIF2, MTIF3, TSFM, TUFM, GFM1, GFM2, MTRF1, MTRF1L and MRRF - were screened for mutations in our patient group. One patient harbored the Arg333Trp mutation in elongation factor mtEFTs (TSFM) that was published earlier (see chapter 4 for the

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first report and see the discussion section of chapter 5 for a note on the finding in the current patient cohort). This mutation caused severe mitochondrial disease leading to death within the first few months after birth in three unrelated patients. The phenotypic expression of the mutation was remarkably different between patients. Furthermore, we identified a new mutation in elongation factor mtEFG1 (GFM1) and demonstrated its role in the combined OXPHOS deficiency and mitochondrial translation defect that ultimately resulted in fatal mitochondrial encephalopathy in one patient (chapter 5). In stark contrast to previously described GFM1 cases, decreased enzyme activities were detected only in fibroblasts and not in muscle tissue of this patient, underscoring that comprehensive biochemical investigation is critical in the diagnosis of mitochondrial disease. The reports on mutations in mitochondrial translation factors emphasize the heterogeneity and complexity of mitochondrial disorders: a) an identical mutation, e.g. Arg333Trp in TSFM, can result in strikingly different clinical presentations in individual patients; b) tissues are selectively affected despite ubiquitous expression of the translation factors; c) in which tissues the biochemical defect manifests itself can differ between patients with distinct mutations in the same gene, e.g. GFM1. Currently, one can primarily speculate on the causes for the biochemical and clinical variability associated with mutations in mitochondrial translation factors. Even though the core mitochondrial protein synthesis machinery has been characterized (see chapter 2), much remains to be unraveled concerning details of the mechanisms of mitochondrial translation, the exact function of some of the proteins involved, and especially regulation of the entire system. Antonicka et al showed that tissue-specific differences exist not only in the basal mitochondrial translation machinery, but also in its response to dysfunction.28 The efficiency of mitochondrial translation partly depends on congruous ratios of the translation Chapter elongation factors (mtEFTs, mtEFTu and mtEFG1) and these relative ratios differ between tissues. In line with this, over-expression of certain translation elongation 9 factors can be beneficial in patient cells but can be detrimental to mitochondrial translation in control cells. Furthermore, the residual level of mutant protein was found to parallel the severity of the OXPHOS deficiency caused by compound heterozygous GFM1 mutations, suggesting it as the major factor in the tissue specificity of the biochemical and clinical phenotypes.28 The differences in mtEFG1 protein expression are likely the result of post-transcriptional regulatory mechanisms as mRNA steady-state levels did not correlate with protein levels in the various tissues. Moreover, changes in the translation factor ratios, for instance through transcriptional upregulation of mtEFTu in heart tissue of a patient with mutations in GFM1, can occur in response to dysfunction of part of the mitochondrial translation apparatus. Such adaptive mechanisms appear to function differently between tissues and plausibly also between patients,

198 General discussion and future perspectives thereby contributing to the observed biochemical and clinical variability. It would be interesting to investigate whether similar observations as the ones reported by Antonicka et al would be made in patients with other GFM1 mutations and with TSFM and TUFM mutations. Therefore, the levels of all OXPHOS complexes and translation elongation factors as well as the effect of over-expression of the elongation factors on these levels and on the rate of mitochondrial protein synthesis should be determined in multiple patient tissues. This could shed more light on, for instance, whether heart indeed uniquely possesses compensatory mechanisms to avoid deleterious effects on mitochondrial translation when one of the proteins involved in this process is impaired, as speculated by Antonicka et al, or whether this was a patient-, mutation- or gene-specific response. The last option seems most probable since mutations in TSFM and MRPS22 have subsequently been associated with hypertrophic cardiomyopathy (chapters 4 and 8), whereas two other GFM1 cases again did not show any signs of heart involvement (chapter 5).29 Other explanations for the biochemical and clinical variability, besides differences in (compensatory changes in) translation factor ratios, could be the presence of genetic modifiers and threshold effects. Genetic modifiers are likely to play a role in the variation among individuals with mitochondrial translation defects, however, they will be difficult to identify because usually large numbers of patients are needed to obtain reliable results. The term threshold effect refers to the finding that genetic defects are manifested only when a threshold value is exceeded.30 These threshold levels vary between individuals, e.g. due to dissimilarities in OXPHOS capacity related to mtDNA haplotypes.31 Threshold effects also differ between tissues within an individual and can occur at various levels: transcription, translation, enzyme complex, biochemical, and cellular, finally resulting in the phenotypic threshold effect. Differences between tissues are based on quantitative properties of the basal Chapter mitochondrial machineries and adaptive mechanisms that are at hand, which together lead to distinct threshold values beyond which pathology ensues. For 9 example, if the basal OXPHOS or mitochondrial translation system, which both have been shown to vary quantitatively between tissues, contains an excess of a protein, this excess could be used as a reserve to compensate for a deficit in that particular system (of either the same protein or another component). Consequently, the threshold value for the amount of mutant protein above which deleterious effects arise would be higher in a tissue with reserves present in that system. To illustrate, over-expression of mtEFTu has been shown to rescue a mtEFTs defect (chapter 4), and interestingly, over-expression of mtEFTu or mtEFG2 can partially suppress a mitochondrial translation deficit due to a mtDNA mutation (m.3243A>G, in tRNALeu).32 Thus, the rate of mitochondrial respiration or translation can be maintained when the activity of an OXPHOS complex,

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translation factor or tRNA is diminished. There are numerous threshold effects, for both mtDNA and nDNA mutations,30 with many of them poorly understood or perhaps still unknown. Further research is required to better comprehend the molecular bases of the various mitochondrial threshold effects and thereby improve our knowledge on the complex regulation of mitochondrial translation. Model organisms such as the long-lived Caenorhabditis elegans mitochondrial mutants could be used to study the compensatory pathways that are activated to delay human mitochondrial disease appearance.33 So far, no pathogenic mutations have been identified in factors involved in the initiation or termination phase of mitochondrial protein synthesis. During the mutational screen in our patient cohort, we came across one sequence variant in mitochondrial initiation factor MTIF2 and two sequence variants in ribosome recycling factor MRRF. All three variations were heterozygous and not detected in 100 control samples. Table 1 shows predictions of the functional consequence of the substitutions. It additionally lists a rare polymorphism (Ser695Gly, rs34174245) that is located at the same position as our MTIF2 sequence variant (Ser695Cys).

Table 1. Predictions of functional impact of substitutions in MTIF2 and MRRF Gene Substitution (Genbank Polyphen SIFT Accession number) PSIC score Predicted effect Score Predicted effect difference MTIF2 Ser695Cys 1.764 Possibly 0.04 Affect protein (NP_001005369.1) damaging function Ser695Gly 0.025 Benign 0.46 Tolerated (NP_001005369.1) MRRF Thr70Ile 1.553 Possibly 0.23 Tolerated (NP_620132.1) damaging Chapter Pro141Ser 1.288 Benign 0.06 Tolerated 9 (NP_620132.1) Both prediction methods, Polyphen and SIFT, clearly designate polymorphism Ser695Gly in MTIF2 as a benign variant and Ser695Cys as a possibly pathogenic mutation. Notably, the patient harboring this mutation has additional heterozygous changes in POLG2, MRPS14 and MRPS22, which suggests digenic disease. This is considered to be unlikely, though, because of the silent nature of the other changes. Based on sequence homology and physical properties of the amino acids, the substitutions in MRRF are predicted not to affect protein function, albeit the slight inconsistency of the two prediction methods. Furthermore, the heterozygous Pro141Ser mutation was found in a patient born of consanguineous parents, making it a less plausible cause for the patient's mitochondrial disorder. The pathogenicity of substitutions Ser695Cys (MTIF2) and Thr70Ile (MRRF) could

200 General discussion and future perspectives be elucidated by screening family members for the presence of the change and investigating the effects of over-expression of the wild-type proteins in patient cells. Unless deleterious mutations in mitochondrial translation initiation or termination factors are incompatible with life, they are good candidates for involvement in mitochondrial translation defects.

Mitochondrial ribosomal proteins Of the more than 80 mitochondrial ribosomal proteins (MRPs) currently known, mutations have been found in merely two of them: MRPS16 and MRPS22.34,35 In principle, all MRPs are candidate genes for mitochondrial disorders. To facilitate the selection of which MRPs to screen for mutations first, we performed extensive homology searches to reconstruct the evolutionary history of the mitochondrial ribosomal proteome across different eukaryotic lineages (chapter 6). MRPs could thereafter be prioritized based on their location and function in the mitochondrial ribosome (mitoribosome) and the outcome of yeast knock-out studies, if this information was available, as well as on their evolutionary conservation. What is more, two new human MRPs were detected in this comparative genomics analysis: CHCHD1 and METT11D1, orthologs of yeast mitoribosomal small subunit proteins MRP10 and RSM22, respectively. These novel MRPs as well as MRPS12, MRPS14, MRPS16, MRPS22, MRPL11, MRPL12 and MRPL45 were subsequently screened for mutations in our patient cohort. This revealed three possibly pathogenic and not previously reported sequence variations in MRPS14, MRPS22 and MRPL11. Chapter 8 reports the mutation in MRPS22 and evinces its pathogenicity by transfection of patient cells with wild-type MRPS22. This study confirms that not only well-conserved core proteins, such as MRPS16, but also phylogenetically less conserved proteins, such as MRPS22, can be implicated in disease. Essential genes are generally more evolutionary conserved than non-essential genes and Chapter consequently, well-conserved proteins are obvious candidates for involvement in disease. Nevertheless, supernumerary proteins, which typically do not show 9 a wide phylogenetic distribution, are functionally important, as has clearly been shown by knock-out studies and mutational reports. When prioritizing proteins for their involvement in mitochondrial disease, one should therefore be careful when using evolutionary conservation as a criterion. Table 2 lists predictions of the functional impact of the substitutions detected in MRPS14 and MRPL11. Both PolyPhen and SIFT predict the change in MRPS14 not to be deleterious, even though the properties of glycine and arginine are considerably dissimilar. This discrepancy may be attributed to the low conservation level of this position, making the predictions less reliable. The substitution is located in the mitochondrial targeting sequence and could thus affect targeting of the protein to the mitochondrion. Mitochondrial translation

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was found to be impaired in patient fibroblasts. However, this sequence variant was heterozygous in a patient born of consanguineous parents, making it a less plausible cause for the patient's mitochondrial disorder. To ascertain its functional importance, additional research is necessary, starting with investigation of control DNAs to determine whether this concerns a polymorphism. The sequence variant in MRPL11 is evidently predicted to affect protein function and was absent from 99 control samples. Nonetheless, the mutation was heterozygous on both DNA and RNA level and did not result in a clear disruption of mitochondrial translation. Introduction of wild-type MRPL11 cDNA into patient cells could shed more light on the pathogenicity of this mutation. No mutations were found in our patient cohort in the other MRP genes that were analyzed (see chapter 7 for the report on CHCHD1 and METT11D1).

Table 2. Predictions of functional impact of substitutions in MRPS14 and MRPL11 Gene Substitution (Genbank Polyphen SIFT Accession number) PSIC score Predicted effect Score Predicted effect difference MRPS14 Gly7Arg 1.478 Benign 0.35 Tolerated (NP_071383.1) MRPL11 Arg142Cys 2.165 Probably 0.00 Affect protein (NP_057134.1) damaging function

Recently, two additional components of the mitoribosome were identified, bringing the total number of human MRPs on 83. Richter et al convincingly showed that ICT1 is actually an integral component of the human mitoribosome, unlike the other members of the release factor family to which ICT1 belongs.36 It Chapter is thought to be essential for hydrolysis of prematurely terminated peptidyl-tRNA species in stalled mitoribosomes, releasing the mitoribosome for recycling. The 9 other members of the mitochondrial release factor family are mtRF1a and mtRF1, both suggested to be class I release factors, and C12orf65. Notably, C12orf65 mutations were detected in patients with encephalopathy and were demonstrated to be responsible for the OXPHOS system and mitochondrial translation defects.37 Although the specific role of C12orf65 in mitochondrial protein synthesis has not yet been elucidated, Antonicka et al speculate that it functions in recycling abortive peptidyl-tRNAs, which would explain why over-expression of ICT1 can partially rescue the C12orf65 defect. The other newly found MRP is ERAL1, a constituent of the small subunit.38 Knock-down of ERAL1 leads to impairments in small subunit assembly, mitochondrial translation and cell growth. In addition to functions in mitoribosome assembly and mitochondrial translation, ERAL1 is also posited to be involved in other processes, including mitochondrial transcription

202 General discussion and future perspectives through association with nucleoid proteins. Furthermore, recent experimental data confirmed that NOA1 is an indispensible GTPase specifically required for mitochondrial protein synthesis, probably through a role in mitoribosome biogenesis.39 NOA1 deficiency was shown to be lethal in mid-gestation in mice, which was accompanied by a severe developmental defect and malfunctioning of mitochondrial translation and all OXPHOS enzymes except for complex II. Interestingly, NOA1 knock-out also hampered mitochondria-dependent apoptosis. This is not surprising given that NOA1 interacts with, among other MRPs, MRPS29 or DAP3 (Death-Associated Protein 3),40 which is an integral part of the mitoribosome and involved in programmed cell death.41 These findings illustrate that our understanding of the mitochondrial translation system is still far from complete and that this system and its relationships to other pathways are probably much more complex than previously anticipated.

Other candidate genes Summarizing, in our cohort of 33 patients with combined OXPHOS complex I, III and IV deficiencies, the causative genetic defect was detected in 4 patients (12%) in the mitochondrial genome and also in 4 patients (12%) in the nuclear genome. Of these patients in whom the genetic cause was elucidated, half harbored mutations that were known and half had mutations that were not reported previously. MtDNA mutations thus appear to account for mitochondrial disease in 12% of patients with combined complex I, III and IV deficits, whereas the remaining 88% is consequently due to nDNA mutations. Since a nuclear gene defect was identified in merely 12% of all cases, 76% of patient disease currently remains unresolved, disregarding the possibly deleterious mutations that were found in some of these cases. In conclusion, hardly one quarter of patients with combined complex I, III and IV deficiencies are genetically diagnosed using the Chapter current strategy. The presently unresolved cases of combined OXPHOS deficiencies in our 9 patient cohort are due to either new mutations in known mitochondrial disease genes or, which is likely the larger contributor, mutations in genes that have not yet been linked with mitochondrial disease. Many candidate genes exist for mitochondrial disorders in general and combined OXPHOS deficiencies in particular. When multiple OXPHOS enzymes are affected, the genetic defect is presumed not to be located in genes encoding OXPHOS subunits but rather in genes needed for mtDNA maintenance, mitochondrial transcription (including transcript processing), mitochondrial translation, import of nDNA-encoded proteins into the mitochondrion, quality control, processing of mitochondrial proteins, insertion of OXPHOS components into the inner membrane, mitochondrial membrane biogenesis, or assembly of OXPHOS supercomplexes

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(see Chapter 2 for more details). When OXPHOS complex II activities are normal, the genetic defect should be sought mainly in the first three gene classes as the others theoretically influence all OXPHOS complexes. Several candidate genes, in addition to the ones mentioned in the sections above, were screened for mutations in the current study: two genes that code for presumed nucleoid proteins (NIPSNAP1 and GBAS, chapter 7), 7 transcription factors (POLRMT, TFAM, TFB1M, TFB2M, MTERF1, MTERFD1 and MTERFD3), and OXA1L, which facilitates the insertion of proteins from the mitochondrial matrix into the inner membrane. Unfortunately, no pathogenic sequence variations were found in any of these genes in our patient cohort. This could be attributed to 1) erroneous inclusion of patients in the cohort, 2) the genes not being good candidates for combined complex I, III and IV deficiencies, 3) the genetic defect not being perceived, or 4) the multitude of other genes that could theoretically harbor the disease-causing mutation and the scarcity of mutations in most disease genes. These points, except for number 2, of course also apply to the genes screened before. First of all, patient selection. In several patients that were included in the cohort, not all OXPHOS enzyme activities were measured. Complex II was missing in most of these cases, although a normal activity of this enzyme is actually an inclusion criterion. In some other patients, complex II was (slightly) decreased. Its diminished activity could simply be secondary to the combined OXPHOS deficiency via disturbance of supercomplex formation, however, many supercomplexes appear to not contain complex II.42,43 Furthermore, conflicting results were obtained in three patients: when repeating the measurements, normal activities of (nearly) all complexes were found. Considering the above, it is not unlikely that some patients were erroneously included in the study. And as mentioned before, there is still a small chance that the mitochondrial Chapter genome harbors the deleterious mutation since heteroplasmic mtDNA mutations might have been overlooked. Secondly, let us evaluate whether the candidate 9 genes were selected appropriately. The potential roles of NIPSNAP1 and GBAS in mtDNA maintenance have not yet been substantiated by functional studies. NIPSNAP1 has been suggested to be essential for OXPHOS complex I based on a large-scale proteomics and bioinformatics study44 and more specifically to be required for complex I assembly using protein correlation profiling.45 The involvement of GBAS in ATP production has recently been confirmed via knock down experiments, however, the enzyme activities of complexes I and IV were not affected by GBAS knock-down and so the exact role of GBAS in oxidative phosphorylation remains to be ascertained.46 Consequently, NIPSNAP1 and GBAS are possible but not definite candidate genes. Although the mechanisms of mitochondrial transcription - transcription termination in particular - have not been fully elucidated, the transcription machinery components that were

204 General discussion and future perspectives analyzed for mutations are evidently needed to maintain adequate transcript levels and are thus good candidates for association with combined OXPHOS deficiencies.47 The potential function of MTERFD2, which was not included in our screen, in transcription termination still awaits determination. Most of the knowledge on OXA1L is based on studies in yeast. Knock-down of OXA1L in human cells disrupted the assembly and activity of OXPHOS complexes I and V but surprisingly did not adversely affect complexes III and IV.48 Furthermore, since OXA1L mediates membrane insertion of not only proteins synthesized in the mitochondrion but also nuclear-encoded proteins, OXA1L might not be the best candidate for the current patient cohort, considering the normal activities of complex II. Explanation 3 - that the defect is located in a gene that was subjected to mutational analysis, but that this defect did not get identified - is a reasonable cause for the seemingly absence of mutations. Using standard PCR and DNA sequencing methods, heterozygous deletions of (part of) a gene can be missed. This can be overcome by performing MLPA for all genes.21 In addition, besides the splicing signals at the exon-intron boundaries, which are usually included in the mutational screen of a gene, an extensive array of intronic and exonic splicing enhancer and suppressor elements are necessary for proper splicing.4950 Intriguingly, 15% up to 60% of disease-causing mutations are estimated to disrupt splicing. Sequencing of the open reading frame and exon-intron boundaries is not sufficient to exclude deleterious mutations in a gene as mutations in intronic splicing signals are thereby missed. Furthermore, synonymous and non- synonymous mutations can have severe effects on the structure of the encoded protein when they inactivate an exonic splicing enhancer (ESE), which will result in exon skipping and thus a profound effect on protein level. Therefore, when predicting the effect of an exonic mutation, one should always assess the possible consequences on pre-mRNA processing, either by computational prediction Chapter or better yet by RT-PCR analysis. Lastly, we fail to identify the disease-causing mutation in more than half of patients with mitochondrial disorders partly as a 9 result of the vast number of genes required for mitochondrial function. There is a large degree of genetic heterogeneity among patients, each mutation accounting for disease in often not more than a few families. Additionally, due to the lack of a clear genotype-phenotype correlation in most cases, which candidate genes to investigate in a patient is not always easily determined and generally many genes need to be analyzed before a genetic diagnosis can be made. Moreover, as our comprehension of the genes related to the function of mitochondria is far from complete, a substantial number of patients remain without a molecular diagnosis merely because we are not aware of the role in oxidative phosphorylation for the gene in which they harbor mutations. Before discussing the evidently needed expansion of our knowledge of the genes associated with mitochondrial function,

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the candidate genes for combined OXPHOS deficiencies that are presently known will be further considered. As previously indicated, all genes necessary for mtDNA maintenance, mitochondrial transcription (including transcript processing) and mitochondrial translation are the most likely candidates for involvement in combined complex I, III and IV deficits. These include all genes that were screened in the current study, except for OXA1L, as well as the many other genes related to these processes (see Chapter 2). The list of candidate genes continues to grow as the functions of more and more genes are being elucidated. The above-mentioned newly identified components of the mitochondrial translation machinery are good examples.37,36,38,39 Several other genes that were recently demonstrated to play a role in mitochondrial protein synthesis will be highlighted below (see also Table 3). First of all, mitochondrial methyltransferase HEMK1 was shown to methylate a glutamine residue in the region of translation termination factor MTRF1L that interacts with the mitoribosomal peptidyl transferase center.51 It is thereby advantageous for mitochondrial translation, especially when cells are exposed to stress. Secondly, during mitochondrial translation initiation, N-terminal formylation occurs. Downstream processing of nascent mitochondrial translation products involves a peptide deformylase (PDF) that removes the formyl group and a methionine aminopeptidase (METAP1D) that subsequently excises the initial methionine residue.52 Inhibition of PDF caused disruption of mitochondrial protein synthesis, OXPHOS assembly and ATP production,53 and similar results are expected for METAP1D dysfunction. Third, 5S rRNA forms an essential part of the large (mito)ribosomal subunit in many organisms. Nuclear-encoded 5S rRNA was found to be present in human mitochondria,54 however, it has only been detected in cytoplasmic ribosomes and not in mitoribosomes. Part of the cytoplasmic 5S Chapter rRNA molecules are imported into human mitochondria and their mitochondrial levels are comparable with the predicted average number of mitoribosomes.55 9 The precise location and function of 5S rRNA inside mitochondria remains unclear, but the significance for mitochondrial translation was demonstrated by silencing the gene coding for thiosulfate sulfurtransferase (rhodanese), which is required for import of 5S rRNA.56 A change in rhodanese expression caused proportional changes in 5S rRNA import as well as in mitochondrial translation rates. Notwithstanding the fast pace at which the intricacies of mitochondrial biology and disease are being unraveled, many areas still remain obscure or even unexplored. There is a great need to unequivocally identify all requisite factors and to better understand the regulation and interrelations of all processes ultimately related to oxidative phosphorylation.

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Table 3. Recently identified candidate genes for combined complex I, III and IV deficiencies Gene Protein (Predicted) function ICT1 Peptidyl-tRNA hydrolase Mitoribosomal large subunit protein; hydrolysis ICT1, mitochondrial of prematurely terminated peptidyl-tRNA species f C 2 0 6 1 r 5 Probable peptide chain Recycling abortive peptidyl-tRNAs release factor C12orf65, mitochondrial ERAL1 GTP-binding protein Mitoribosomal small subunit protein; involvement era homolog in transcription through association with nucleoid proteins C4orf14 Mitochondrial nitric Mitoribosome biogenesis; apoptosis oxide synthase (NOA1) HEMK1 HemK methyltransferase Methylation of translation termination factor family member 1 MTRF1L PDF Peptide deformylase Removal of the formyl group from the initiating (mitochondrial) methionine of nascent peptides METAP1D Methionine aminopeptidase Removal of the initial methionine residue type 1D (mitochondrial) of nascent peptides TST Thiosulfate sulfurtransferase Import of 5S rRNA into mitochondria (rhodanese)

F u t u r e perspectives This thesis demonstrates that locating the disease-causing mutation in patients with combined OXPHOS complex I, III and IV deficiencies remains a complicated and laborious undertaking in many cases. The current approach yielded up till now elucidation of the genetic defect in 8 out of 33 patients, including 4 novel mutations. Chapter It allows for focus on the most promising genes and for a rapid reaction to reports on newly identified candidate genes. However, there are several negative aspects, making it not the favored approach for future research. First of all, our patient cohort 9 was rather heterogeneous: patients with deficiencies in either fibroblasts or muscle tissue or in both, with often not all enzyme activities being measured. Besides more stringent selection criteria for the OXPHOS enzyme activities, analysis of the mitochondrial translation rate - which was performed in approximately half of the present cohort, either to direct mutational analysis or to indicate the pathogenicity of a mutation - could be used as an additional selection step. Thereby, a better focus on deficiencies originating from mitochondrial translation defects could be achieved and thus the number of candidate genes could be limited. On the other hand, implementation of this extra criterion could turn out problematic because of the need for multiple controls per translation assay and/or repeated assays to be

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able to properly assign the ambiguous cases. In addition, it would surely lead to exclusion of patients that do harbor mutations in genes implicated in mitochondrial translation since, for instance, tRNA mutations do not always result in a clearly measurable translation defect.5758 Further drawbacks of the approach that was used for this thesis are the facts that it is quite time-consuming and that a relatively small number of cases gets resolved. This is due to the multitude of candidate genes for combined OXPHOS defects (with many yet to be identified), the paucity of common mutations in most of these genes, the poor genotype-phenotype relationships (complicating the selection of genes for mutational analysis), and the inability to detect copy number variations and intronic mutations (except for those bordering exons). Linkage analysis or homozygosity mapping could facilitate disease-gene discovery, as demonstrated in chapter 8, but usually homozygosity mapping is not an option because of the lack of consanguineous families in Northern Europe. And lastly, perhaps the major disadvantage is that uncharacterized genes that may function in OXPHOS will not be identified via this research strategy. The elucidation of the complete human genome and the considerable progress in high-throughput DNA sequencing platforms open up new avenues for future research into the molecular genetic origins of mitochondrial diseases. Next-generation sequencing technologies permit more rapid and cost-effective sequencing of larger DNA volumes in comparison with the commonly used Sanger method59,60 and thus overcome many drawbacks of the research strategy used for this thesis. They have proven effective by detecting defects in genes not previously predicted to be involved in disease pathology.6162 Despite the major advances in this field in recent years, there still remains much to be accomplished. Most importantly, further cost reductions and improvements in bioinformatics tools63 that can cope with and interpret the huge quantities of sequence data are Chapter needed for high-throughput sequencing to become a feasible option for routine mutation detection. Targeted sequencing, e.g. of all protein-coding regions or 9 exomes,64 markedly curtails costs and the amount of data generated compared with whole-genome sequencing. Eventually, these powerful sequencing technologies will enable complete genome sequencing as a standard approach to disease-gene discovery. The key hurdle will then be the interpretation of data: the great natural variation within the human population makes it difficult to establish which of the many mutations in an individual patient is causative. Large-scale DNA sequencing will thus shift the focus from prioritizing candidate genes for sequencing to prioritizing sequence variants for functional follow-up. In the coming couple of decades, the traditional strategies to uncover nuclear OXPHOS disease genes - i.e. sequencing candidate genes sequentially by the Sanger method, if possible based on data from linkage analysis or homozygosity mapping - are anticipated to become obsolete. The accumulating data obtained

208 General discussion and future perspectives in the increasing number of high-throughput sequencing projects should permit better differentiation between benign and deleterious variations. Additionally, advances in the field of systems biology will be of great value. Systems biology has enabled the development of catalogs that integrate data from multiple sources to systematically characterize the complement of proteins that comprise the mitochondrial proteome.65 Currently, the most comprehensive mitochondrial proteome databases are MitoCarta and MitoMiner, with the latter containing a total of 3025 human proteins predicted to be located in the mitochondrion.4466 These compendia, together with either next-generation sequencing platforms or the present-day mapping methods, provide powerful tools for novel disease gene identification. Furthermore, integration of information available on disease genes, clinical features and biological pathways could reveal new candidate genes for OXPHOS disorders and facilitate the discovery of new pathways underlying their pathogenesis,65 ultimately leading to a more thorough understanding of the enigmatic genetic, biochemical and clinical diversity of disorders due to mitochondrial dysfunction. Several other strategies that offer great potential to further our insights into the molecular mechanisms of mitochondrial disease are the following: 1) comparative mitochondrial proteomics, i.e. identification of differentially expressed mitochondrial proteins in patient material,67 2) using evolutionary data of multiple species to predict new disease genes and functional links between human mitochondrial proteins,68 3) computational expression screening, which uses a compilation of microarray data to find uncharacterized genes that could be involved in a certain pathway based on co-expression.69 The recent technological developments and our rapidly expanding knowledge of the genetic defects and pathogenic mechanisms underlying mitochondrial disorders will undoubtedly improve our diagnostic capabilities for these often devastating diseases. Whole mitochondrial genome screening can now easily Chapter and reliably be performed using a microarray-based analysis, the MitoChip.70 However, microarray chips for the detection of nuclear gene mutations will not 9 be very valuable because of the great and ever-expanding numbers of nuclear genes involved in OXPHOS deficiencies and mutations in these genes, demanding constant updating of the chips. Moreover, most mutations are present in just a small subset of patients. Instead, whole-exome sequencing will likely become the genetic diagnostic method of choice in the not too distant future. In addition to being successfully applied to OXPHOS disorders in research settings,71 whole- exome sequencing presently is a feasible option for diagnostics when merely the variations in genes known to be implicated in the particular OXPHOS deficiency are considered. As soon as bioinformatics tools are optimized and more sequence data are gathered to facilitate downstream filtering and interpretation of gene variants, also novel causative genes can be discovered by routine diagnostic whole-

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exome sequencing. Consequently, the percentage of patients that receive a genetic diagnosis will increase, which will aid prevention of mitochondrial disease through genetic counseling and prenatal diagnosis. This is of great importance as most cases of mitochondrial disease are currently untreatable, besides relieving certain symptoms, despite the impressive progress made in the field of mitochondrial biology and disease. Existing treatments (e.g. exercise training and coenzyme Q supplementation) and novel therapeutic strategies (e.g. gene therapy and pharmacological induction of mitochondrial biogenesis) are being explored for their applicability and effectiveness.7273 Although genetic-based therapies are far from being ready for clinical practice, some show promise.74 To illustrate, mitochondrial translation defects caused by mutations in tRNAs could be corrected by importing yeast or cytosolic tRNAs into mitochondria.757677 Nevertheless, many hurdles are to be overcome and shifting heteroplasmy towards wild-type mtDNA appears an option easier to attain for mtDNA mutations than gene therapy.7879 A promising approach to treating nuclear as well as mitochondrial gene defects, including those leading to combined OXPHOS deficiencies, is promotion of mitochondrial biogenesis through activation of the peroxisome proliferator-activated receptor (PPAR)/PPARy coactivator-1a (PGC-1a) pathway.8081 This pathway regulates energy metabolism by controlling expression levels of numerous genes involved in mitochondrial respiration and biogenesis. Increasing expression of PGC-1a, e.g. via the PPAR agonist bezafibrate (a widely used hypolipidemic drug), improved respiratory function in both control cells and cells with OXPHOS deficiencies. These are exciting times for patients, medical practitioners and scientists dealing with mitochondrial disorders now that our deeper understanding of these errors of metabolism is bringing us closer to the development of effective therapies. Future research will surely yield the much-needed further insights into Chapter the intricate and somewhat enigmatic processes fundamental to proper OXPHOS system function. This will hopefully shed more light on relations between 9 mitochondrial disease genotype and phenotype, among others. Additional factors involved in the critical step of mitochondrial protein synthesis are bound to be uncovered. Moreover, the currently known nuclear gene defects underlying combined OXPHOS deficiencies are quite likely just the tip of the iceberg. This thesis is a contribution to the field devoted to unraveling the etiology of mitochondrial disorders - combined OXPHOS complex I, III and IV deficiencies in particular - and exemplifies the challenge this endeavor presents.

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R e f e r e n c e s 1. Tucker EJ, Compton AG, Thorburn DR: Recent advances in the genetics of mitochondrial encephalopathies. Curr Neurol Neurosci Rep 2010; 10: 277-285. 2. Coenen MJ, Antonicka H, Ugalde C et al: Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency. N Engl J Med 2004; 351: 2080 2086. 3. Smits P, Smeitink JA, van den Heuvel LP, Huynen MA, Ettema TJ: Reconstructing the evolution of the mitochondrial ribosomal proteome. Nucleic Acids Res 2007; 35: 4686 4703. 4. Virgilio R, Ronchi D, Bordoni A et al: Mitochondrial DNA G8363A mutation in the tRNA Lys gene: clinical, biochemical and pathological study. J Neurol Sci 2009; 281: 85-92. 5. Enriquez JA, Chomyn A, Attardi G: MtDNA mutation in MERRF syndrome causes defective aminoacylation of tRNA(Lys) and premature translation termination. Nat Genet 1995; 10: 47-55. 6. Malfatti E, Cardaioli E, Battisti C et al: A novel point mutation in the mitochondrial tRNA(Trp) gene produces late-onset encephalomyopathy, plus additional features. J Neurol Sci 2010; 297: 105-108. 7. Mkaouar-Rebai E, Chamkha I, Kammoun F et al: Two new mutations in the MT-TW gene leading to the disruption of the secondary structure of the tRNA(Trp) in patients with Leigh syndrome. Mol Genet Metab 2009; 97: 179-184. 8. McFarland R, Elson JL, Taylor RW, Howell N, Turnbull DM: Assigning pathogenicity to mitochondrial tRNA mutations: when "definitely maybe" is not good enough. Trends Genet 2004; 20: 591-596. 9. Scaglia F, Wong LJ: Human mitochondrial transfer RNAs: role of pathogenic mutation in disease. Muscle Nerve 2008; 37: 150-171. Chapter 10. Chan SS, Copeland WC: DNA polymerase gamma and mitochondrial disease: understanding the consequence of POLG mutations. Biochim Biophys Acta 2009; 1787: 312-319. 9 11. de Vries MC, Rodenburg RJ, Morava E et al: Multiple oxidative phosphorylation deficiencies in severe childhood multi-system disorders due to polymerase gamma (POLG1) mutations. Eur J Pediatr 2007; 166: 229-234. 12. de Vries MC, Rodenburg RJ, Morava E et al: Normal biochemical analysis of the oxidative phosphorylation (OXPHOS) system in a child with POLG mutations: A cautionary note. J Inherit Metab Dis 2008; May 20th, Short Report #108. 13. Nguyen KV, Sharief FS, Chan SS, Copeland WC, Naviaux RK: Molecular diagnosis of Alpers syndrome. J Hepatol 2006; 45: 108-116. 14. Nguyen KV, Ostergaard E, Ravn SH et al: POLG mutations in Alpers syndrome. Neurology 2005; 65: 1493-1495.

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15. Gonzalez-Vioque E, Blazquez A, Fernandez-Moreira D et al: Association of novel POLG mutations and multiple mitochondrial DNA deletions with variable clinical phenotypes in a Spanish population. Arch Neurol 2006; 63: 107-111. 16. Luoma P, Melberg A, Rinne JO et al: Parkinsonism, premature menopause, and mitochondrial DNA polymerase gamma mutations: clinical and molecular genetic study. Lancet 2004; 364: 875-882. 17. Blok MJ, van den Bosch BJ, Jongen E et al: The unfolding clinical spectrum of POLG mutations. J Med Genet 2009; 46: 776-785. 18. Schulte C, Synofzik M, Gasser T, Schols L: Ataxia with ophthalmoplegia or sensory neuropathy is frequently caused by POLG mutations. Neurology 2009; 73: 898-900. 19. Horvath R, Hudson G, Ferrari G et al: Phenotypic spectrum associated with mutations of the mitochondrial polymerase gamma gene. Brain 2006; 129: 1674-1684. 20. Wong LJ, Naviaux RK, Brunetti-Pierri N et al: Molecular and clinical genetics of mitochondrial diseases due to POLG mutations. Hum Mutat 2008; 29: E150-E172. 21. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G: Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002; 30: e57. 22. Ramensky V, Bork P, Sunyaev S: Human non-synonymous SNPs: server and survey. Nucleic Acids Res 2002; 30: 3894-3900. 23. Ng PC, Henikoff S: SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res 2003; 31: 3812-3814. 24. Kasiviswanathan R, Longley MJ, Young MJ, Copeland WC: Purification and functional characterization of human mitochondrial DNA polymerase gamma harboring disease mutations. Methods 2010; 51: 379-384. 25. Van Goethem G, Lofgren A, Dermaut B, Ceuterick C, Martin JJ, Van Broeckhoven Chapter C: Digenic progressive external ophthalmoplegia in a sporadic patient: recessive mutations in POLG and C10orf2/Twinkle. Hum Mutat 2003; 22: 175-176. 9 26. Longley MJ, Clark S, Yu Wai MC et al: Mutant POLG2 disrupts DNA polymerase gamma subunits and causes progressive external ophthalmoplegia. Am J Hum Genet 2006; 78: 1026-1034. 27. Walter MC, Czermin B, Muller-Ziermann S et al: Late-onset ptosis and myopathy in a patient with a heterozygous insertion in POLG2. J Neurol 2010; 257: 1517-1523. 28. Antonicka H, Sasarman F, Kennaway NG, Shoubridge EA: The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1. Hum Mol Genet 2006; 15: 1835-1846. 29. Valente L, Tiranti V, Marsano RM et al: Infantile encephalopathy and defective mitochondrial DNA translation in patients with mutations of mitochondrial elongation factors EFG1 and EFTu. Am J Hum Genet 2007; 80: 44-58.

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30. Rossignol R, Faustin B, Rocher C, Malgat M, Mazat JP, Letellier T: Mitochondrial threshold effects. Biochem J 2003; 370: 751-762. 31. Gomez-Duran A, Pacheu-Grau D, Lopez-Gallardo E et al: Unmasking the causes of multifactorial disorders: OXPHOS differences between mitochondrial haplogroups. Hum Mol Genet 2010; 19: 3343-3353. 32. Sasarman F, Antonicka H, Shoubridge EA: The A3243G tRNALeu(UUR) MELAS mutation causes amino acid misincorporation and a combined respiratory chain assembly defect partially suppressed by overexpression of EFTu and EFG2. Hum Mol Genet 2008; 17: 3697-3707. 33. Ventura N, Rea SL, Testi R: Long-lived C. elegans mitochondrial mutants as a model for human mitochondrial-associated diseases. Exp Gerontol 2006; 41: 974-991. 34. Miller C, Saada A, Shaul N et al: Defective mitochondrial translation caused by a ribosomal protein (MRPS16) mutation. Ann Neurol 2004; 56: 734-738. 35. Saada A, Shaag A, Arnon S et al: Antenatal mitochondrial disease caused by mitochondrial ribosomal protein (MRPS22) mutation. J Med Genet 2007; 44: 784-786. 36. Richter R, Rorbach J, Pajak A et al: A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome. EMBO J 2010; 29: 1116-1125. 37. Antonicka H, Ostergaard E, Sasarman F et al: Mutations in C12orf65 in patients with encephalomyopathy and a mitochondrial translation defect. Am J Hum Genet 2010; 87: 115-122. 38. Uchiumi T, Ohgaki K, Yagi M et al: ERAL1 is associated with mitochondrial ribosome and elimination of ERAL1 leads to mitochondrial dysfunction and growth retardation. Nucleic Acids Res 2010; 38: 5554-5568. 39. Kolanczyk M, Pech M, Zemojte T et al: NOA1 is an essential GTPase required for mitochondrial protein synthesis. Mol Biol Cell 2011; 22: 1-11. 40. Tang T, Zheng B, Chen SH et al: hNOA1 interacts with complex I and DAP3 and Chapter regulates mitochondrial respiration and apoptosis. J Biol Chem 2009; 284: 5414-5424. 41. Cavdar KE, Ranasinghe A, Burkhart W et al: A new face on apoptosis: death-associated 9 protein 3 and PDCD9 are mitochondrial ribosomal proteins. FEBS Lett 2001; 492: 166 170. 42. Acin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, Enriquez JA: Respiratory active mitochondrial supercomplexes. Mol Cell 2008; 32: 529-539. 43. Dudkina NV, Sunderhaus S, Boekema EJ, Braun HP: The higher level of organization of the oxidative phosphorylation system: mitochondrial supercomplexes. J Bioenerg Biomembr 2008; 40: 419-424. 44. Pagliarini DJ, Calvo SE, Chang B et al: A mitochondrial protein compendium elucidates complex I disease biology. Cell 2008; 134: 112-123.

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45. Wessels HJ, Vogel RO, van den Heuvel L et al: LC-MS/MS as an alternative for SDS- PAGE in blue native analysis of protein complexes. Proteomics 2009; 9: 4221-4228. 46. Martherus RS, Sluiter W, Timmer ED, VanHerle SJ, Smeets HJ, Ayoubi TA: Functional annotation of heart enriched mitochondrial genes GBAS and CHCHD10 through guilt by association. Biochem Biophys Res Commun 2010; 402: 203-208. 47. Shutt TE, Shadel GS: A compendium of human mitochondrial gene expression machinery with links to disease. Environ Mol Mutagen 2010; 51: 360-379. 48. Stiburek L, Fornuskova D, Wenchich L, Pejznochova M, Hansikova H, Zeman J: Knockdown of human Oxa1l impairs the biogenesis of F1Fo-ATP synthase and NADH:ubiquinone oxidoreductase. J Mol Biol 2007; 374: 506-516. 49. Cartegni L, Chew SL, Krainer AR: Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 2002; 3: 285-298. 50. Wang GS, Cooper TA: Splicing in disease: disruption of the splicing code and the decoding machinery. Nat Rev Genet 2007; 8: 749-761. 51. Ishizawa T, Nozaki Y, Ueda T, Takeuchi N: The human mitochondrial translation release factor HMRF1L is methylated in the GGQ motif by the methyltransferase HMPrmC. Biochem Biophys Res Commun 2008; 373: 99-103. 52. Serero A, Giglione C, Sardini A, Martinez-Sanz J, Meinnel T: An unusual peptide deformylase features in the human mitochondrial N-terminal methionine excision pathway. J Biol Chem 2003; 278: 52953-52963. 53. Escobar-Alvarez S, Gardner J, Sheth A et al: Inhibition of human peptide deformylase disrupts mitochondrial function. Mol Cell Biol 2010; 30: 5099-5109. 54. Magalhaes PJ, Andreu AL, Schon EA: Evidence for the presence of 5S rRNA in mammalian mitochondria. Mol Biol Cell 1998; 9: 2375-2382. 55. Entelis NS, Kolesnikova OA, Dogan S, Martin RP, Tarassov IA: 5 S rRNA and tRNA Chapter import into human mitochondria. Comparison of in vitro requirements. J Biol Chem 2001; 276: 45642-45653. 9 56. Smirnov A, Comte C, Mager-Heckel AM et al: Mitochondrial enzyme rhodanese is essential for 5 S ribosomal RNA import into human mitochondria. J Biol Chem 2010; 285: 30792-30803. 57. Janssen GM, Maassen JA, van den Ouweland JM: The diabetes-associated 3243 mutation in the mitochondrial tRNA(Leu(UUR)) gene causes severe mitochondrial dysfunction without a strong decrease in protein synthesis rate. J Biol Chem 1999; 274: 29744-29748. 58. Toompuu M, Tiranti V, Zeviani M, Jacobs HT: Molecular phenotype of the np 7472 deafness-associated mitochondrial mutation in osteosarcoma cell cybrids. Hum Mol Genet 1999; 8: 2275-2283. 59. Ansorge WJ: Next-generation DNA sequencing techniques. N Biotechnol 2009; 25: 195-203.

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60. Metzker ML: Sequencing technologies - the next generation. Nat Rev Genet 2010; 11: 31-46. 61. Choi M, Scholl UI, Ji W et al: Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci U S A 2009; 106: 19096-19101. 62. Ng SB, Buckingham KJ, Lee C et al: Exome sequencing identifies the cause of a mendelian disorder. Nat Genet 2010; 42: 30-35. 63. Horner DS, Pavesi G, Castrignano T et al: Bioinformatics approaches for genomics and post genomics applications of next-generation sequencing. Brief Bioinform 2010; 11: 181-197. 64. Ng SB, Turner EH, Robertson PD et al: Targeted capture and massively parallel sequencing of 12 human exomes. Nature 2009; 461: 272-276. 65. Calvo SE, Mootha VK: The mitochondrial proteome and human disease. Annu Rev Genomics Hum Genet 2010; 11: 25-44. 66. Smith AC, Robinson AJ: MitoMiner, an integrated database for the storage and analysis of mitochondrial proteomics data. Mol Cell Proteomics 2009; 8: 1324-1337. 67. Chen X, Li J, Hou J, Xie Z, Yang F: Mammalian mitochondrial proteomics: insights into mitochondrial functions and mitochondria-related diseases. Expert Rev Proteomics 2010; 7: 333-345. 68. Huynen MA, de Hollander M, Szklarczyk R: Mitochondrial proteome evolution and genetic disease. Biochim Biophys Acta 2009; 1792: 1122-1129. 69. Baughman JM, Nilsson R, Gohil VM, Arlow DH, Gauhar Z, Mootha VK: A computational screen for regulators of oxidative phosphorylation implicates SLIRP in mitochondrial RNA homeostasis. PLoS Genet 2009; 5: e1000590. 70. Maitra A, Cohen Y, Gillespie SE et al: The Human MitoChip: a high-throughput sequencing microarray for mitochondrial mutation detection. Genome Res 2004; 14: 812-819. Chapter 71. Haack TB, Danhauser K, Haberberger B et al: Exome sequencing identifies ACAD9 mutations as a cause of complex I deficiency. Nat Genet 2010; 42: 1131-1134. 9 72. Hassani A, Horvath R, Chinnery PF: Mitochondrial myopathies: developments in treatment. Curr Opin Neurol 2010; 23: 459-465. 73. Kerr DS: Treatment of mitochondrial electron transport chain disorders: a review of clinical trials over the past decade. Mol Genet Metab 2010; 99: 246-255. 74. Kyriakouli DS, Boesch P, Taylor RW, Lightowlers RN: Progress and prospects: gene therapy for mitochondrial DNA disease. Gene Ther 2008; 15: 1017-1023. 75. Kolesnikova OA, Entelis NS, Jacquin-Becker C et al: Nuclear DNA-encoded tRNAs targeted into mitochondria can rescue a mitochondrial DNA mutation associated with the MERRF syndrome in cultured human cells. Hum Mol Genet 2004; 13: 2519 2534.

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76. Mahata B, Mukherjee S, Mishra S, Bandyopadhyay A, Adhya S: Functional delivery of a cytosolic tRNA into mutant mitochondria of human cells. Science 2006; 314: 471-474. 77. Rubio MA, Rinehart JJ, Krett B et al: Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import. Proc Natl Acad Sci U S A 2008; 105: 9186-9191. 78. Dimauro S, Rustin P: A critical approach to the therapy of mitochondrial respiratory chain and oxidative phosphorylation diseases. Biochim Biophys Acta 2009; 1792: 1159-1167. 79. Koene S, Smeitink J: Mitochondrial medicine: entering the era of treatment. J Intern Med 2009; 265: 193-209. 80. Bastin J, Aubey F, Rotig A, Munnich A, Djouadi F: Activation of peroxisome proliferator- activated receptor pathway stimulates the mitochondrial respiratory chain and can correct deficiencies in patients' cells lacking its components. J Clin Endocrinol Metab 2008; 93: 1433-1441. 81. Srivastava S, Diaz F, Iommarini L, Aure K, Lombes A, Moraes CT: PGC-1alpha/beta induced expression partially compensates for respiratory chain defects in cells from patients with mitochondrial disorders. Hum Mol Genet 2009; 18: 1805-1812.

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216 General discussion and future perspectives

Chapter 9

217 When your dreams turn to dust, vacuum. Author unknown S um m ary S amenvatting S amenvatting v o o r leken Summary

220 Summary

S u m m a r y Mitochondrial disorders are a heterogeneous group of often multisystemic and early fatal errors of metabolism, and are amongst the most common inherited human diseases. These disorders are generally caused by defects in the main energy-generating system, the oxidative phosphorylation (OXPHOS) system. It comprises five multisubunit enzyme complexes coded for by both the nuclear (n) and the mitochondrial (mt) genomes; only complex II is encoded entirely by the nDNA. Due to the multitude of proteins and intricacy of the processes required for a properly functioning OXPHOS system, identifying the genetic defect that underlies OXPHOS impairment is a daunting task, especially in the case of combined OXPHOS deficiencies. Whenever the activities of multiple enzymes are diminished, the molecular cause is presumed not to be located in genes encoding OXPHOS subunits but rather in genes implicated in processes such as mtDNA maintenance, mitochondrial transcription and mitochondrial translation. Chapter 2 of this thesis gives an extensive overview of these processes, highlighting proteins involved in the biogenesis or maintenance of multiple OXPHOS complexes and implicated in mitochondrial disorders. To date, numerous mutations associated with combined OXPHOS deficits have been reported - most in mtDNA, but the number of nDNA mutations is increasing rapidly - yet many cases remain unresolved.

The main objective of the research presented in this thesis was to discover new nuclear gene defects that cause combined OXPHOS complex I, III and IV deficiencies, with the focus on deficiencies originating from mitochondrial translation impairments. This could consequently improve diagnostic possibilities, our understanding of underlying disease mechanisms, and hopefully curative therapy in the future. For this purpose, we assembled a cohort of 33 patients with decreased enzyme activities of complex I, III and IV - and normal complex II activities - in fibroblasts and/or muscle tissue. Mutations in the mtDNA were excluded by sequencing the entire mitochondrial genome, when available in affected tissue. Subsequently, nuclear genes were screened for mutations: 1) genes known to be implicated in combined complex I, III and IV deficiencies, namely the genes coding for polymerase gamma (POLG and POLG2), mitochondrial translation factors mtEFTs, mtEFTu and mtEFG1, and mitochondrial ribosomal proteins (MRPs) MRPS16 and MRPS22; 2) candidate genes for combined OXPHOS deficits, namely the genes encoding the 6 remaining mitochondrial translation factors, 7 more MRPs, 2 potential mtDNA maintenance proteins, mitochondrial inner membrane transport protein OXA1L, and 7 mitochondrial transcription factors. Additionally, the rate of mitochondrial protein synthesis was determined

221 Summary in 18 patients. The results were used either to direct mutational analysis or to indicate the pathogenicity of a mutation. Furthermore, homozygosity mapping was performed for 7 patients. This aided in the detection of the genetic defect in one patient (see chapter 8), but it was uninformative in the other cases.

MtDNA sequencing in our patient cohort revealed pathogenic tRNA mutations in four patients, including novel mutations in tRNAArg (m.10450A>G) and tRNATrp (m.5556G>A). Chapter 3 demonstrates that these latter two mutations affect the conformation and steady-state levels of the tRNAs, causing mitochondrial translation impairment and consequently combined OXPHOS deficiencies. The fact that the tRNATrp mutation was previously listed as a polymorphism underscores the importance of functional studies in defining the pathogenicity of tRNA mutations. Furthermore, the findings emphasize the rapid progress that is still being made in the identification of mitochondrial tRNA mutations and that such mutations are important contributors to mitochondrial disease. Mutation analysis of a total of 30 nuclear genes yielded deleterious mutations (including two new mutations) in four genes previously reported to be implicated in combined OXPHOS deficits - POLG, TSFM, GFM1 and MRPS22- as well as several sequence variants with presently slightly ambiguous functional significance. With over 150 pathogenic mutations, POLG is a major locus for a wide range of mitochondrial disorders. The most common POLG mutation, Ala467Thr, was found compound heterozygous with Gly848Ser in one of our patients. Up to date, three out of nine mitochondrial translation factors - all involved in the elongation phase - have been associated with human mitochondrial disease. The previously described Arg333Trp mutation in elongation factor mtEFTs (TSFM) was detected in one patient (see chapter 4 for the first report and see the discussion section of chapter 5 for a note on the finding in the current patient cohort). The combined OXPHOS system and mitochondrial translation defects could be rescued by over-expression of either mtEFTs or mtEFTu. In control cells, on the contrary, over-expressing either elongation factor resulted in a dominant negative effect. This can be explained by the observation that the relative ratios of all three translation elongation factors, which are finely tuned and differ between tissues, are critical determinants of translation efficiency. Furthermore, we discovered a novel mutation in elongation factor mtEFG1 (GFM1), Arg250Trp, and demonstrated its role in the malfunctioning of the OXPHOS system and mitochondrial protein synthesis in one patient (chapter 5). In stark contrast to previously reported GFM1 cases, decreased enzyme activities were detected only in fibroblasts and not in muscle tissue of this patient, stressing that comprehensive biochemical investigation is critical in the diagnosis of mitochondrial disease. These and other reports on mutations in mitochondrial translation factors

222 Summary emphasize the heterogeneity and complexity of mitochondrial disorders: a) an identical mutation, e.g. Arg333Trp in TSFM, can result in strikingly different clinical presentations in individual patients; b) tissues are selectively affected despite ubiquitous expression of the translation factors; c) in which tissues the biochemical defect manifests itself can differ between patients with distinct mutations in the same gene, e.g. GFM1. Currently, one can primarily speculate on the causes for the biochemical and clinical variability associated with mutations in mitochondrial translation factors. Even though the core mitochondrial protein synthesis machinery has been characterized, much remains to be unraveled concerning details of the mechanisms of mitochondrial translation, the exact function of some of the proteins involved, and especially regulation of the entire system. Moreover, additional proteins involved in mitochondrial translation, such as MRPs, are still being uncovered. Of the more than 80 MRPs presently known, mutations have been found in merely two of them: MRPS16 and MRPS22. In principle, all MRPs are candidate genes for mitochondrial disorders. To facilitate the selection of which MRPs to screen for mutations first, we performed extensive homology searches to reconstruct the evolutionary history of the mitochondrial ribosomal proteome across different eukaryotic lineages (chapter 6). What is more, two new human MRPs were detected in this comparative genomics analysis: CHCHD1 and METT11D1. These two MRPs, together with two other candidate genes that were selected based on their potential function in mtDNA maintenance (NIPSNAP1 and GBAS), were screened for mutations in our group of patients with OXPHOS complex I, III and IV deficiencies. Chapter 7 contains the results of this study, i.e. many non- pathogenic sequence variations. Sequence analysis of 7 additional MRP genes (MRPS12, MRPS14, MRPS16, MRPS22, MRPL11, MRPL12 and MRPL45) revealed a new mutation in MRPS22. Its pathogenic role in the patient's mitochondrial disorder is reported in chapter 8. Evidently, MRPS22 is indispensable for effective mitochondrial protein synthesis, even though it is evolutionary not well conserved. Thus, supernumerary proteins such as MRPS22, lacking homologs in bacteria and other lower species, should not be disregarded during investigations of mitochondrial disorders.

Finally, chapter 9 reflects on all results obtained and discusses directions for future research with respect to combined OXPHOS complex I, III and IV deficiencies. This thesis demonstrates that locating the disease-causing mutation in patients with combined complex I, III and IV deficits remains a complicated and laborious undertaking in many cases, despite the substantial progress in this area. The current approach yielded up till now elucidation of the genetic defect in 8 out of 33 patients, including 4 novel mutations. MtDNA mutations accounted for

223 Summary

mitochondrial disease in 12% of patients, whereas the residual 88% consequently harbor nDNA mutations. Future research using high-throughput DNA sequencing platforms will undoubtedly provide the much-needed further insights into the intricate and somewhat enigmatic processes fundamental to proper OXPHOS system function. Moreover, many more disease genes are expected and required to be found to explain the numerous as yet unsolved cases. The currently known nuclear gene defects underlying combined OXPHOS deficiencies are quite likely just the tip of the iceberg.

224 Samenvatting

S amenvatting Mitochondriële aandoeningen zijn een heterogene groep van veelal multisysteem en vroeg fatale energiestofwisselingsziekten, en zijn een van de meest frequente erfelijke humane ziekten. Deze aandoeningen worden doorgaans veroorzaakt door defecten in het belangrijkste energiegenererende systeem, het oxidatieve fosforyleringssysteem (OXFOS systeem). Dit systeem bestaat uit vijf multisubunit enzymcomplexen die gecodeerd worden door zowel het nucleaire (n) als het mitochondriële (mt) genoom; alleen complex II wordt volledig gecodeerd door het nDNA. Het enorme aantal eiwitten en de gecompliceerdheid van de processen die benodigd zijn voor het goed functioneren van het OXFOS systeem bemoeilijken de opsporing van het genetische defect dat ten grondslag ligt aan een verstoring van het OXFOS systeem, in het bijzonder in het geval van gecombineerde OXFOS deficiënties. Wanneer de activiteiten van meerdere enzymen verlaagd zijn, wordt verondersteld dat de moleculaire oorzaak niet gelegen is in genen die coderen voor OXFOS subunits maar juist in genen die betrokken zijn bij processen als mtDNA onderhoud, mitochondriële transcriptie en mitochondriële translatie. Hoofdstuk 2 van dit proefschrift geeft een uitgebreid overzicht van deze processen, waarbij de nadruk gelegd wordt op eiwitten die vereist zijn voor de biogenese of het in stand houden van meerdere OXFOS complexen en welke geassocieerd worden met mitochondriële ziekten. Tot op heden zijn er talrijke mutaties in verband gebracht met gecombineerde OXFOS deficiënties - de meeste in het mtDNA, maar het aantal nDNA mutaties neemt snel toe - doch vele gevallen blijven onopgelost.

De voornaamste doelstelling van het onderzoek dat beschreven wordt in dit proefschrift was het identificeren van nieuwe nucleaire gendefecten die gecombineerde OXFOS complex I, III en IV deficiënties veroorzaken, met daarbij de focus leggend op deficiënties die voortkomen uit een stoornis in de mitochondriële translatie. Dit zou de diagnostische mogelijkheden, ons begrip van de onderliggende ziektemechanismen, en hopelijk toekomstige curatieve behandelingsstrategieën kunnen bevorderen. Met dit doel voor ogen hebben we een cohort van 33 patiënten samengesteld met verlaagde enzymactiviteiten van complex I, III en IV - en normale complex II activiteiten - in fibroblasten en/of spierweefsel. Mutaties in het mtDNA zijn uitgesloten door het volledige mitochondriële genoom te sequencen, wanneer beschikbaar in aangedaan weefsel. Vervolgens zijn nucleaire genen gescreend op mutaties: 1) genen die bekend zijn betrokken te zijn bij gecombineerde complex I, III en IV tekorten, namelijk de genen die coderen voor polymerase gamma (POLG en POLG2), mitochondriële translatiefactoren mtEFTs, mtEFTu en mtEFG1, en mitochondriële

225 Samenvatting

ribosomale eiwitten (MRP's) MRPS16 en MRPS22; 2) kandidaatgenen voor gecombineerde OXFOS deficiënties, namelijk de genen die coderen voor de 6 resterende mitochondriële translatiefactoren, 7 andere MRP's, 2 potentiële mtDNA nucleoid eiwitten, mitochondrieel binnenmembraan transporteiwit OXA1L, en 7 mitochondriële transcriptiefactoren. Bovendien is de snelheid van de eiwitsynthese in de mitochondriën bepaald bij 18 patiënten. De resultaten werden gebruikt of om richting te geven aan de screening van nucleaire genen of om de pathogeniteit van een mutatie aan te tonen. Voorts is er voor 7 patiënten homozygosity mapping uitgevoerd. Dit droeg bij tot de vondst van het genetische defect in één patiënt (zie hoofdstuk 8), maar in de overige gevallen waren de resultaten niet van nut.

Het sequencen van mtDNA in ons patiëntencohort bracht bij vier patiënten pathogene tRNA mutaties aan het licht, waaronder nieuwe mutaties in tRNAArg (m.10450A>G) en tRNATrp (m.5556G>A). Hoofdstuk 3 bewijst dat deze twee mutaties de conformatie en steady-state niveaus van de tRNA's negatief beïnvloeden, hetgeen gebreken in de mitochondriële translatie en daardoor gecombineerde OXFOS deficiënties tot gevolg heeft. Het feit dat de tRNATrp mutatie tevoren als polymorfisme vermeld stond benadrukt dat functionele studies belangrijk zijn bij het karakteriseren van de pathogeniteit van tRNA mutaties. De bevindingen onderstrepen de snelle vooruitgang die nog steeds geboekt wordt in de opsporing van mitochondriële tRNA mutaties en dat zulke mutaties een aanzienlijke bijdrage leveren aan mitochondriële aandoeningen. Middels mutatieanalyse vanin totaal 30nucleairegenenzijnziekteverwekkende mutaties (inclusief twee nieuwe mutaties) gedetecteerd in vier genen die eerder in verband gebracht waren met gecombineerde OXFOS tekorten - POLG, TSFM, GFM1 en MRPS22 - evenals verscheidene sequentievarianten waarvan de functionele betekenis momenteel enigszins onduidelijk is. Met meer dan 150 pathogene mutaties is POLG een locus die van groot belang is voor een breed scala aan mitochondriële ziekten. De meest voorkomende POLG mutatie, Ala467Thr, werd compound heterozygoot met Gly848Ser aangetroffen in één van onze patiënten. Tot dusver zijn drie van de negen mitochondriële translatiefactoren - alle drie betrokken bij de elongatiefase - in verband gebracht met humane mitochondriële syndromen. De reeds beschreven Arg333Trp mutatie in elongatiefactor mtEFTs (TSFM) werd gedetecteerd in één patiënt (zie hoofdstuk 4 voor de eerste publicatie en zie de discussie sectie van hoofdstuk 5 voor een aantekening over de bevinding in het huidige patiëntencohort). De gecombineerde OXFOS systeem en mitochondriële eiwitsynthese defecten konden hersteld worden door overexpressie van ofwel mtEFTs ofwel mtEFTu. In controle cellen daarentegen

226 Samenvatting resulteerde overexpressie van ieder van beide elongatiefactoren in een dominant negatief effect. Dit kan verklaard worden met de observatie dat de relatieve ratio's van alle drie de translatie elongatiefactoren, welke nauwkeurig afgestemd zijn en variëren tussen weefsels, kritische determinanten zijn van de translatie efficiëntie. Tevens hebben we een nieuwe mutatie, Arg250Trp, in elongatiefactor mtEFG1 (GFM1) ontdekt in één patiënt en de rol in de disfunctie van het OXFOS systeem en de mitochondriële translatie aangetoond (hoofdstuk 5). In schril contrast met eerder gerapporteerde GFM1 gevallen werden verlaagde enzymwaarden alleen waargenomen in fibroblasten en niet in spierweefsel van deze patiënt, hetgeen benadrukt dat uitvoerig biochemisch onderzoek cruciaal is bij het stellen van de diagnose voor mitochondriële ziekten. Deze en andere publicaties over mutaties in mitochondriële translatiefactoren maken wederom duidelijk hoe heterogeen en complex mitochondriële aandoeningen zijn: a) een identieke mutatie, bv. Arg333Trp in TSFM, kan zeer uiteenlopende klinische fenotypes teweeg brengen in afzonderlijke patiënten; b) weefsels zijn selectief aangedaan ondanks de ubiquitaire expressie van de translatiefactoren; c) in welke weefsels het biochemische defect tot uiting komt kan verschillen tussen patiënten met diverse mutaties in hetzelfde gen, bv. GFM1. Momenteel kan men voor het merendeel slechts speculeren over de oorzaken van de biochemische en klinische variabiliteit ten gevolge van mutaties in mitochondriële translatiefactoren. Alhoewel de basiscomponenten van de mitochondriële eiwitsynthese machinerie gekarakteriseerd zijn, moet er nog veel opgehelderd worden omtrent de details van de mitochondriële translatie mechanismen, de exacte functie van bepaalde betrokken eiwitten en in het bijzonder de regulering van het hele systeem. Daarnaast worden er nog altijd additionele eiwitten die benodigd zijn voor mitochondriële translatie, zoals MRP's, geïdentificeerd. Van de meer dan 80 thans bekende MRP's zijn er slechts in twee mutaties gevonden: MRPS16 en MRPS22. In principe zijn alle MRP's kandidaatgenen voor mitochondriële ziekten. Om de selectie van MRP's voor mutatieanalyse te vergemakkelijken, hebben we omvangrijke homologie zoekopdrachten uitgevoerd om de evolutionaire geschiedenis van het mitochondriële ribosomale proteoom over verscheidene eukaryote geslachten te reconstrueren (hoofdstuk 6). Bovendien werden twee nieuwe humane MRP's aan het licht gebracht in deze vergelijkende genomics analyse: CHCHD1 en METT11D1. Deze twee MRP's zijn samen met twee andere kandidaatgenen (NIPSNAP1 en GBAS), welke werden geselecteerd op basis van hun vermeende functie in mtDNA onderhoud, gescreend op mutaties in onze groep van patiënten met OXFOS complex I, III en IV deficiënties. Hoofdstuk 7 bevat de resultaten van deze studie, d.w.z. veel niet pathogene sequentievariaties. Sequentieanalyse van 7 additionele MRP genen (MRPS12, MRPS14, MRPS16, MRPS22, MRPL11, MRPL12 en MRPL45) onthulde een

227 Samenvatting

nieuwe mutatie in MRPS22. De pathogene rol van de mutatie in de mitochondriële aandoening van de patiënt wordt beschreven in hoofdstuk 8. MRPS22 is klaarblijkelijk onmisbaar voor effectieve mitochondriële eiwitsynthese, ook al is het eiwit evolutionair niet goed geconserveerd. Dus boventallige eiwitten zoals MRPS22, waarvoor homologen in bacteriën en andere lagere soorten ontbreken, zouden niet buiten beschouwing gelaten moeten worden tijdens onderzoek naar mitochondriële ziekten.

Tot slot reflecteert hoofdstuk 9 op de verkregen resultaten en bespreekt de richting van toekomstig onderzoek naar gecombineerde OXFOS complex I, III en IV deficiënties. Dit proefschrift toont aan dat het lokaliseren van de ziekteveroorzakende mutatie bij patiënten met gecombineerde complex I, III en IV tekorten in veel gevallen nog immer een ingewikkelde en moeizame onderneming is, ondanks de aanzienlijke vooruitgang op dit gebied. De huidige aanpak zorgde tot nu toe voor opheldering van het genetische defect bij 8 van de 33 patiënten, inclusief 4 nieuwe mutaties. MtDNA mutaties waren verantwoordelijk voor mitochondriële ziekte in 12% van de patiënten, terwijl de resterende 88% dientengevolge nDNA mutaties herbergen. Toekomstig onderzoek middels high- throughput DNA sequencing platforms zal ons ongetwijfeld de broodnodige verdere inzichten bieden in de complexe en enigszins raadselachtige processen die ten grondslag liggen aan het adequaat functioneren van het OXFOS systeem. Tevens verwacht en dient men beduidend meer ziektegenen te vinden om de talrijke nog onopgeloste zaken te kunnen verklaren. De nucleaire gendefecten die vooralsnog gerelateerd zijn aan gecombineerde OXFOS deficiënties zijn zeer waarschijnlijk slechts het topje van de ijsberg.

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S amenvatting v o o r l e k e n Achtergrondinformatie Energie en mitochondriën Zonder energie is er geen leven mogelijk. Voor al onze acties, van rennen tot slapen, voor de opbouw en het onderhoud van het lichaam en voor de normale werking van weefsels en organen is energie nodig. Deze energie verkrijgen we uit onze voeding door middel van vele stofwisselingsreacties, waarin enzymen de voedingsstoffen verwerken en omzetten in energie. De energie wordt opgeslagen in de vorm van het molecuul ATP. Verreweg de meeste ATP wordt geproduceerd in de mitochondriën, ook wel de 'energiecentrales' van de cel genoemd. Dit zijn organellen ('celorganen') die voorkomen in vrijwel elke cel van ons lichaam. Het aantal mitochondriën is afhankelijk van de energiebehoefte van de cel en loopt uiteen van enkele tot meer dan duizend per cel. Mitochondriën bestaan uit een binnen- en een buitenmembraan, met binnenin de zogenaamde matrix. In de binnenmembraan, welke sterk geplooid is, bevindt zich het oxidatieve fosforyleringssysteem (OXFOS systeem). Oxidatieve fosforylering vormt de laatste schakel in het stofwisselingsproces en levert de hoogste opbrengst aan ATP

Mitochondriële ziekten Wanneer de energieproductie verstoord is, kan dit leiden tot energiestofwisselingsziekten ofwel mitochondriële ziekten. De meeste patiënten met een mitochondriële ziekte hebben een defect in een of meerdere enzymcomplexen van het OXFOS systeem. Een van de gevolgen is een tekort aan energie, waardoor voornamelijk weefsels en organen met een hoge energiebehoefte, zoals hart, hersenen en spieren, niet optimaal kunnen functioneren. Maar aangezien mitochondriën in nagenoeg alle cellen aanwezig zijn, kan een stoornis hierin elk deel van het lichaam aantasten. De leeftijd waarop een mitochondriële aandoening zich openbaart, de symptomen die hierbij optreden en het verloop van de ziekte variëren sterk tussen patiënten, ook tussen patiënten met eenzelfde defect. Deze vaak ernstige en eveneens frequente aandoeningen (met ongeveer één nieuw geval per week in Nederland) zijn over het algemeen helaas nog niet te genezen. Symptoombestrijding en het voorkomen van nieuwe gevallen, door prenatale diagnose en erfelijkheidsadvisering, zijn daarom van groot belang.

Vorming van het OXFOS systeem De oorzaak van mitochondriële ziekten ligt in het erfelijke materiaal van het lichaam, het DNA. Bij de vorming en werking van mitochondriën zijn een paar duizend genen betrokken. Het merendeel van deze genen ligt op het nucleaire genoom (het DNA in de celkern, het nDNA), maar de mitochondriën zelf bezitten

229 Samenvatting voor leken ook DNA. Dit mitochondriële DNA (mtDNA) bevat slechts 37 genen, welke coderen (de genetische informatie bevatten) voor 2 rRNA's, 22 tRNA's en 13 mRNA's (zie Figuur 1). Het DNA van een gen wordt omgezet in ribosomaal (r), transfer (t) of messenger (m) RNA, afhankelijk van het type gen; dit proces heet transcriptie. Vervolgens wordt het mRNA molecuul vertaald naar een eiwit, ook wel eiwitsynthese of translatie genoemd. Voor dit proces zijn onder meer rRNA en tRNA moleculen benodigd. Het OXFOS systeem bestaat uit vijf enzymcomplexen: complex I tot en met IV van de ademhalings- of electronentransportketen en complex V (ATP synthase). Ieder van deze complexen bestaat uit meerdere eiwitten die samen als enzym functioneren. In de mitochondriën worden met behulp van het mtDNA en de mitochondriële transcriptie- en translatiesystemen 13 eiwitten gemaakt voor de bouw van de complexen I, III, IV en V. De genetische informatie voor de overige 76 eiwitten van de complexen I-V van het OXFOS systeem en alle andere eiwitten die aanwezig zijn in mitochondriën ligt op het nDNA; deze eiwitten worden in het cytosol (celvloeistof) gemaakt en daarna naar het mitochondrion getransporteerd.

Figuur 1. Vorming van het OXFOS systeem (een bewerking van Florentz et al. 2003 Cell Mol Life Sci). Het mitochondriële genoom codeert voor 2 rRNA's, 22 tRNA's en 13 mRNA's (in groen). Het nucleaire genoom codeert voor alle overige eiwitten die in het mitochondrion voorkomen, waaronder eiwitten van het OXFOS systeem en eiwitten die betrokken zijn bij de mitochondriële transcriptie en translatie (in paars).

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Oorzaken van OXFOS deficiënties Een afwijking in de genetische informatie, in de celkern of het mitochondrion, kan ervoor zorgen dat het OXFOS systeem niet goed werkt met uiteindelijk een mitochondriële aandoening tot gevolg. Wanneer slechts één van de enzymcomplexen van het OXFOS systeem een verlaagde activiteit vertoont, zogenoemde geïsoleerde deficiënties of tekorten, is er meestal iets mis met een bouwsteen of met een eiwit dat alle onderdelen samenvoegt tot één geheel. Wanneer meerdere OXFOS complexen zijn aangetast, zogenoemde gecombineerde deficiënties, is de situatie gecompliceerder. Talrijke processen zijn noodzakelijk voor de vorming van de vijf complexen van het OXFOS systeem, zoals de aanmaak van de afzonderlijke bouwstenen, het transport van deze eiwitcomponenten naar het mitochondrion, de plaatsing in het binnenmembraan en de assemblage tot enzymcomplexen. Hierbij zijn honderden eiwitten en andere stoffen betrokken en een fout in de genetische code voor ieder van deze elementen kan resulteren in een gecombineerde OXFOS deficiëntie. Hoofdstuk 2 van dit proefschrift geeft een uitgebreid overzicht van al deze processen. Hierbij is de nadruk gelegd op eiwitten die vereist zijn voor de vorming of het in stand houden van meerdere OXFOS complexen en welke geassocieerd worden met mitochondriële ziekten. Tot op heden zijn tal van mutaties (foutjes in het DNA) in verband gebracht met gecombineerde OXFOS tekorten. De meeste van deze afwijkingen zijn gevonden in het mtDNA, maar het aantal nDNA mutaties neemt snel toe. Toch kan bij veel patiënten de genetische oorzaak van hun ziekte nog niet worden ontdekt.

Mijn promotieonderzoek Doelstelling en aanpak De voornaamste doelstelling van het onderzoek dat beschreven wordt in dit proefschrift was het opsporen van nieuwe nucleaire gendefecten die gecombineerde OXFOS deficiënties veroorzaken, om zo ons begrip van de onderliggende ziektemechanismen, de diagnostische mogelijkheden en hopelijk toekomstige curatieve behandelingsstrategieën te kunnen bevorderen. De focus van het onderzoek is gelegd op gecombineerde tekorten van de complexen I, III en IV, welke kunnen voortkomen uit een stoornis in de mitochondriële eiwitsynthese. Wanneer de enzymcomplexen I, III en IV niet optimaal functioneren, maar complex II wel een normale activiteit heeft, kan er verondersteld worden dat er een afwijking is in één van de processen betrokken bij de vorming van eiwitten in het mitochondrion. Complex II bestaat namelijk volledig uit eiwitten die door het nDNA gecodeerd worden, terwijl de overige vier complexen voor hun bouwstenen afhankelijk zijn van het nDNA én het mtDNA. Een normale activiteit van complex II duidt er dus op dat de aanmaak van eiwitten in het cytosol en het transport naar het

231 Samenvatting voor leken

mitochondrion correct verlopen. In het geval van gecombineerde complex I, III en IV deficiënties moet daarom het mankement gezocht worden in processen als het onderhoud van het mitochondriële genoom, de mitochondriële transcriptie (van DNA naar RNA) en de mitochondriële translatie (van RNA naar eiwit). Complex V wordt hier overigens buiten beschouwing gelaten, aangezien dit enzym in weinig patiënten wordt gemeten. Met bovenstaande doel voor ogen hebben we een groep van 33 patiënten samengesteld met verlaagde enzymactiviteiten van OXFOS complexen I, III en IV - en normale complex II activiteiten - in huidcellen en/of spierweefsel. Mutaties in het mtDNA zijn uitgesloten door het complete mitochondriële genoom te sequencen bij deze patiënten. Sequencen is een methode voor het bepalen van de DNA sequentie, de volgorde van nucleotiden ofwel bouwstenen van het DNA. Vervolgens zijn een totaal van 30 nucleaire genen gescreend op mutaties. Hieronder waren zowel genen waarin afwijkingen gevonden waren bij andere patiënten met gecombineerde OXFOS deficiënties als genen waarbij dit niet het geval was, maar waarvan men op basis van hun functie een rol in gecombineerde OXFOS tekorten verwacht. Bovendien is de snelheid van eiwitsynthese in de mitochondriën bepaald bij 18 patiënten. De resultaten werden gebruikt om richting te geven aan de screening van nucleaire genen of om vast te stellen of een bepaald gendefect effect had op de mitochondriële eiwitsynthese. Voorts is er voor 7 patiënten homozygosity mapping uitgevoerd. Deze methode berust op het feit dat patiënten van ouders met een hoge graad van bloedverwantschap vaak dezelfde mutatie op beide chromosomen hebben, welke de ouders van een gezamenlijke voorouder hebben geërfd. De stukken DNA rondom de mutatie zijn dan ook identiek en kunnen we zichtbaar maken met deze techniek. Homozygosity mapping droeg bij tot de vondst van het genetische defect in één patiënt (zie hoofdstuk 8), maar in de overige gevallen waren de resultaten niet van nut.

Resultaten Mitochondrieel DNA Het sequencen van het mtDNA in onze patiëntengroep bracht bij vier patiënten S ziekteveroorzakende veranderingen in tRNA genen aan het licht, waaronder nieuwe mutaties in tRNAArg en tRNATrp. Deze tRNA's dragen respectievelijk de aminozuren arginine (Arg) en tryptofaan (Trp) bij zich, die ze tijdens het translatieproces afleveren om toegevoegd te worden aan de groeiende aminozuurketen (die zich later vouwt tot een eiwit). H oofdstuk 3 bewijst dat de mutaties in deze twee tRNA genen de structuur van en gehalte aan de tRNA moleculen negatief beïnvloeden, hetgeen gebreken in de mitochondriële eiwitsynthese en daardoor gecombineerde OXFOS deficiënties tot gevolg heeft. Het feit dat de tRNATrp mutatie tevoren als polymorfisme vermeld stond - een polymorfisme is een vaker

232 Samenvatting voor leken voorkomende genafwijking die niet tot ziekte leidt - benadrukt dat functionele studies belangrijk zijn bij het karakteriseren van de gevolgen van tRNA mutaties voor de cel en de patiënt. De bevindingen onderstrepen de snelle vooruitgang die nog steeds geboekt wordt in de opsporing van mitochondriële tRNA mutaties en dat zulke mutaties een aanzienlijke bijdrage leveren aan mitochondriële aandoeningen.

Nucleair DNA Middels mutatieanalyse van in totaal 30 nucleaire genen zijn ziekteverwekkende mutaties (inclusief twee nieuwe mutaties) gedetecteerd in vier genen die eerder in verband gebracht waren met gecombineerde OXFOS tekorten - POLG, mtEFTs, mtEFG1 en MRPS22 - evenals verscheidene veranderingen in de DNA sequentie waarvan de functionele betekenis momenteel enigszins onduidelijk is. Hieronder zullen de mutaties in deze vier genen en andere belangrijke bevindingen besproken worden. Met meer dan 150 ziekteveroorzakende mutaties is POLG een gen dat van groot belang is voor een breed scala aan mitochondriële ziekten. De twee genen POLG en POLG2 coderen samen voor het enzym polymerase gamma, wat zorgt voor de replicatie (verdubbeling) en het in stand houden van het mitochondriële genoom. De meest voorkomende POLG mutatie werd samen met een andere mutatie aangetroffen in één van onze patiënten. Tot dusver zijn drie van de negen mitochondriële translatiefactoren (eiwitten die een belangrijke rol spelen in de eiwitsynthese in mitochondriën) gerelateerd aan mitochondriële aandoeningen. Er zijn drie soorten translatiefactoren die elk bij een bepaalde fase van de translatie betrokken zijn: initiatie-, elongatie- en terminatiefactoren. Een reeds gepubliceerde mutatie in elongatiefactor mtEFTs werd gedetecteerd in één patiënt (zie hoofdstuk 4 voor de eerste publicatie en zie de discussie sectie van hoofdstuk 5 voor een aantekening over de bevinding in de huidige patiëntengroep). Om te bewijzen dat deze mutatie leidt tot een mitochondrieel translatiedefect en een gecombineerde OXFOS systeem deficiëntie bij de patiënt, hebben we viruscellen die elongatiefactor mtEFTs produceren in gekweekte patiëntcellen gebracht. Op deze manier konden de stoornissen grotendeels hersteld worden. In controle cellen daarentegen had deze zogenaamde overexpressie van elongatiefactor mtEFTs een negatief effect op de mitochondriële eiwitsynthese. Dit komt doordat de relatieve ratio's van alle drie de bestaande elongatiefactoren, welke variëren tussen weefsels, van groot belang zijn voor de efficiëntie van de eiwitsynthese. Tevens hebben we een nieuwe mutatie in elongatiefactor mtEFG1 ontdekt in één patiënt en de rol in de disfunctie van het OXFOS systeem en de mitochondriële translatie aangetoond (hoofdstuk 5). In schril contrast met eerder gerapporteerde gevallen met een

233 Samenvatting voor leken afwijking in mtEFG1, werden verlaagde enzymwaarden alleen waargenomen in huidcellen en niet in spierweefsel van deze patiënt. Dit benadrukt dat uitvoerig biochemisch onderzoek, bij voorkeur in meer dan één weefseltype, naar het functioneren van het OXFOS systeem cruciaal is bij het stellen van de diagnose voor mitochondriële ziekten. Deze en andere publicaties over mutaties in mitochondriële translatiefactoren maken wederom duidelijk hoe gevarieerd en complex mitochondriële aandoeningen zijn. Zo zijn er de volgende situaties te noemen: a) een identieke mutatie, bv. die in mtEFTs, kan zeer uiteenlopende klinische verschijningsvormen teweeg brengen in afzonderlijke patiënten; b) weefsels zijn selectief aangedaan ondanks dat de translatiefactoren in alle cellen aangemaakt worden; c) in welke weefsels de deficiëntie van het OXFOS systeem tot uiting komt kan verschillen tussen patiënten met diverse mutaties in hetzelfde gen, bv. mtEFG1. Momenteel kan men voor het merendeel slechts speculeren over de oorzaken van de biochemische en klinische variabiliteit ten gevolge van mutaties in mitochondriële translatiefactoren. Hoewel de basiscomponenten van de mitochondriële eiwitsynthese machinerie bekend zijn, moet er nog veel opgehelderd worden omtrent de details van de mitochondriële translatie mechanismen, de exacte functie van bepaalde betrokken eiwitten en in het bijzonder de regulering van het hele systeem. Daarnaast worden er nog altijd additionele eiwitten die benodigd zijn voor mitochondriële translatie geïdentificeerd. Tot op heden zijn er meer dan 80 mitochondriële ribosomale eiwitten (MRP's) bekend. Deze eiwitten vormen samen met de twee mitochondriële rRNA moleculen het mitochondriële ribosoom, een groot complex dat essentieel is voor de eiwitsynthese. Vooralsnog zijn in slechts twee MRP's mutaties gevonden: MRPS16 en MRPS22. In principe zou elk van de MRP's aan de basis kunnen liggen van een mitochondriële ziekte. Om de selectie van MRP's voor mutatieanalyse te vergemakkelijken, hebben we de evolutionaire geschiedenis van de mitochondriële ribosomale eiwitten gereconstrueerd (hoofdstuk 6). Er is bepaald welke MRP's in welke organismen, van eencelligen tot de mens, aanwezig zijn. Vervolgens kon een prioritering aangebracht worden voor het screenen van de MRP's op mutaties aan de hand van hun evolutionaire conservering en andere beschikbare informatie. Evolutionaire conservering betekent dat een gen behouden is gebleven in de loop van de evolutionaire ontwikkeling. Genen die evolutionair goed geconserveerd zijn, en dus voorkomen in veel verschillende organismen, zijn vaak van bijzonder belang en afwijkingen in zulke genen worden daarom verwacht tot ziekte te leiden. Dit onderzoek bracht bovendien twee nieuwe humane MRP's aan het licht. Deze twee MRP's zijn samen met twee andere genen, welke werden geselecteerd op basis van hun vermeende functie in mtDNA onderhoud, gescreend op mutaties in onze groep van patiënten met OXFOS complex I, III en IV deficiënties. Hoofdstuk

234 Samenvatting voor leken

7 bevat de resultaten van deze studie, dat wil zeggen veel DNA veranderingen die geen ziekte veroorzaken. Sequentieanalyse van 7 additionele MRP genen onthulde een nieuwe mutatie in MRPS22. De ziekteverwekkende rol van deze mutatie in de mitochondriële aandoening van de patiënt wordt beschreven in hoofdstuk 8. MRPS22 is klaarblijkelijk onmisbaar voor effectieve mitochondriële eiwitsynthese, ook al is het eiwit evolutionair niet goed geconserveerd. Dus eiwitten zoals MRPS22, waarvoor overeenkomstige eiwitten in bacteriën en andere lagere soorten ontbreken, zouden niet buiten beschouwing gelaten moeten worden tijdens onderzoek naar mitochondriële ziekten.

Conclusies Tot slot reflecteert hoofdstuk 9 op de verkregen resultaten en bespreekt de richting van toekomstig onderzoek naar gecombineerde OXFOS complex I, III en IV deficiënties. Dit proefschrift toont aan dat het lokaliseren van de ziekteveroorzakende mutatie bij patiënten met gecombineerde complex I, III en IV tekorten in veel gevallen nog immer een ingewikkelde en moeizame onderneming is, ondanks de aanzienlijke vooruitgang op dit gebied. De huidige aanpak zorgde tot nu toe voor opheldering van het genetische defect bij 8 van de 33 patiënten, inclusief 4 nieuwe mutaties. MtDNA mutaties waren verantwoordelijk voor mitochondriële ziekte in 12% van de patiënten, terwijl de resterende 88% derhalve nDNA mutaties herbergen. Toekomstig onderzoek middels grootschalige DNA sequencing technieken zal ons ongetwijfeld de broodnodige verdere inzichten bieden in de complexe en enigszins raadselachtige processen die ten grondslag liggen aan het adequaat functioneren van het OXFOS systeem. Tevens verwacht en dient men beduidend meer ziektegenen te vinden om de talrijke nog onopgeloste zaken te kunnen verklaren. De nucleaire gendefecten die tot dusver gerelateerd zijn aan gecombineerde OXFOS deficiënties zijn zeer waarschijnlijk slechts het topje van de ijsberg.

235 The only people with whom you should try to get even are those who have helped you. John E. Southard D a n kw o o rd A cknowledgements Dankwoord / Acknowledgements

Eindelijk is mijn proefschrift af, althans bijna, want zonder dankwoord is het natuurlijk niet compleet. Dit is misschien wel het belangrijkste en meest gelezen onderdeel van het proefschrift. Ondanks dat promoveren soms eenzaam kan zijn, met name het schrijfwerk, zou dit proefschrift niet tot stand zijn gekomen zonder de bijdragen van vele mensen. Daarom wil ik bij dezen iedereen bedanken die op de een of andere manier betrokken is geweest bij mijn promotietraject. Graag noem ik hierbij een aantal personen in het bijzonder. Mocht jouw naam niet vermeld worden, dan ben ik je vandaag misschien vergeten, maar morgen denk ik ongetwijfeld wel aan je.

Allereerst wil ik mijn promotors bedanken voor de kans die ze mij hebben geboden om onderzoek te doen naar deze intrigerende ziekten en in een stimulerende werkomgeving waar diagnostiek, research en patiëntenzorg samenkomen. Ik heb als junior onderzoeker een leerzame en leuke tijd gehad. Beste Bert, je begeleiding heb ik erg gewaardeerd. Ondanks je drukke agenda hadden we wekelijkse werkbesprekingen en kon ik tussendoor altijd langskomen met vragen. Naast nuttige adviezen en ideeën, gaf je me voldoende vrijheid in de uitvoering van mijn promotieonderzoek. Tevens wil ik je bedanken voor het feit dat je me toestond om mijn laatste jaar te onderbreken voor vrijwilligerswerk in Zuid-Amerika, wat niet bepaald gebruikelijk is tijdens een promotietraject. Beste Jan, bedankt voor je steun en kritische blik. Hoewel ons contact gedurende de jaren wat verminderde door je vele taken, van kinderarts tot organisator van popconcerten voor zieke kinderen, bleef je altijd betrokken bij mijn onderzoek en goed bereikbaar via email. Bedankt ook dat ik een keer mee mocht lopen op de poli; dat was interessant en heeft mij het belang van dit onderzoek alleen maar meer doen beseffen. Ik hoop van harte dat je het streven naar een medicijn voor je pensioen gaat halen!

Ten tweede wil ik al mijn (oud-)collega's van Laboratorium Kindergeneeskunde en Neurologie bedanken voor hun getoonde belangstelling, hulpvaardigheid en bovenal voor de gezelligheid. Jullie hebben het onderzoekswerk tot een aangename bezigheid gemaakt. Op het lab heerste altijd een goede sfeer en al leidden het vele geklets, de (twee verschillende) radio(zenders) en de vals zingende kerstboom soms af, ik had me toch geen leukere werkplek kunnen wensen. Ook denk ik met veel plezier terug aan de vele activiteiten buiten werktijd - etentjes, afdelingsuitjes, feesten, borrels, Sinterkerst, BOTS - en de congressen in San Diego en Stockholm. Verscheidene groepen en personen wil ik naast bovenstaande nog extra bedanken. ■ De DNA diagnostiek groep, welke tijdens mijn promotietraject flink is uitgebreid: Heleen, Roel, Edwin, Jitske, Marieke, Marthe, de twee Maaike's,

238 Dankwoord / Acknowledgements en tijdelijke medewerkers Ilse, Alexandra en Brenda. Ontzettend bedankt voor de plezierige samenwerking en dat ik op jullie kon rekenen als ik enige ondersteuning nodig had. Maaike, fijn dat je de 3 'mammoetgenen' binnen de deadline hebt kunnen screenen, zodat mijn artikel toch gepubliceerd kon worden. Heleen, mijn stagebegeleidster en vraagbaak tijdens het eerste deel van mijn promotieonderzoek. Dankjewel voor je prettige gezelschap, alle hulp en de heldere uitleg. Jouw enthousiaste begeleiding heeft mijn interesse voor het onderzoek zeker aangewakkerd. Saskia, mijn overbuurvrouw en medepromovenda. We begonnen gelijktijdig aan ons promotietraject op soortgelijke onderwerpen en hebben daarom veel kunnen delen. Het was erg fijn om een klankbord te hebben voor de dagelijkse beslommeringen. Mijn dank hiervoor en voor de gezellige tijd tijdens het vele pipetteerwerk. Leuk dat je mijn paranimf wilt zijn en dat we binnenkort misschien weer collega's worden! Iedereen van de eiwitgroep hartelijk bedankt voor jullie assistentie en tips met betrekking tot SDS- en BN-PAGE, western blotting, etc. Speciale dank aan Mariël voor het uitvoeren van meerdere experimenten, waarvan een aantal ook gebruikt zijn voor publicatie. Leo, bedankt voor je hulp bij het opzetten van de translatie assay. Zonder patiëntencellen om DNA uit te isoleren zou ik niet ver gekomen zijn met mijn onderzoek. Daarom ben ik de weefselkweek dankbaar voor het kweken van de patiëntencellen en hulp wat betreft mijn eigen celkweken. Frans bovendien bedankt voor het maken en opkweken van cybride cellijnen, welke onmisbaar waren voor de bewijsvoering van twee mutaties. Verder mag de spiergroep natuurlijk niet ontbreken in dit rijtje. De uitslagen van jullie enzymactiviteitsbepalingen vormden de basis voor de selectie van mijn patiëntengroep. Daarnaast kon ik altijd bij jullie terecht met vragen of verzoeken om extra metingen. Mijn dank daarvoor. En Richard, bedankt voor de ondersteuning en je waardevolle commentaar tijdens werkbesprekingen en op mijn manuscripten. Met veel plezier heb ik gedurende mijn promotie vijf studenten begeleid: Sandra, Marieke, Benigna, Samanta en Ramon, bedankt voor jullie inzet en bijdragen aan mijn onderzoek. De genen wilden niet altijd meewerken, maar toch zetten jullie door. Sandra, mijn eerste stagiaire, je was erg gemotiveerd maar stelde mijn geduld wel extra op de proef. Helaas verliep je stage niet zonder problemen. Marieke, als enige heb jij een pathogene mutatie gevonden, wat was ook ik daar blij mee! Benigna, je werkte erg netjes en had, als je iets meer tijd had gehad, de MRPS22 mutatie gevonden. Samanta, net als ik een BMWer en ondanks de weinige praktijkervaring heb je toch snel de handigheid gekregen. Ramon, de enige mannelijke stagiair, een aangename afwisseling. Je hebt heel wat werk verzet.

239 Dankwoord / Acknowledgements

En als laatste wil ik graag de huidige promovendi bij LKN heel veel succes wensen met hun onderzoek en het afronden van hun proefschrift.

Wie ik ook beslist dank verschuldigd ben zijn alle anderen die hun medewerking hebben verleend aan mijn onderzoek, zowel binnen het UMC St Radboud als daarbuiten. Bedankt voor jullie hulp, thank you for your contributions! Een aantal van hen verdienen een aparte vermelding in dit dankwoord. In het eerste jaar van mijn promotietraject heb ik tijdelijk onderzoek gedaan bij het CMBI (Center for Molecular and Biomolecular Informatics). Dit was heel wat anders dan het labwerk: hele dagen achter de computer wijs te zien worden uit evolutionaire data en bijbehorende technieken, materie die grotendeels nieuw was voor mij. Thijs, dankjewel voor je uitgebreide begeleiding bij het opzetten, uitvoeren en verwerken van de analyses; ik heb veel van je geleerd. Martijn, bedankt voor de mogelijkheid om me te verdiepen in de bioinformatica en voor de goede ondersteuning. Dank ook aan beiden voor jullie input voor mijn allereerste publicatie. Iedereen van het CMBI: bedankt voor de gezelligheid naast het (soms wat slaapverwekkende) werk. Herman en Peter, helaas heb ik geen complementatie kunnen krijgen met de baculovirussen, maar dat doet niets af aan mijn dank voor jullie medewerking. Peter, ik heb bovendien onze jaarlijkse mentorgesprekken erg gewaardeerd. Dear Hana, thank you for your support in setting up the translation assay in Nijmegen and for the fruitful collaboration on the EFG1 project. Dear Ann, thank you for our pleasant collaboration on the MRPS22 mutation. Saskia Wortmann, bedankt voor alle moeite die je gedaan hebt voor het case report voor het MRPS22 manuscript. Dit bleek heel wat meer werk te zijn dan oorspronkelijk gedacht, maar uiteindelijk is het allemaal goed gekomen.

Naast werkgerelateerde ondersteuning zijn mentale steun en aangename afleiding ook belangrijk tijdens een promotietraject. Daarvoor wil ik graag mijn familie, vrienden en kennissen hartelijk bedanken. Al had ik het met de meesten van jullie nooit uitvoerig over mijn onderzoek, jullie getoonde interesse stelde ik erg op prijs en ik vond het leuk om toch zo goed mogelijk uit te leggen wat mijn onderzoek inhield. Maar voornamelijk bedankt voor de gezellige tijden. Lieve pap en mam, M&M, ik ben enorm dankbaar voor alles wat jullie voor mij hebben gedaan. Voor jullie liefde, dat jullie altijd voor me klaar staan, het volste vertrouwen in me hebben en me vrijlaten in mijn keuzes. Hoewel het niet altijd duidelijk was waar ik precies mee bezig was, probeerden jullie het toch vaak zo goed mogelijk uit te leggen aan anderen en mijn artikelen te lezen via de I vertaaloptie op internet (wat het er volgens mij alleen maar onbegrijpelijker op maakt). Onder andere door mijn bezigheden voor mijn promotie zagen jullie mij

240 Dankwoord / Acknowledgements

soms weinig, maar ik zal mijn best doen om dat nu te veranderen. Rob, mijn grote broer. Vroeger kon ik je wel achter het behang plakken, maar nu ben ik blij met mijn lieve broer en je grappen en grollen. Bedankt dat je mijn paranimf wilt zijn. Dear Brian, my ex-dushi. I don't know whether you will ever read this - I wouldn't know where to send my thesis: Curaçao, Argentina, Canada, or Nijmegen after all - but that doesn't keep me from devoting part of the acknowledgements to you. Thank you for the great time we had together and of course for your support during my PhD. You checked the English of some of my manuscripts, even though most of the text was gibberish to you, and helped with any computer troubles. Our volunteer work in South America was an inconvenient but amazing experience; thanks for encouraging this adventure. Lieve Jordy, mijn prrr-mannetje. Na eerst voor veel (welkome) afleiding van mijn schrijfwerk te hebben gezorgd in de zomer van 2009, heb je me vooral gestimuleerd. Samen zaten we een groot deel van de tijd op de computerkamer te werken (of zoveel mogelijk andere 'nuttige' dingen te doen om maar niet aan onze promotie/studie te hoeven werken). Het was niet altijd even gezellig en leuke dingen doen schoot er vaak bij in, maar we konden in ieder geval elkaar gezelschap houden en motiveren (of juist van het werk afhouden...). Bedankt voor je mentale en praktische steun, dat je er gewoon altijd voor me bent! Nog even doorzetten en dan ben jij ook klaar, ik ben trots op je.

Tot slot wil ik alle patiënten en hun ouders bedanken voor hun toestemming tot deelname aan het onderzoek. Zonder jullie had ik dit onderzoek natuurlijk nooit kunnen doen!

Iedereen nogmaals bedankt en de allerbeste wensen voor de toekomst. Ik hoop ieder van jullie nog eens tegen het lijf te lopen.

'PaubeA

241 A life without cause is a life without effect. Barbarella

We can never be more than we believe is possible, so start to believe in possibilities. Lindsey Agness C urriculum V itae Curriculum vitae

244 Curriculum vitae

Paulien Smits werd geboren op 3 augustus 1981 te Krimpen aan den IJssel en groeide op in Alkmaar en Oss. In Oss heeft zij de basisschool afgerond en in 1999 haar Gymnasium diploma behaald aan het Titus Brandsmalyceum. Na een jaar gereisd te hebben in Australië, begon zij in 2000 aan de studie Biomedische Wetenschappen aan de Katholieke Universiteit Nijmegen (tegenwoordig Radboud Universiteit Nijmegen). Als onderdeel van haar master heeft ze drie vakken gevolgd aan de University of Victoria, Canada. Tijdens de opleiding liep zij een drietal stages: 1) een bachelorstage van ruim 4 maanden bij de afdeling Matrix Biochemie van het Nijmegen Centre for Molecular Life Sciences naar de invloed van groeifactoren op biomaterialen ten behoeve van tissue engineering; 2) een masterstage van officieel 4 maanden, welke op eigen initiatief is verlengd, bij de afdeling Laboratorium Kindergeneeskunde en Neurologie van het UMC St. Radboud naar nucleaire gendefecten bij gecombineerde oxidatieve fosforyleringsdeficiënties; 3) een masterstage van 8 maanden bij Solvay Pharmaceuticals BV te Weesp naar een nieuw model om het leergedrag van ratten te meten en farmacologisch te beïnvloeden. In december 2005 behaalde zij het doctoraal diploma, met als specialisatie pathobiologie. Haar tweede stage, op de afdeling Laboratorium Kindergeneeskunde en Neurologie (thans Laboratorium voor Genetische, Endocriene en Metabole ziekten), werd in januari 2006 voortgezet met een aanstelling als junior onderzoeker, wederom onder leiding van Prof. dr. Bert van den Heuvel en Prof. dr. Jan Smeitink. De resultaten van dit promotieonderzoek hebben geleid tot een aantal wetenschappelijke artikelen, welke gebundeld zijn in dit proefschrift. Sinds november 2009 is Paulien werkzaam als Regulatory Affairs project manager bij Synthon BV te Nijmegen, een Nederlands farmaceutisch bedrijf dat generieke humane geneesmiddelen ontwikkelt en produceert.

245 Publication is to thinking as childbirth is to the first kiss. Karl Wilhelm Friedrich Schlegel

If I'm trying to sleep, the ideas won't stop. If I'm trying to write, there appears a barren nothingness. Carrie Latet List of P ublications List of publications List of publications

Sm its P,* Saada A,* Wortmann SB, Heister A, Brink M, Pfundt R, Miller C, Haas D, Hantschmann R, Rodenburg RJT, Smeitink JAM, van den Heuvel LP: Mutation in mitochondrial ribosomal protein MRPS22 leads to Cornelia de Lange-like phenotype, brain abnormalities and hypertrophic cardiomyopathy. Eur J Hum Genet 2011; 19(4): 394-399.

Sm its P, Antonicka H, van Hasselt PM, Weraarpachai W, Haller W, Schreurs M, Venselaar H, Rodenburg RJ, Smeitink JA, van den Heuvel LP: Mutation in subdomain G' of mitochondrial elongation factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle. Eur J Hum Genet 2011; 19(3): 275-279.

Sm its P, Smeitink J, van den Heuvel L: Mitochondrial translation and beyond: processes implicated in combined oxidative phosphorylation deficiencies. J Biomed Biotechnol 2010; Article ID 737385, 24 pages.

Sm its P, Mattijssen S, Morava E, van den Brand M, van den Brandt F, Wijburg F, Pruijn G, Smeitink J, Nijtmans L, Rodenburg R, van den Heuvel L: Functional consequences of mitochondrial tRNA Trp and tRNA Arg mutations causing combined OXPHOS defects. Eur J Hum Genet 2010; 18(3): 324-329.

Sm its P, Rodenburg RJ, Smeitink JAM, van den Heuvel LP: Sequence variants in four candidate genes (NIPSNAP1, GBAS, CHCHD1 and METT11D1) in patients with combined oxidative phosphorylation system deficiencies. J Inherit Metab Dis 2009; January 24th, Short Report #149.

Sm its P, Smeitink JAM, van den Heuvel LP, Huynen MA, Ettema TJG: Reconstructing the evolution of the mitochondrial ribosomal proteome. Nucleic Acids Res 2007; 35(14): 4686-4703.

Smeitink JAM,* Elpeleg O,* Antonicka H, Diepstra H, Saada A, Sm its P, Sasarman F, Vriend G, Jacob-Hirsch J, Shaag A, Rechavi G, Welling B, Horst J, Rodenburg RJ, van den Heuvel B, Shoubridge EA: Distinct clinical phenotypes associated with a mutation in the mitochondrial translation elongation factor EFTs. Am J Hum Genet 2006; 79(5): 869-877.

* Joined first authorship

Publicationofthisthesiswasfinanciallysupported by PTC-ICT, Sigma-tau BV, and the Radboud University Nijmegen Medical Centre:

ö| sigma-tau Aa Dynamics AX training, -ICTadvies en consulting