Mitochondrial diseases: Translation matters

Molecular and Cellular Neuroscience 55 (2013) 1–12

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Mitochondrial diseases: Translation matters Sarah Pearce a, 1, Catherine Laura Nezich a, b, 1, Antonella Spinazzola a,⁎ a b

MRC Mitochondrial Biology Unit, Wellcome Trust-MRC Building, Hills Road Cambridge, CB2 0XY, UK National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA

a r t i c l e

i n f o

Article history: Received 11 May 2012 Accepted 25 August 2012 Available online 7 September 2012 Keywords: Mitochondrial encephalopathies Translation Ribosome Assembly factors

a b s t r a c t Mitochondrial diseases comprise a heterogeneous group of disorders characterized by compromised energy production. Since the early days of mitochondrial medical genetics, it has been known that these can be caused by defects in mitochondrial protein synthesis. However, only in recent years have we begun to develop a broader picture of the array of proteins required for mitochondrial translation. With this new knowledge has come the realization that there are many more neurological and other, diseases attributable to impaired mitochondrial translation than previously thought. Perturbation of any part of this intricate machinery, from the primary sequence of transfer or ribosomal RNAs, to the proteolytic processing of ribosomal proteins, can cause mitochondrial dysfunction and disease. In this review we discuss the current understanding of the mechanisms and factors involved in mammalian mitochondrial translation, and the diverse pathologies resulting when it malfunctions. This article is part of a Special Issue entitled ‘Mitochondrial function and dysfunction in neurodegeneration’. © 2012 Published by Elsevier Inc.

Introduction

The mitochondrial genome

Mitochondria are essential double-membrane organelles, whose primary function is energy production through the oxidative phosphorylation (OXPHOS) process. Additionally, mitochondria serve as the crossroads for many important biochemical pathways and perform crucial roles in regulating iron and calcium homeostasis, nitrogen metabolism, and apoptosis. OXPHOS catalyzes the oxidation of fuel molecules and the concomitant energy transduction into ATP via five complexes located in the inner mitochondrial membrane (IMM). Briefly, a series of redox reactions result in the reduction of oxygen to water while complexes I, III and IV of the respiratory chain pump protons across the IMM into the intermembrane space (IMS) generating a ‘proton gradient’ across the IMM. This proton gradient provides the driving force for the synthesis of ATP from ADP and inorganic phosphate (Pi) by complex V (or ATP synthase). Notably, the formation of the OXPHOS system is under the control of two separate genetic systems, the nuclear and the mitochondrial genomes. As a result, genetic defects of either mitochondrial or nuclear DNA can compromise ATP production and potentially cause human pathology at any age, with any symptoms, and by any mode of inheritance. In this review we will focus on protein synthesis in human mitochondria and on the role it plays in mitochondrial disorders, after recapitulating the processes required before mitochondrial protein synthesis can take place.

Human mitochondrial DNA (mtDNA) is a circular, double-stranded molecule with a striking economy of sequence organization, compressing 37 genes into 16.6 kilobase pairs of DNA. Unsurprisingly, there are no introns and the genes are arranged end to end with little or no intergenic regions. However, there is one sizeable non-coding region (NCR) that contains a number of important regulatory elements of replication and transcription (Fig. 1A). Mitochondrial DNA is devoted to the synthesis of 13 subunits of respiratory complexes I, III, IV, and V. In addition to the 13 proteins of the OXPHOS system, mtDNA encodes the 22 transfer RNAs and two ribosomal RNAs necessary for their translation within the organelle. The two strands of human mtDNA have different nucleotide compositions and are designated heavy (H) and light (L), according to their buoyant density on cesium chloride gradients. The H-strand contains most of the coding material, including 12 of the 13 protein-coding genes, both rRNAs, and 14 of the 22 tRNAs, whereas the DNA L-strand encodes a single protein and eight tRNAs. The fact that mtDNA is a compartmentalized extrachromosomal element contributes to its unique genetic features. 1) A typical cell contains hundreds or thousands of mtDNA molecules. 2) Although in normal circumstances almost all copies share the same sequence (homoplasmy), some individuals harbor two (or more) mitochondrial genotypes (heteroplasmy). 3) Because there are many copies of mtDNA in a cell, a deleterious sequence variant can be tolerated until, or unless, the abundance of the defective mtDNAs exceeds a threshold, at which point mitochondrial and cellular dysfunction manifests. 4) Mitochondria are not partitioned equally at cell division, and so the proportion of mutant mtDNAs may shift in daughter cells,

⁎ Corresponding author. Fax: +44 12 23 25 28 45. E-mail address: [email protected] (A. Spinazzola). 1 The first two authors made an equal contribution to the article. 1044-7431/$ – see front matter © 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.mcn.2012.08.013

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S. Pearce et al. / Molecular and Cellular Neuroscience 55 (2013) 1–12 HS P1

A

B. REPLICATION

OH T CY

B

NCR

T

P

F

LS P

A1555G (SNHL)

12S

mRNA

V

HS P 2

5’

ND 6

S 16

mtSSB

E

L

ND5

A3243G (MELAS)

Q

MI

POLRMT

ND2

R

4L ND

D3

3’ 5’

TOPO

POLG

W

N

A

Y

C

ND4

OL N

Twinkle

ND 1

H

S

L

Human Mitochondrial DNA 16569bp

(d)NTPs

G

S CO

III

K AT P6 AT P8

I CO

5’ 3’

D

CO II

A8344G (MERRF)

C. TRANSCRIPTION POLRMT TEFM

TFB2M TFAM HSP2 HSP1 mTERF1

NCR LSP

Phe

12S

16S

Leu ND1

RNA Full-length RNA

Fig. 1. The human mitochondrial genome and factors involved in mtDNA replication and transcription. A) A schematic diagram of human mitochondrial DNA encoding 13 protein, 2 rRNA, and 22 tRNA coding genes. The location of three of the most common mitochondrial mtDNA gene mutations with their associated clinical phenotypes are indicated. B) The core mtDNA replication machinery in mammals includes DNA polymerase POLG, Twinkle helicase, and mtSSB. Replication also involves RNA priming by POLRMT, mtDNA topology alteration by topoisomerases, and possibly RNA incorporation into the lagging strand during leading strand synthesis. C) Mammalian mtDNA transcription initiates from three promoters, one for the light strand (LSP) and two for the heavy strand (HSP1, HSP2). The core mtDNA transcription machinery includes RNA polymerase POLRMT and its accessory subunit TEFM, transcription factor TFB2M, and transcription activator TFAM. RNA synthesis beginning from HSP2 is terminated by mTERF1 within the tRNALeu(UUR) gene while transcription started at HSP1 results in near genome-length polycistronic transcripts. The building blocks of nucleic acid are (deoxy)nucleotides ((d)NTPs).

affecting the phenotype accordingly. Although phenotypic selection will favor fully functional mitochondria, some mutant mtDNAs possess a replicative advantage and this may be key to their selection and persistence. 5) The mitochondria of spermatozoa degenerate after fertilization and so mtDNA is exclusively maternally inherited (Al Rawi et al., 2011). 6) There is a marked restriction in mitochondrial numbers during oogenesis and this ‘bottleneck’ can explain the striking generational shifts in the levels of different mtDNA variants, which were first described in cows (Olivo et al., 1983) and later in humans (Blok et al., 1997). Mitochondrial DNA maintenance, replication and transcription Mitochondrial DNA replication is an ongoing process in most cells and tissues that have been examined and, unlike nuclear DNA, mtDNA synthesis occurs independently of the cell cycle, and postmitosis. Two modes of mtDNA replication have been proposed to operate in mammalian cells and tissues (Pohjoismäki and Goffart, 2011). In one mode, the two strands of DNA are synthesized concurrently as in nuclear DNA replication, whereas in the other there is a considerable delay between the initiation of synthesis of the two strands. The latter mode of replication exposes the lagging-strand template, which is either coated with protein or hybridized to RNA. The mtDNA replication apparatus is known to include a DNA polymerase (POLG), a DNA helicase (Twinkle) and mitochondrial-specific single-stranded DNA binding protein (mtSSB) (Fig. 1B). Other factors

critical for mtDNA replication are RNase H1 and DNA ligase III, which are shared with the nucleus. Notwithstanding this knowledge, we are far from having a complete inventory of proteins required for mtDNA replication and maintenance; however it is of particular note that mitochondrial nucleoprotein complexes, or nucleoids, are closely associated with the machinery of mitochondrial protein synthesis (He et al., 2012b). Some of the nuclear-encoded factors that are important for mtDNA maintenance have a prolonged interaction with mtDNA, as epitomized by the major mtDNA packaging protein TFAM, whereas others do not interact directly with either mtDNA or mtDNA-binding proteins. The latter group includes enzymes involved in de novo synthesis of nucleotides or nucleotide salvage pathways, and some, such as thymidine phosphorylase and the accessory subunit of ribonucleotide reductase, RRM2B, even reside outside mitochondria (Spinazzola, 2011). The mitochondrial genome relies on only three promoters to produce its 11 mRNAs, 22 tRNAs, and two rRNAs. Two are on the heavy strand (HSP1 and HSP2), whereas there is one lone light strand promoter (LSP). LSP and HSP1 are located in the NCR, while in vitro techniques have recently corroborated the earlier mapping of HSP2 to the tRNA Phe gene adjacent to the NCR (Fig. 1A) (Zollo et al., 2012). All three produce polycistronic transcripts, and the products of HSP1 and HSP2 partly overlap. The primary purpose of HSP1 is to produce rRNAs for mitochondrial ribosomes (mitoribosomes), and transcription beyond the rRNA genes is prevented by the mitochondrial transcription termination factor, mTERF1. HSP2 additionally

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so tRNA processing would in most cases yield fully processed mRNAs as a by-product (Ojala et al., 1981). This process is coordinated by a characterized RNase P and an RNase Z activity, which cleave tRNAs at their 5′ and 3′ termini, respectively (Fig. 2A) (Shutt and Shadel, 2010). After cleavage, messenger RNAs are polyadenylated and stabilized through their association with certain factors, including PPR-containing proteins such as LRPPRC, that then direct them to mitochondrial ribosomes for translation.

yields 10 mRNAs and 14 tRNAs from a near-genome length primary transcript (Bonawitz and Clayton, 2006). The sole L-strand promoter gives rise to a primary transcript two-thirds of the genome in length, which encodes a single protein (ND6) and the remaining eight tRNAs. The core elements of the mitochondrial transcription machinery consist of a limited number of nuclear-encoded proteins: the dedicated mitochondrial RNA polymerase (POLRMT) and its accessory subunit (TEFM) (Minczuk et al., 2011), the transcription activator and DNA packaging protein TFAM, and the transcription factor TFB2M (Fig. 1C). Like TFB2M, the protein TFB1M is similar to bacterial rRNA methyltransferases, and both methylate 12S rRNA in vitro. However, they appear to have distinct physiological roles: TFB2M is the primary transcription factor, whereas the principal function of TFB1M is the modification of 12S rRNA (Cotney et al., 2007, 2009). With the complete sequence of human mtDNA came the immediate realization that almost all the mRNAs were flanked by tRNA genes, and

Rnase P

3

Mitochondrial translation Our current understanding of the mechanistic details of human mitochondrial translation depends heavily on extrapolation from bacterial and cytosolic protein synthesis. As yet, we lack an in vitro mitochondrial translation system and an atomic resolution structure of the mitochondrial ribosome. Nevertheless, there has been a veritable explosion in our

tRNase Z

A

Cleavage of primary polycistronic transcripts

LRPPRC

mtPAP 12S

16S

AUG Ribosomal rRNA AAAAA

CCA

AA MRPs

39S

28S

tRNA modifying factors

ARSs

PUS1

MTFMT

TRMU

Mature mRNA

MTO1

Mature ribosomal subunits Mature aminoacyl-tRNA

B Initiation

Elongation 39S

EF-Tumt

fM

fM

Direction of ribosome

EF-Tsmt IF2 IF3

AUG

AUG 28S

P

AAAAA IF3mt

Termination

A

P

A

EFG1

IF2mt

Mature polypeptide

Ribosome recycling mtRRF1

mtRF1a UA(A/G)(AAAA)n UA(A/G)(AAAA)n

UA(A/G)(AAAA)n ICT1? mtRF1?

UA(A/G)(AAAA)n mtRRF2/ EFG2

C12ORF65?

Fig. 2. Post-transcriptional processing and translation of human mitochondrial transcripts. A) Post-transcriptional processes leading to maturation of mitochondrial RNA species. Factors shown in black/red are known to cause mitochondrial translation disorders. B) The four stages of mitochondrial translation. Initiation: an initiation complex consisting of the 28S subunit, fMet-tRNA, mRNA, and IF2/3mt forms and associates with the 39S subunit to form a 55S monosome and then initiation factors are released. Elongation: after a charged aminoacyl-tRNA enters the A site of the monosome (in a ternary complex with GTP-bound EF-TUmt), the growing polypeptide chain is passed from the tRNA in the P site to the new aminoacyl-tRNA in the A site by intrinsic peptidyltransferase activity of the 39S subunit. The monosome translocates along the mRNA in a 5′ to 3′ direction via the action of EFG1 to allow a new codon to enter the A site. Termination: termination factor mtRF1a binds when a stop codon (either UAA or UAG) on the mRNA is recognized. Binding of mtRF1a leads to hydrolysis of the peptide bond between the nascent chain and the tRNA in the P site. Additional factors homologous to mtRF1a, namely ICT1, C12ORF65, and mtRF1, may also be involved in mitochondrial translation termination. Ribosome recycling: the 55S monosome is dissociated via binding of mtRRF1 and mtRRF2 (also known as EFG2) to the A site, releasing the mRNA and tRNA. The ribosome can now commence a new protein synthesis cycle. Factors shown in black/red are known to cause mitochondrial translation disorders.

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knowledge compared to a decade ago. In addition to the RNA elements of mitochondrial translation that are produced in mitochondria from mtDNA, there are a host of factors that are transcribed in the nucleus, synthesized on cytosolic ribosomes, and imported into the organelle. Many of these will be familiar to anyone with even a passing acquaintance with protein synthesis: ribosomal proteins (80 or more), aminoacyl synthetases that deliver amino acids to the ribosome, tRNA modifying proteins, initiation factors (IF2mt and IF3mt), elongation factors (EFG1, EFG2, EF-Tsmt, and EF-Tumt), four termination release factors, mtRF1a, mtRF1, C12ORF65, and ICT1 (although their individual roles in mitochondrial termination remain to be confirmed), and two ribosomal recycling factors, mtRRF1 and mtRRF2. A related fast developing area is that of mitochondrial biogenesis. The formation of cytosolic ribosomes requires in the order of 200 factors, and whilst the number will almost certainly be lower in mitochondria it will nevertheless run to scores of proteins. Those identified or implicated in ribosomal biogenesis to date include the aforementioned TFB1M, C7ORF30, ERAL1, C4ORF14, GTPBP5, and GTPBP10 (Dennerlein et al., 2010; He et al., 2012a, 2012b; Rorbach et al., 2012; Wanschers et al., 2012). Mechanism of mitochondrial translation Mitochondrial protein synthesis can be broken down into four major steps: initiation, elongation, termination and ribosome recycling. These are described below and in Fig. 2B, and have recently been reviewed in greater detail (Christian and Spremulli, 2011). Mitochondrial translation begins with IF3mt prizing apart the two subunits of the 55S mitoribosome and forming a complex with the smaller, 28S subunit. Entrance of the mRNA into the IF3mt:28S subunit complex is followed by pausing to allow the presence of an appropriate start codon to be confirmed before formylmethionyl-tRNA (fMet-tRNA), the first residue in all mitochondrial polypeptide chains, can bind to the first codon. If this step fails, the complex will dissociate. To proceed further, IF2mt must bind to the complex and this facilitates binding to the large 39S subunit, whereupon GTP bound to IF2mt is hydrolyzed. The initiation factors then depart company with the newly restored 55S ribosome, allowing elongation of the polypeptide chain to commence. During elongation, ternary complexes comprised of mitochondrial elongation factor Tu (EF-Tumt), bound GTP, and aminoacyl-tRNAs enter the A site of the mitoribosome to coordinate specific codon:anticodon pairing of the tRNA and mRNA. Correct codon:anticodon pairing leads to EF-Tumt mediated GTP hydrolysis and the release of newly formed EF-Tumt-GDP. Intrinsic enzymatic activity of the ribosome catalyzes the transfer of the peptidyl chain on the tRNA in the P site to the new aminoacyl-tRNA in the A site, thus extending the growing polypeptide chain. While EFG1 facilitates translocation of the ribosome

along the mRNA in a GTP-dependent manner, EF-Tumt-GTP is regenerated by EF-Tsmt and complexes with a new aminoacyl-tRNA. The elongation cycle repeats until a stop codon is encountered. Translation termination occurs when the translocating ribosome encounters a mitochondrial stop codon of either UAA or UAG. In addition, it has recently been reported that upon encountering either an AGA or AGG codon, the mitoribosome performs a −1 frameshift to bring a classical UAG stop codon into the A site, triggering standard termination (Temperley et al., 2010). Once the stop codon has entered the A site, mtRF1a recognizes the codon and binds to the ribosome in its GTP-bound form. This binding event induces hydrolysis of the peptidyl‐tRNA bond in the A site, via the intrinsic peptidyltransferase activity of the 39S subunit, thereby releasing the mature polypeptide from the ribosome. All that remains is to admit the recycling factors mtRRF1 and mtRRF2 to the A-site of the ribosome to induce release of the mRNA. Mitochondrial protein synthesis and human diseases Human disorders associated with protein synthesis deficiency can arise either from mutation in the mitochondrial genome itself (maternal inheritance) or from mutation in the nuclear gene products required for the translation of mRNAs encoded in the mtDNA (Mendelian inheritance) (see Tables 1 and 2). Mitochondrial tRNA mutations The mammalian mitochondrial translation system uses 22 tRNAs to decipher the mitochondrial genetic code, two each for serine and leucine and one each for the other amino acids. Although two-thirds of the mtDNA sequence is allocated to protein-encoding genes, the majority of pathogenic mtDNA mutations causing human disease affect tRNA genes, which account for only 10% of the genome (Levinger et al., 2004). Transfer RNA mutations are associated with a diverse array of clinical phenotypes, including encephalopathies, (cardio)myopathies, hearing impairment, progressive external ophthalmoplegia (PEO), and diabetes mellitus (Scaglia and Wong, 2008). The most frequent and best studied mitochondrial tRNA mutations are the A3243G mutation in tRNALeu(UUR) and the A8344G missense mutation in tRNALys (Fig. 1A). The former is responsible for over 80% of patients with mitochondrial encephalopathy, lactic acidosis and stroke-like episode (MELAS) syndrome [MIM 540000] (Goto et al., 1990, 1992). This syndrome was first described by Pavlakis et al. (1984) and usually presents in children or young adults after normal early development. Symptoms include recurrent vomiting, migraine-like headache, hemiparesis or

Table 1 Classification of common mtDNA mutations and associated disorders. Mutation Protein synthesis genes tRNA

A3243G (tRNALeu(UUR)) Lys

rRNA Large-scale rearrangements

Protein coding genes Multisystemic

Tissue specific

Phenotype

Reference

MELAS, MIDD

Goto et al. (1990); van den Ouweland et al. (1992) Shoffner et al. (1990) Prezant et al. (1993) Dunbar et al. (1993); Holt et al. (1988); Martin Negrier et al. (1998); Moraes et al. (1989); Poulton et al. (1989, 1991, 1994); Rötig et al. (1991, 1992); Zeviani et al. (1988)

A8344G (tRNA ) A1555G (12S rRNA) Deletion/duplication/triplication

MERRF SNHL KSS, CPEO, Pearson syndrome, MIDD, ataxia

G3460A (ND1) G11778A (ND4) T14484C (ND6) T8993C/G (ATPase 6) Point mutations or deletion (Cyt b)

LHON

MILS/NARP Exercise intolerance

Wallace et al. (1988) Howell et al. (1991) Johns et al. (1992) de Vries et al. (1993); Holt et al. (1990) Andreu et al. (1999)

CPEO, chronic progressive external ophthalmoplegia; Cyt b, cytochrome b; KSS, Kearns–Sayre syndrome; LHON, Leber hereditary optic neuropathy; MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; MERRF, myoclonic epilepsy with ragged-red fibers; MIDD, maternally-inherited diabetes and deafness; MILS, maternally-inherited Leigh syndrome; NARP, neuropathy, ataxia, and retinitis pigmentosa; ND1/4/6, NADH dehydrogenase subunits 1/4/6; SNHL, sensorineural hearing loss.

S. Pearce et al. / Molecular and Cellular Neuroscience 55 (2013) 1–12

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Table 2 Nuclear genes associated with impaired mitochondrial protein synthesis in humans. Gene

Protein

tRNA-modifying enzymes PUS1 Pseudouridine synthase 1 TRMU MTO1 MTFMT

tRNA 5-methylaminomthyl2-thiouridylate methyltransferase Mitochondrial translation optimization 1 homolog Mitochondrial methionyl-tRNA formyltransferase

Aminoacyl-tRNA synthetases DARS2 Aspartyl-tRNA synthetase 2 RARS2 EARS2

Arginyl-tRNA synthetase 2 Glutamyl-tRNA synthetase 2

MARS2 YARS2

Methionyl-tRNA synthetase 2 Tyrosyl-tRNA synthetase 2

HARS2

Histidyl-tRNA synthetase 2

AARS2 SARS2

Alanyl-tRNA synthetase 2 Seryl-tRNA synthetase 2

FARS2

Phenylalanyl-tRNA synthetase 2

Ribosomal proteins MRPL3 Mitochondrial ribosomal protein L3 MRPS16 Mitochondrial ribosomal protein S16 MRPS22 Mitochondrial ribosomal protein S22 Elongation factors GFM1 Elongation factor G 1, mitochondrial (EFG1mt) TUFM TSFM

Clinical phenotype

Age of onset

OMIM

Myopathy, lactic acidosis, and sideroblastic anemia (MLASA1) Acute liver failure

Juvenile

600462 Bykhovskaya et al. (2004a)

Infantile

Hypertrophic cardiomyopathy and lactic acidosis Leigh syndrome

Infantile

613070 Schara et al. (2011); Zeharia et al. (2009) 614702 Ghezzi et al. (2012)

Leukoencephalopathy with brainstem and spinal cord involvement and high lactate (LBSL) Pontocerebellar hypoplasia type 6 (PCHD-6) Leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) Autosomal recessive spastic ataxia with leukoencephalopathy Myopathy, lactic acidosis, and sideroblastic anemia (MLASA2) Perrault syndrome (sensorineural deafness, ovarian dysgenesis) Hypertrophic cardiomyopathy HUPRA syndrome (hyperuricemia, pulmonary hypertension, renal failure in infancy, and alkalosis) Alpers syndrome, encephalopathy, epilepsy, lactic acidosis

Juvenile (early childhood)

–

Juvenile

611105 Scheper et al. (2007)

Neonatal Infantile

611523 Edvardson et al. (2007) – Steenweg et al. (2012)

Juvenile or adulthood Infantile

– Bayat et al. (2012) 613561 Riley et al. (2010)

Juvenile or adulthood

–

Infantile Infantile

614096 Götz et al. (2011) 613845 Belostotsky et al. (2011)

Neonatal and infantile –

Tucker et al. (2011)

Pierce et al. (2011)

Elo et al. (2012); Shamseldin et al. (2012)

Hypertrophic cardiomyopathy and psychomotor retardation Infantile

614582 Galmiche et al. (2011)

Agenesia of the corpus callosum, hypotonia, and fatal neonatal lactic acidosis Edema, fatal cardiomyopathy, and tubulopathy Cornelia de Lange-like syndrome

Neonatal

610498 Miller et al. (2004)

Neonatal

611719 Saada et al. (2007); Smits et al. (2011b)

Neonatal

Neonatal

609060 Coenen et al. (2004); Smits et al. (2011a); Valente et al. (2007) 610678 Valente et al. (2007)

Neonatal

610505 Smeitink et al. (2006)

Infantile

613559 Antonicka et al. (2010)

Juvenile

–

Infantile

220111 Mootha et al. (2003)

Juvenile (early childhood)

613672 Crosby et al. (2010)

Encephalopathy with or without liver involvement

Elongation factor Tu, Lactic acidosis, leukoencephalopathy and polymicrogyria mitochondrial (EF-TUmt) Elongation factor Ts, mitochondrial Encephalomyopathy, hypertrophic cardiomyopathy (EF-Tsmt)

Termination factors C12orf65 Chromosome 12 open reading frame 65

Reference

Leigh syndrome, optic atrophy, ophthalmoplegia

mRNA stability factors and translation activators TACO1 Translational activator of Late-onset Leigh syndrome cytochrome c oxidase 1 LRPPRC Leucine-rich PPR-motif containing Leigh syndrome French–Canadian variant (LSFC) protein MTPAP Mitochondrial poly-A polymerase Progressive spastic ataxia with optic atrophy

hemianopsia, hyperlactacidemia, and stroke-like episodes. Magnetic resonance imaging (MRI) of the brain shows infarcts that do not correspond to the distribution of major vessels. Mitochondrial cytopathy, mitochondrial angiopathy, and endothelial dysfunction are all considered possible underlying causes of these stroke-like lesions (Koga et al., 2010). The clinical features associated with the A3243G mutation are heterogeneous, with some individuals developing diabetes and deafness rather than suffering from any evident neurological dysfunction (van den Ouweland et al., 1992). In other cases, the same mutation is recognized as a cause of cardiomyopathy (Wortmann et al., 2007). This highlights one of the enduring paradoxes of mtDNA diseases: how can the same primary mutation cause such tissue-specific and distinct clinical syndromes? The A8344G mutation is associated chiefly with MERRF (myoclonic epilepsy with ragged red fibers) syndrome [MIM 545000]. This

Weraarpachai et al. (2009)

presents as a multisystem disorder characterized by myoclonus, followed by generalized epilepsy, ataxia, weakness, and dementia. Onset is usually during childhood, occurring after normal early development. Common findings are hearing loss, short stature, optic atrophy, and cardiomyopathy with Wolff–Parkinson–White (WPW) syndrome. Occasionally, pigmentary retinopathy and lipomatosis are observed (Rötig, 2011; Shoffner et al., 1990). Notably, the A8344G substitution was the first tRNA mutation shown to directly cause a defect of mitochondrial protein synthesis (Chomyn et al., 1991). Mutation in mt-tRNAs can lead to translation defects by numerous means, including impaired tRNA processing (Levinger et al., 2004), decreased aminoacylation (Enriquez et al., 1995), inability of the mutant aminoacylated tRNA to interact with the translation elongation factor EF-Tumt (Hino et al., 2004), or inhibition of taurine modification in the anticodon region (Kirino et al., 2004). A common end point of

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these abnormalities is tRNA instability. Stability of tRNAs depends on having a constant interaction with proteins, such as CCA-adding enzyme, aminoacyl-tRNA synthetases, modification enzymes, EF-Tumt, or the mitoribosome, which are collectively involved in tRNA synthesis, maturation, and function (Belostotsky et al., 2012). Hence, any mutation that affects one of these steps can lead to tRNA instability and degradation. It is not surprising, therefore, that many pathological mitochondrial tRNA mutations are associated with a decrease in individual tRNA steady-state level. The importance of these ancillary proteins is demonstrated by their ability to partially rescue mutant tRNA-associated phenotypes. For example, over-expression of either leucyl-tRNA synthetase, or elongation factors EF-Tumt and EFG2, ameliorate the effects of the pathological A3243G mutation in cultured cells (Park et al., 2008; Sasarman et al., 2008). Although mutations that suppress phenotype in mtDNA can occur spontaneously (El Meziane et al., 1998), there is currently no robust method for site-directed mutagenesis of mtDNA. Thus, elevated expression of tRNA interacting proteins represents one of the few potential therapeutic approaches for treating tRNA-related mitochondrial DNA disorders.

(Metodiev et al., 2009). Speculated causative polymorphisms in this locus were not within the coding region of the gene, and therefore it is possible that they affect expression of the protein. However, it is presumably reduced, as opposed to increased, expression of TFB1M that is the key to avoiding hearing loss, as the A1555G mtDNA mutation leads to hypermethylation of 12S rRNA (Cotney et al., 2009). This hypermethylation is purported to be part of a retrograde signal from mitochondria to the nucleus stimulating an increase in mitochondrial biogenesis, but also predisposing the cells to apoptosis (Raimundo et al., 2012). Alternatively, the A1555G mutation may affect ribosomal assembly or stability, and TFB1M might compensate by boosting ribosomal biogenesis. The key question is whether the TFB1M polymorphisms proposed to be associated with increased penetrance of A1555G-linked SNHL increases or decreases TFB1M expression. If the hearing impaired individuals have increased levels of TFB1M in cochlear hair cells, this would provide compelling evidence for the ‘hypermethylation hypothesis’ as the underlying cause of the disease. mRNA stability

Mitochondrial rRNA mutations Mitochondrial ribosomes are more closely related to bacterial ribosomes than their cytosolic counterparts. Unfortunately, this means that a number of antibiotics interfere with mitochondrial protein synthesis, a notable example being chloramphenicol. Mutations in 16S rRNA confer chloramphenicol resistance on human cultured cells (Blanc et al., 1981), demonstrating that the ribosome in mitochondria is the ‘victim’ of the drug, as in bacteria. Although chloramphenicol resistant mutations in the 16S rDNA gene were found in cultured cells over three decades ago, there is as yet no report of a pathogenic 16S rRNA mutation. In contrast, individual mutations at five nucleotide positions within the mitochondrial 12S rRNA gene (827, 961, 1095, 1494, and 1555) have been linked to human disease. These mutations all result in non-syndromic sensorineural hearing loss (SNHL), which in many cases manifests only following exposure to aminoglycoside antibiotics (Rötig, 2011; Xing et al., 2007). The A1555G substitution was the first 12S rDNA mutation to be described (Prezant et al., 1993), and it is the single most common cause of aminoglycoside-induced SNHL (Fig. 1A), accounting for 17% of the US and Spanish cases of aminoglycoside ototoxicity, and up to 87% of cases among Asians (del Castillo et al., 2003; Xing et al., 2007). In most individuals the A1555G mtDNA is homoplasmic. When it co-exists with wild-type mtDNA there is a clear correlation between mutant load and hearing loss (del Castillo et al., 2003). Thus, individuals carrying this mutation are particularly sensitive to developing SNHL. The proposed mechanism involves aminoglycoside accumulation in the inner ear where it interacts with 12S rRNA thereby stabilizing mismatched aminoacyl-tRNAs and decreasing mitochondrial protein synthesis. This leads in turn to reduced ATP production in cochlear hair cells (Prezant et al., 1993). Not only is the A1555G mutation associated with congenital SNHL as well as aminoglycoside ototoxicity in some pedigrees, but the penetrance is highly variable and more extensive clinical phenotypes have been described, including Parkinson's disease and cardiomyopathy (Xing et al., 2007). This suggests that the A1555G mutation requires other factors to cause disease, such as aminoglycoside exposure and/or the appropriate variants of particular nuclear genes (TFB1M, TRMU, MTO1, and GTPBP3) (Bykhovskaya et al., 2004b, 2004c; Estivill et al., 1998; Guan et al., 2006; Prezant et al., 1993). Linkage analysis has led to speculation that the TFB1M gene locus on chromosome 6 may modulate the phenotypic penetrance of the A1555G mutation (Bykhovskaya et al., 2004b). TFB1M dimethylates 12S rRNA of the small mitochondrial ribosomal subunit, at a site only 28 residues from nucleotide 1555, and without the addition of two methyl groups at this site the 28S subunit cannot be assembled

As in other systems, polyadenylation of messenger RNAs in animal mitochondria is a key element of gene expression, yet the mitochondrial procedure has its own unique features. Some mtmRNA coding regions terminate with U or a single A after processing and so polyadenylation is required to create a UAA stop codon (Anderson et al., 1981). Thus, this process is essential for the generation of certain functional mRNAs that can be translated in mitochondria. Polyadenylation in mitochondria may also be involved in editing the 3′-acceptor region of some tRNAs and has been shown to increase the stability of several mt-mRNAs, while decreasing the stability of others (Rorbach et al., 2011). In human mitochondria, polyadenylation of mRNA is performed by the mitochondrial poly(A) RNA polymerase (mtPAP) (Nagaike et al., 2005; Tomecki et al., 2004). Recently, a homozygous A1432G substitution in the nuclear-encoded MTPAP gene was discovered in an Amish family exhibiting progressive spastic ataxia with optic atrophy, associated with severely truncated poly(A) tails [MIM 613672] (Crosby et al., 2010). The newly solved structure for mtPAP indicates that the mutated residue is highly conserved and lies within the ‘fingers’ domain of the polymerase (Bai et al., 2011). The aberration in mtPAP function results in a loss of polyadenylation of specific mitochondrial mRNAs. However, residual oligoadenylation is retained, suggesting either some residual function of mtPAP or an alternative means of oligoadenylation in mitochondria. Abnormal tRNA modifications In the tRNA maturation process, a subset of the nucleotide bases within a nascent tRNA molecule undergoes specific modifications, which are critical for folding and codon recognition. One of the best understood modifications is pseudouridylation, common to both nuclear and mitochondrial tRNAs. It is proposed that pseudouridylation facilitates base-pairing and base-stacking within the tRNA secondary structure, increasing the overall stability and functional conformation of tRNAs (Patton et al., 2005). Pseudouridine synthase I (PUS1) has been implicated in this process in both cellular compartments. Mutations in PUS1 gene are causative of mitochondrial myopathy and sideroblastic anemia (MLASA) [MIM 600462], a rare autosomal recessive disorder characterized by defects in oxidative phosphorylation and iron metabolism, with symptoms specific to skeletal muscle and bone marrow (Bykhovskaya et al., 2004a). The disease is clinically heterogeneous, which has in part been attributed to the dual function of PUS1 in nuclear and mitochondrial tRNA maturation, and so perturbation of PUS1 function can impact differentially on the two translation systems (Fernandez-Vizarra et al., 2007).

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Modifications to the first base of the anticodon, the so-called wobble position, enable tRNAs to recognize multiple codons. Three mitochondrial enzymes, TRMU, MTO1, and GTPBP3, are responsible for the highly conserved 5-carboxymethylaminomethylation (mnm 5s 2U34) modification of the wobble uridine base in glutamine, glutamate, and lysine tRNAs in bacteria, and yeast and human mitochondria (Shutt and Shadel, 2010; Suzuki et al., 2001; Umeda et al., 2005; Wang et al., 2010; Yan et al., 2005). Moreover, in humans these three enzymes act as nuclear-modifier genes that influence the penetrance of the deafness-associated A1555G mutation (Bykhovskaya et al., 2004c; Guan et al., 2006). In particular, when a TRMU missense A10S mutation is co-inherited with the A1555G mutation the likelihood of an individual developing hearing impairment increases. The mutant form of TRMU lowers the steady-state levels of lysine, glutamate, and glutamine tRNAs owing to their instability, which is due to deficient wobble-position modification. The A1555G mutation is located in the aminoacyl-tRNA decoding A site of the mitochondrial ribosome, and is thought to induce a conformational change in 12S rRNA (Prezant et al., 1993). Hence, the combination of mutant 12S rRNA and a shortage of certain tRNAs compromises mitochondrial translation to such an extent that it leads to cellular dysfunction and cochlear hair cell death (Guan et al., 2006). Recently, additional mutations in TRMU have been implicated in acute fatal infantile liver failure [MIM 613070] (Schara et al., 2011; Zeharia et al., 2009). This has been ascribed to a transient lack of cysteine, the sulfur donor for the thiouridylation reaction, resulting in a mitochondrial translation defect early in development. Although the severe reduction in 2-thiolation of mitochondrial tRNA Lys, tRNA Glu, and tRNA Gln was recapitulated in fibroblasts of a TRMU patient, and the TRMU protein was undetectable by immunoblotting, mitochondrial translation was not measurably different from control cells (Sasarman et al., 2011). However, limiting cysteine in the growth medium might reveal a defect. Impaired tRNA modification is also predicted to underlie the hypertrophic cardiomyopathy and lactic acidosis associated with mutations in human MTO1 [MIM 614702] (Ghezzi et al., 2012). Mutations in methionyl-tRNA formyltransferase (MTFMT) are another cause of defective mitochondrial translation (Tucker et al., 2011). Analogous to the bacterial pathway, mitochondrial translation initiates with N-formylmethionine (fMet) forming the first residue of newly translated peptides. The requisite fMet-tRNAMet is generated from aminoacylated tRNAMet by MTFMT. Tucker and colleagues described two patients presenting with Leigh syndrome and combined OXPHOS deficiency, whose fibroblasts lacked detectable formylated fMet-tRNAMet. Thus, some residual mitochondrial translation must occur in its absence, as there would be no OXPHOS activity without the protein products of mtDNA. Abnormal aminoacyl-tRNA synthetases An early essential step of protein translation involves covalently attaching an amino acid to its cognate transfer RNA. This process, often referred to as tRNA charging, is performed by a highly specialized group of enzymes, the aminoacyl-tRNA synthetases (ARSs). Reflecting their fundamental importance, ARSs are ubiquitously expressed enzymes that are found in all domains of life. Thirty-six ARSs are sufficient to aminoacylate all tRNAs in humans: 16 act exclusively in the cytoplasm, 17 act exclusively in the mitochondria, and three are bi-compartmental. Although few mutations have been reported in the cytosolic ARSs, an increasing number of mitochondrial ARS mutations have been linked to highly tissue-specific diseases in both humans and mice. An intriguing observation is that mutations of genes encoding mitochondrial ARSs are usually associated with specific clinical syndromes. For instance, leukoencephalopathy with brainstem and spinal cord involvement and high lactate (LBSL) is caused by DARS2 mutations (440 published cases) [MIM 611105] (Scheper et al., 2007), pathogenic RARS2 mutations cause pontocerebellar hypoplasia type 6 (PCHD-6)

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(seven published cases) [MIM 611523] (Edvardson et al., 2007), and leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) is associated with EARS2 mutations (Steenweg et al., 2012). Furthermore, complex rearrangements in MARS2 were recently shown to cause autosomal recessive spastic ataxia with leukoencephalopathy (ARSAL) in 54 individuals (Bayat et al., 2012). In this report, complex I activity, MARS2 protein levels and mitochondrial protein synthesis were reduced in both patients and Drosophila mutants. However, in patient-derived fibroblasts steady-state levels of the cognate tRNA were similar to controls. Interestingly, the patients and the flies displayed increased production of reactive oxygen species. The resulting progressive neurodegeneration and myopathy in the flies was partially suppressed by antioxidant treatment, implying a potential therapeutic avenue for ARSAL patients, and possibly other ARS deficiencies. Although currently far fewer in number, patients with mutations in YARS2, HARS2, AARS2, SARS2 and FARS2 also display distinct phenotype– genotype relationships. YARS2 was identified as the affected gene in two families with myopathy, lactic acidosis, and sideroblastic anemia (MLASA) [MIM 613561] (Riley et al., 2010). HARS2 has been found mutated in one family with Perrault syndrome, a sex-influenced disorder characterized by sensorineural deafness in both males and females, and ovarian dysgenesis in females (Pierce et al., 2011). Two mutations in AARS2 have been reported to cause fatal perinatal or infantile hypertrophic cardiomyopathy [MIM 614096] (Götz et al., 2011). A recessive SARS2 mutation was associated with infantile HUPRA syndrome, a multisystem mitochondrial cytopathy defined by hyperuricemia, pulmonary hypertension, renal failure in infancy, and alkalosis [MIM 613845] (Belostotsky et al., 2011). Finally, mutations in FARS2 have been recently associated with mitochondrial encephalopathy Alpers type, thus expanding the genetic cause of Alpers syndrome (previously only associated with mutations in POLG1 or Twinkle) (Elo et al., 2012; Shamseldin et al., 2012). Despite our increasing awareness of pathological ARS mutations, the mechanisms underpinning the specific clinical– genetic associations are unknown. Mitoribosomes and disease Mammalian mitochondrial ribosomes are distinct from both cytosolic and ancestrally-related bacterial ribosomes in both their protein and ribosomal RNA content, and together with the OXPHOS complexes, are the only cellular structures containing components encoded by both the mitochondrial and nuclear genomes. Mitoribosomes have a lower sedimentation coefficient (55S) than the ribosomes of prokaryotes, due to smaller rRNA species, but are actually larger and heavier owing to their higher protein content. They comprise a 28S small subunit (SSU) made of 12S RNA and about 30 proteins, and a 39S large subunit composed of 16S RNA and approximately 50 proteins (Koc et al., 2001a, 2001b). The differences in protein and RNA content between prokaryote ribosomes and eukaryote mitoribosomes reflect major structural differences between the two ribonucleoprotein complexes. Consequently, it is assumed that eukaryotic mitochondrial ancestors have lost some of their mitochondrial rRNA and replaced it with novel proteins, bi-functional proteins, or N- or C-terminal extensions of existing proteins (O'Brien, 2002; Smits et al., 2007). Given this input of new protein it is not surprising that several mammalian ribosomal proteins exhibit extra-ribosomal functions, most intriguingly apoptosis (Koc et al., 2001c). Of the roughly 80 identified ribosomal proteins, only 3 have been firmly linked to mitochondrial disease, MRPS16, MRPS22, and MRPL3. A nonsense mutation in MRPS16 was found to cause agenesia of the corpus callosum, hypotonia, and fatal neonatal lactic acidosis [MIM 610498] (Miller et al., 2004). Two mutations in MRPS22 have been reported and linked to different phenotypes characterized by edema, fatal cardiomyopathy, and tubulopathy in one case (Saada et al., 2007) and Cornelia de Lange-like syndrome in the second [MIM 611719] (Smits et al., 2011b). Both defects resulted in an impaired

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assembly of small ribosomal subunits, leading to a marked decrease in 12S rRNA and of several protein components of the SSU. Proper assembly of MRPs into ribosomes is critical for efficient translation. However, our current knowledge of the factors involved and the sequence of events for 55S monosome formation is limited. Recent reports have identified several proteins important for the process, including C7ORF30, ERAL1, and C7ORF14 (NOA1) (Dennerlein et al., 2010; He et al., 2012b; Rorbach et al., 2012; Wanschers et al., 2012). Another recent study identified an mTERF family member, mTERF4, as an essential factor in mitochondrial ribosome assembly where its precise role appears to be targeting of the rRNA methyltransferase NSUN4 to the 39S subunit (Cámara et al., 2011). Previously, the mTERF family members had been implicated in mtDNA transcriptional regulation (Guja and Garcia-Diaz, 2011). Recently, a mutation in MRPL3 was identified in a patient with hypertrophic cardiomyopathy and psychomotor retardation. The mutation altered the stability of MRPL3 and resulted in defective assembly of the large ribosomal subunit with a severe decrease of mitochondrial translation [MIM 614582] (Galmiche et al., 2011). Other work has identified proteolytic processing as a key element in mitochondrial ribosomal biogenesis, and this is particularly relevant from the clinical perspective. Members of the AAA family use their ATPase domain to perform diverse cellular functions. For example, the matrix-AAA (m-AAA) protease of mitochondria both degrades misfolded proteins and regulates mitochondrial biogenesis by controlling proteolytic maturation of the ribosomal subunit MRPL32 for proper incorporation into, and functioning of, mitochondrial ribosomes in yeast (Nolden et al., 2005). Synthesis of mitochondrially-encoded proteins is strongly impaired in cells lacking the m-AAA protease, indicating that mitochondrial translation depends on maturation of MrpL32. Both subunits of the m-AAA protease are conserved in higher eukaryotes (AFG3L2 and SGP7/paraplegin) (Atorino et al., 2003; Nolden et al., 2005) and the mitochondrial dysfunction associated with paraplegin ablation in yeast is mirrored in liver mitochondria of paraplegindeficient mice (Nolden et al., 2005). In humans, mutations in SPG7 cause hereditary spastic paraplegia (HSP) [MIM 607259] (Casari et al., 1998), whereas those in AFG3L2 cause spinocerebellar ataxia (SCA) [MIM 610246] (Di Bella et al., 2010) and Spastic ataxia neuropathy syndrome (Pierson et al., 2011). Thus, while there is no report of reduced mitochondrial protein synthesis in patients with SPG7 or AFG3L2 mutations, aberrant processing of MRPs might impair mitochondrial ribosomal biogenesis and translation, provoking the neurodegenerative phenotype observed in HSP and SCA patients. If true, this would still leave unanswered the basis of the selective involvement of specific neurons in HSP and SCA. Mutations in elongation factors Mutations in genes encoding components of the mitochondrial translation elongation machinery are yet another established cause of encephalopathy and other organ failures. These include elongation factors EF-Tumt (TUFM) (Valente et al., 2007), EF-Tsmt (TSFM) (Smeitink et al., 2006), and EFG1 (GFM1) (Antonicka et al., 2006; Coenen et al., 2004; Smits et al., 2011a; Valente et al., 2007). Affected patients typically present in infancy and die young. They often have profound OXPHOS deficiencies, which are invariably associated with decreased mitochondrial translation. Nevertheless, clinical symptoms vary greatly between patients, even between those with mutations in the same gene. Mutations in EFG1 result in encephalopathy presenting either with (Antonicka et al., 2006; Coenen et al., 2004) or without liver involvement [MIM 609060] (Smits et al., 2011a; Valente et al., 2007). Remarkably, in one patient mitochondrial protein synthesis was impaired in fibroblasts but not in muscle. The phenotypic differences might result from mutation-specific effects on the stability of the protein in different tissues, or variation in the abundance of other elongation factors (Antonicka et al., 2006).

Indeed, two studies have shown that the relative abundance of elongation factors is important in determining mitochondrial translation efficiency (Antonicka et al., 2006; Smeitink et al., 2006). A fatal mutation in TUFM was identified in a patient who presented with lactic acidosis, a diffuse cystic leukoencephalopathy, and polymicrogyria [MIM 610678] (Valente et al., 2007). The mutation, located in the tRNA binding region of EF-Tumt, impairs the formation of a ternary complex with GTP and aminoacyl-tRNA, leading to a severe defect in protein synthesis (Valente et al., 2009). Three unrelated pediatric cases share the same mutation in TSFM, encoding EF-Tsmt, yet the clinical features were quite different, being associated with either mitochondrial encephalomyopathy or hypertrophic cardiomyopathy, or a combination of both [MIM 610505] (Smeitink et al., 2006). Translation termination Once synthesis of the polypeptide is complete, translation termination factors catalyze its release from the mitoribosome. In mitochondria, three proteins are involved in translation termination: a single class I release factor mtRF1a/mtRF1L and two ribosome recycling factors (Christian and Spremulli, 2011). All known class I release factors have a GGQ motif at the active site. Based on sequence similarity, three additional putative family members with the GGQ motif were recently identified: mtRF1, ICT1, and C12orf65 (Richter et al., 2010). ICT1 is a peptidyl-tRNA hydrolase, but as yet little is known about the other two factors. However, C12orf65 has been identified as a disease gene in two unrelated families with Leigh syndrome, optic atrophy, and ophthalmoplegia (Antonicka et al., 2010). Although C12orf65 does not exhibit peptidyl-tRNA hydrolase activity in vitro, overexpression of ICT1 partially rescued the cytochrome c oxidase (COX) defect in patient fibroblasts suggesting the two proteins have overlapping functions. Translation activators The mechanisms that regulate mitochondrial translation remain largely unexplored in mammalian systems. Yeast mitochondrial mRNAs contain untranslated sequences upstream of the open-reading frame (so-called 5′ UTRs), to which bind specific translation activators (Herrmann et al., 2012). Mammalian mitochondrial mRNAs have no such 5′ UTRs, suggesting that alternative mechanisms must exist to promote and regulate translation. Recently, two mammalian proteins have been proposed to act as translation activators. TACO1 is necessary for the efficient translation of COXI, and mutations in the corresponding gene are associated with late-onset Leigh syndrome and COX deficiency [MIM 612958] (Weraarpachai et al., 2009). The second potential translation activator is LRPPRC (leucine-rich pentatricopeptide repeat containing protein), which is a loose homolog of Pet309, the yeast mitochondrial translation activator of COXI and COXIII. Mutations in LRPPRC have been identified as the cause of French–Canadian variant of Leigh syndrome (LSFC) [MIM 220111] (Mootha et al., 2003; Xu et al., 2004). A recent study outlines the effects of a cardiac muscle-specific knockout mouse model of LRPPRC (Ruzzenente et al., 2012). Results indicated that the lack of LRPPRC activity leads to reduced steady-state levels of most mitochondrial mRNAs and suggested that LRPPRC functions to maintain the stability of pools of untranslated mRNAs. Conclusions and prospects Impairment of mitochondrial protein synthesis is a medically important area of biology. Gene sequencing and proteomic approaches have recently succeeded in identifying many of the factors involved in normal and aberrant mitochondrial translation. We anticipate that exome and whole genome sequencing will provide us with many of the missing pieces of the jigsaw within a few years. Although the details of how mitochondrial protein synthesis leads to neurological dysfunction can only come from studying animal models and humans, yeasts and bacteria

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will continue to advance our understanding of protein synthesis in mitochondria because of the strong evolutionary links. Defects in mitochondrial translation produce diverse clinical phenotypes, with the most severe presenting in infancy as metabolic disorders. However, neurological features tend to manifest later in life. For example, one child who survived the metabolic phase went on to develop severe neurological dysfunction (McShane et al., 1991). Thus, while treatments for mitochondrial diseases are currently scarce, the employment of drugs that stimulate mitochondrial biogenesis such as bezafibrate (Wenz et al., 2008), or as yet to be developed compounds that elevate the expression of the aforementioned genes (AARs and elongation factors), could actually bring problems in their wake. Specifically, shepherding children through the metabolic crises of early life could herald severe neurological problems. Adult onset cases are likely to fare much better from such treatments, and will therefore provide the more appropriate testing ground for new compounds for mitochondrial translation disorders. Gene therapy should be considered as an alternative strategy, and the delivery methods being developed for other Mendelian diseases will be equally applicable to mitochondrial disorders due to mutations in nuclear DNA. However, the challenge is more complicated in the case of disorders due to primary mutations of mtDNA, due to the heteroplasmic nature of many of these conditions. It may prove possible that manipulation of the level of mutant and wild-type mtDNA may be achieved through modification of nuclear gene expression (Malena et al., 2009; Suen et al., 2010). Reversing the pathological mutations directly is a distant prospect as altering the sequence of mtDNA in a heritable manner has not yet been achieved. It has proved possible to alleviate the mitochondrial dysfunction of human cells with mutant mitochondrial tRNA by importing a functional version from the cytoplasm, via the tRNA import machinery of a protozoan (Mahata et al., 2006). Proteins that can bypass defects in respiratory chain complexes, such as Ndi1 and alternative oxidase also hold promise (Bai et al., 2001; Hakkaart et al., 2006; Seo et al., 1998). Of more immediate practical value, genetic counseling and preimplantation diagnosis (PGD) has a fundamental role to play in preventing the transmission of mitochondrial DNA disorders (Poulton et al., 2010). The proportion of mutant mtDNA in chorionic villi or amniotic fluid can be determined and provides a measure of risk of disease transmission dependent on the particular mutation (Thorburn and Dahl, 2001). However, the predictive value of these analyses remains uncertain: the mutant load in amniocytes or chorionic villi might not correspond to that of other fetal tissues, and the mutant load may shift in utero, or postnatally. On the other hand, these approaches offer early and reliable prenatal diagnosis if the mitochondrial disease is the result of a nuclear gene mutation. Pathogenic mtDNA mutations can also be eliminated by transferring an in vitro-fertilized nucleus from the ooplasm of a woman carrying the mutation to an enucleated oocyte of a normal donor, so-called pronuclear transfer. The resulting embryo, containing normal nuclei from mother and father and normal mtDNAs from the donor, can be implanted in the mother's uterus (Craven et al., 2010). This approach is most applicable in cases where the mother carries a severe pathological mutation with no wild-type mtDNA (Taylor and Turnbull, 2005). Acknowledgments The authors are supported by the Medical Research Council, UK. A.S. is in receipt of a European Union Marie Curie Fellowship. C.L.N is supported by the National Institutes of Health, Bethesda, USA. References Al Rawi, S., Louvet-Vallée, S., Djeddi, A., Sachse, M., Culetto, E., Hajjar, C., Boyd, L., Legouis, R., Galy, V., 2011. Postfertilization autophagy of sperm organelles prevents paternal mitochondrial DNA transmission. Science 334, 1144–1147.

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