Translation Initiation in Mammalian Mitochondria - a Prokaryotic Perspective

Abstract

ATP is generated in mitochondria of eukaryotic cells by oxidative phosphorylation (OXPHOS). The OXPHOS complex, crucial for cellular metabolism and comprises of both nuclear and mitochondrial encoded subunits. Also, the occurrence of several pathologies because of mutations in the mitochondrial translation apparatus indicates the importance of mitochondrial translation and its regulation. The mitochondrial translation apparatus is similar to its prokaryotic counterpart due to a common origin of evolution. However, the mitochondrial translation has diverged from the prokaryotic translation in many ways by reductive evolution. In this review, we focus on the aspects of the mammalian mitochondrial translation initiation, a highly regulated step of translation, and present a comparison with prokaryotic translation.

Keywords: mammalian mitochondria, ribosome, mitoribosome, protein synthesis, translation initiation, mitochondrial disease

Introduction

Mitochondria are sub-cellular organelles that perform a number of functions, which are crucial for the cellular homeostasis.  According to the endosymbiont theory, mitochondria are descendants of α-proteobacteria endocytosed by pre-eukaryotic cells ¹ and retained, since they produced ATP. A vast majority of the mitochondrial genes moved to the nuclear genome over time, except the genes encoding mitochondrial tRNAs, rRNAs and 13 subunits of the electron transport chain ². Mitochondria have retained a translational apparatus to synthesise the remaining members of the OXPHOS complex from its genome. Broadly, the mitochondrial translational system is similar to its eubacterial counterpart ³, but mitochondrial translation is unique in the structure and composition of ribosomes and tRNAs, presence of leaderless mRNAs, codon assignments and a smaller number of tRNAs ³. The process of translation initiation is the most highly regulated step of protein synthesis, and perturbations of mitochondrial translation initiation have been associated with human disease. In this article, we survey the idiosyncrasies of mammalian mitochondrial translation initiation based on the features of the mitoribosome, initiator tRNA and initiation factors. We compare and contrast prokaryotic and mammalian mitochondrial translation initiation, to understand how mitochondrial translation has diverged from its evolutionary ancestor and adapted to its eukaryotic environment. We also discuss the various pathologies associated with the dysfunction of mammalian mitochondrial translation initiation. Finally, we propose methods of studying mitochondrial translation in vivo. 

1. The Mitochondrial Ribosome

The bovine mitochondrial ribosome or mitoribosome is a 55S molecule composed of a small 28S and a large 33S subunit. It is a much more proteinaceous complex with an RNA: protein ratio of 1:2 as opposed to 2:1 for prokaryotic and eukaryotic cytosolic ribosomes ³. The reversal in ratio is not due to loss of individual nucleotides, but deletion of entire regions, such as the anti-Shine-Dalgarno (aSD) region, which is accompanied by a simultaneous loss in Shine-Dalgarno (SD) sequence in mitochondrial mRNAs. Many regions such as helix 45 and the sarcin/ ricin loop (SRL) are, however, conserved. Some, but not all excised regions have been replaced by ‘new’ ribosomal proteins that have no known homologs outside of the mitoribosomes. Besides, the proteins have extra sequences compared to their bacterial counterparts^{4, 5}. This has led to a rather ‘porous’ structure with low density.

The small  subunit (28S) is composed of 12S rRNA and 29 proteins and the large  subunit (33S) is made up of 16S rRNA and 48 proteins ². About half the mitoribosomal proteins have homologs in prokaryotes while the rest are unique to mitochondria. A 5S rRNA is not encoded by the mitochondrial genome ⁶ and it was thought that it may be imported into mitochondria through the mitochondrial intermembrane enzyme polynucleotide phosphorylase ⁷. Interestingly, recent cryoelectron microscopy (cryoEM) studies have shown that the mitochondrial tRNA^Val (mt-tRNA^Val) is present in mitochondrial LSU (large subunit) and it plays an integral structural role ⁸ (Fig. 1). The mt-tRNA^Val interacts with mL46, mL40, mL48, uL18m, mL38, bL27m in mt-LSU (Fig. 1A), while 5S rRNA interacts with bL25, uL5, uL18 and bL27 of the prokaryotic ribosome LSU (Fig. 1B). The L18 and L27 homologues are conserved as interacting partners of 5S rRNA/ mt-tRNA^Val across bacteria and mammalian mitochondria.

The Ramakrishnan group solved the structure of human mitoribosome to a resolution of 3.5 Å using single particle cryoEM 8, which revealed that the 28S ribosome lacks homologs of the ribosomal proteins uS8, uS13, uS19, and bS20 and, the uS4 has been replaced by a mitochondria-specific counterpart. The most striking observation from this structure was that the mRNA tunnel was distinctly different from that of the prokaryotic ribosomes, due to the absence of uS4 and a shortening of the C-terminal domain of uS3m, which is partially compensated for by uS5m. This has led to a shift and an expansion of the channel. The mRNA exit region in bacterial ribosomes contains the aSD sequence, which is crucial for bacterial translation initiation. As stated earlier, both the SD (mRNA) and aSD (12S rRNA) sequence features are absent from the mitochondrial translation machinery. The mRNA exit tunnel is composed of mS37, bS21m and bS1m, through which the mitochondrial mRNA emerges during translation. The structural conservation of bS1m with bS1, coupled with similar positions on the respective ribosomes and the presence of a large electropositive patch in the direction of the mRNA indicate that bS1m may be involved in RNA binding. The mRNA channel is lined with positively charged conserved amino acids contributed by an extension of uS5m. Nenad Ban’s group has proposed that the small subunit protein mS39 which is present at the mRNA entrance may initially tether mitochondrial mRNAs. Subsequently, the uS5m extension with its positively charged residues may guide the mRNA through the channel and subsequent codon-anticodon interaction may stabilise mRNA binding ⁹. Additionally, the N-terminus of mL45 is shown to be important for mitochondrial translation, by recruiting the translocation machinery after translation initiation.

The P site of the bacterial 30S ribosomal subunit includes parts of the 3’ major domain, central domain and h44 and the C-terminal tails of ribosomal proteins uS13 and uS9 ^10-12 and this region is crucial for initiator tRNA (i-tRNA) discrimination.  Thus, some components of the human mitoribosome, such as uS12m, uS9m, bS1m and the rRNAs could be important for translation initiation. X-ray crystallography studies of prokaryotic ribosomes indicate that IF1 contacts the ribosomal protein uS12 and 16S rRNA helices 18 and 44 in the A site ¹³. Among the proteins of the 30S subunit, IF3 interacts with bS1, uS2, uS3, uS7, uS11, uS13, bS18, uS19, bS21 and particularly strongly with uS12 ^14-16. The ribosomal protein, uS12 serves to modulate translocation of mRNA-tRNA complex through the ribosome ¹⁷ and it is one of the strongest interacting partners of IF3. IF3_mt can cross-link to mitochondrial homologs of the bacterial ribosomal proteins uS5, uS9, uS10, and bS18 and to the unique mitochondrial ribosomal proteins mS29, mL42, mS36 and mS39 ¹⁸. No cross-links were obtained between IF3_mt and uS12m although eubacterial IF3 interacts strongly with uS12 as stated earlier.

A well-studied protein involved in i-tRNA discrimination at the P site is the ribosomal protein uS9. According to the crystal structure of Thermus thermophilus ribosomes, the C-terminal tail of the uS9 protein contacts the i-tRNA at positions 33 and 34 ¹². The C-terminal tail sequence (SKR) of uS9  is highly conserved amongst bacteria and its deletion leads to a modest decrease in cellular growth at 37 °C, but significant increase in cold sensitivity and a decrease in i-tRNA binding to the 30S ribosome ¹⁹. Additionally, in vivo reporter studies from our lab have established that the absence of the C-terminal SKR sequence leads to an increase in initiation with anticodon stem mutants of i-tRNAs ²⁰. In many of the mycoplasmal uS9, SKR sequence is found to be represented by TKR, which correlates with a change of the conserved R131 to P, F or Y in their IF3 ²¹. Multiple sequence alignments of uS9m from mitochondria and its prokaryotic homologue have revealed the presence of KKR instead of the SKR at the C-terminal tail (Fig. 2). A detailed exploration of the role of the C-terminal tail of mitochondrial uS9m would probably serve to reveal a great deal about the mechanism of fidelity of translation initiation.

2. Mitochondrial Initiation Factors

Three initiation factors are present in all prokaryotes: IF1, IF2 and IF3. In the absence of initiation factors, initiation complex can be formed with any elongator tRNA in the P site, but the presence of all the factors distinctly favours the accommodation of fMet-tRNA^fMet (i-tRNA) at the P site ²². Unlike the eubacterial system, mammalian mitochondria have only two initiation factors: IF2_mt and IF3_mt.

a. Mitochondrial Initiation Factor 2

A mitochondrial initiation factor equivalent to the E. coli IF2 was characterised and studied from bovine mitochondria ²³ and yeast ²⁴. The bovine IF2_mt was first identified as an agent that promoted binding of yeast i-tRNAto mitoribosomes, where the efficiency of binding was tremendously enhanced by GTP, but not GDP ²³. Bovine IF2_mt bears 39% sequence identity with EcoIF2 and can stimulate binding of formylated i-tRNAs from bacteria and yeast, much more efficiently than their non-formylated forms.

IF2_mt is a single subunit protein whichis compatible with mitochondrial, chloroplast and prokaryotic ribosomes ²⁵ but E. coli IF2 is not functional on mitochondrial ribosomes ²³.  IF2_mt also consists of a conserved 37 amino acid insertion between domains V and VI (Fig. 3A, red box; Fig. 3B, pink region). Homologs of IF1 have been found in the organellar translational system of chloroplasts but not in mitochondria.  This phenomenon has been explained by Gaur et al. (2008) using an E. coli system wherein the 37 amino acid protrusion in IF2_mt discharges the functions of IF1. Thus, IF2_mt has a bifunctional role in mammalian mitochondria ²⁶. Subsequently, cryoEM studies indicated that the 37 amino acid domain interacts with the same region on the ribosome where IF1 is known to bind ²⁷. Thus, this helical insertion domain blocks the A site of the ribosome, very much like eubacterial IF1. IF2_mt consists of domains III to VI which are homologous to those found in E. coli of which domain VI binds to i-tRNA²⁸. Recent cryoEM studies have identified that H678 of this domain interacts with the formyl group of the i-tRNA while F632 interacts with the amino acid methionine ⁹.

b. Mitochondrial Initiation Factor 3

There is a rather low sequence homology between eubacterial IF3 and IF3_mt (21-26%) and the sequences of IF3_mt from various eukaryotes are not very conserved ²⁹. However, IF3_mt has a central region with homology to the bacterial factors with additional N-terminal and C-terminal extensions (N_ext and C_ext, respectively), which are absent in eubacterial IF3 (Fig. 4). The closest prokaryotic relatives of IF3_mt are from Mycoplasma species ³⁰. Greater homology is however observed between human IF3_mt and many chloroplast IF3s rather than the prokaryotic IF3s. Previously, the NTD of IF3_mt had been modelled on the structure of the Bacillus stearothermophilus IF3NTD, while the structure of the CTD of mouse IF3_mt (without the C_ext) had been solved by NMR ³¹. The extensions were predicted to be disordered. More recently, cryoEM structures of IF3_mt complexed with the 28S mammalian mitochondrial ribosomal subunit  were obtained at 3.3- 3.5 Å resolution ³².The structures indicate that the NTD is composed of an  α-helix and four β-stranded sheets which are packed against two α-helices (Fig. 4B). The two globular domains are joined by a flexible linker region.

The human IF3_mt has been expressed in E. coli and the purified protein (mass 27 kDa) displayed properties similar to those of eubacterial IF3 ²⁹. According to the in vitro experiments performed by Koc and Spremulli, IF3_mt acts as an anti-association factor for the two mitochondrial ribosomal subunits. It can allow the formation of the initiation complex on mitoribosomes when IF2_mt, fMet-tRNA^Met and poly (A, U, G) or transcripts of a mitochondrial gene are present, but not on 70S ribosomes in the presence of E. coli IF2 and IF1. However, upon inclusion of IF2_mt in place of IF2, the IF3_mt becomes active on 70S ribosomes. As EcoIF2 has two more domains at its N-terminus than IF2_mt ³³, it may obstruct the binding site of IF3_mt. IF3_mt can allow binding of eubacterial mRNA to 55S ribosome even though mitochondrial mRNAs have almost no 5’ or 3’ untranslated nucleotides. The Spremulli group has also subsequently demonstrated that the addition of even a few nucleotides to the 5’ end of the AUG reduces the efficiency of translation initiation in the in vitro mitochondrial system ³⁴.

The roles of the isolated NTD and CTD of IF3_mt have been studied in vitro ³⁵. Most of the interactions of IF3_mt with the 28S subunit are mediated by its CTD which binds to the mitoribosome with two to threefold lower affinity than the full length molecule. IF3_mt NTD binds the mitoribosome with an affinity that is only ten-fold lower than that of the full length molecule indicating that it may have contacts with the ribosome. Recent cryoEM studies confirm these findings since the NTD is found to interact with the mitoribosomal protein uS11 and h23 of the 12S rRNA ³². In the presence of the 39S subunit, both the isolated domains of IF3_mt are displaced from the 28S subunit, although the CTD is displaced more readily. The binding site of IF3_mt CTD with the small subunit is in a region where the latter has contacts with the large subunit. The isolated NTD, with or without the linker region is incapable of dissociating the 55S ribosome. The CTD alone has weak dissociation activity, but in the presence of the linker region, it becomes active in dissociating the 55S subunit. Thus, the linker plays an important role in dissociation. In bacteria, the residue Y75 of the linker which is highly conserved, plays an important function in the fidelity function of IF3 and interacts with C701 of 16S rRNA ³⁶. In IF3_mt, the residues R140 and R144 that are conserved amongst mammals allow the linker to bind to the 28S platform and they may have evolved to compensate for the loss of Y75 ³². Only the C-terminal domain in the presence of the linker region is able to reduce fMet-tRNA^Met binding to 28S or 30S ribosomal subunit in the absence of mRNA. The action of the NTD in this regard even in the presence of the linker region is very weak. Both the NTD and CTD play a very significant role in promoting complex formation between fMet-tRNA^Met and IF2_mt. The NTD is more effective than the CTD in this process and formylation of fMet-tRNA^Met is essential.

The N- and C-termini of IF3_mt have 30 amino acid long extensions compared to bacterial IF3. These extensions are not essential for promoting initiation complex formation on mitochondrial 55S ribosomes. However, the C_ext was found to be essential for the dissociation of fMet-tRNA^Met bound in the absence of mRNA ³⁷. The currently accepted evolutionary role of the C_ext is believed to be a mode of generation of an ordered pathway of mRNA binding prior to fMet-tRNA^Met binding during initiation. Full length IF3_mt interacts weakly with 39S subunits. However, when either the N_ext or the C_ext are deleted, the factor has a higher affinity for the 39S subunit ³⁸. Thus, another role for the extensions is to reduce the affinity of IF3_mt for the protein-rich 39S subunit that could lead to regulation of joining of the two subunits during formation of initiation complex. A truncated derivative of IF3_mt missing the extensions crosslinks to the same mitoribosomal proteins as the full length IF3_mt except that no cross-links were detected to mS36. This indicates that the terminal extensions of IF3_mt do not contribute significantly for its interactions with the mitoribosome. The uS10m, which is near the head region of the small subunit, was the only ribosomal protein that interacted with the NTD of IF3_mt indicating that the NTD of IF3_mt may be interacting in this region. Since the CTD of IF3_mt does not interact with the proteins of the platform region such as uS11m and uS15m, it is possible that the binding of IF3_mt to the mitoribosome differs from the binding of eubacterial IF3 to the 30S subunit.

Our group has recently utilized an E. coli strain, in conjunction with plasmid-borne IF3_mt and its truncated derivatives ³⁹ to characterize IF3_mt in vivo. Using the CAT reporter system and by analysing polysome profiles, we have shown that IF3_mt allowed ‘promiscuous’ translation initiation with non-AUG codons such as AUA, AUU and ACG but it did not permit initiation with i-tRNAs that lack the universally conserved 3GC base pairs in their anticodon stems. Our studies have suggested that the extensions of IF3_mt may have evolved to relax the fidelity of mitochondrial translation initiation in order to accommodate classically non-canonical start codons such as AUU and AUA. Our findings also indicated that the NTD adds to the fidelity function of IF3_mt for start codon and i-tRNA (through its anticodon stem) selections, which are reminiscent of the fidelity functions of the NTD of EcoIF3 ⁴⁰.  This finding is supported by structural studies which indicate that the N_ext may interact with A424 (E. coli A790) of h24 of 12S rRNA and thus the NTD may play a role in i-tRNA binding in the mitoribosomal P site, through its extension ³².

3. Mitochondrial mRNA and mitochondrial initiator tRNA

The protein-coding genes of the mammalian mitochondrial genome are transcribed, post-transcriptionally modified and translated within the mitochondria ². The ORFs are separated by very few nucleotides and, in two instances, the genes overlap, as seen in ND4 and ND4L and in ATPase 6 and ATPase 8. The two distinguishing features of the mammalian mitochondrial mRNAs are the usage of non-AUG start codons such as AUU (for NADH dehydrogenase subunit 2 or ND2 mRNA) and AUA (for ND1, ND3 and ND5 mRNAs) and also the presence of very short or non-existent leader sequences. Studies have also shown the efficient usage of the GUG codon in mammalian mitochondria under diseased conditions ⁴¹. Accurate recognition of the AUA codon as an initiation codon is facilitated by methylation of cytosine 34 in the mitochondrial tRNA^Met by NSUN3 ⁴². According to in vitro studies from the Spremulli group, 5′ phosphate group of leaderless mRNAs is not required for their recruitment to the ribosome ³⁴.

Although, mitochondria of lower eukaryotes such as yeast possess distinct initiator and elongator tRNA^Met, mammalian mitochondria are characterized by the presence of a single tRNA^{Met 30} for its functions in initiation and elongation. The mammalian mitochondrial i-tRNA harbours a combination of features present in eukaryotic cytoplasmic and prokaryotic i-tRNAs and also elongator tRNAs ³⁰. (i) Mitochondrial tRNA^Met has three consecutive GC base pairs in the anticodon stem, which is found virtually in all i-tRNAs. (ii)  Quite like cytoplasmic i-tRNAs, mammalian mitochondrial tRNA^Met has an A:U pair at the beginning of the acceptor helix. This serves as an optimal pair to allow interaction with both IF2_mt and EF-Tu_mt, since many mitochondrial elongator tRNAs also have an A:U pair at the end of the acceptor stem. (iii) Prokaryotic i-tRNAs have a purine 11: pyrimidine 24 pair, which mammalian mitochondrial tRNA^Met also possesses. (iv) Mammalian mitochondrial tRNA^Met partially resembles prokaryotic tRNAs due to the presence of U54 and pyrimidine 60, except that the 54^th position is un-methylated. In general, mammalian mitochondrial tRNA^Met has very few modifications compared to the prokaryotic and eukaryotic i-tRNAs. These modifications include Ψ at positions 27 and 50 and f⁵C in the anticodon. To facilitate initiation with the AUA initiation codon, the C of the CAU anticodon of mitochondrial i-tRNA is modified to 5-formylcytidine (f⁵C) by the successive actions of the RNA methyltransferase NSUN3 and the dioxygenase ABH1 ^42-44. Recently, our group has generated and characterised mutants of E. coli tRNA^fMet which sustained E. coli for its requirement of both the initiator and elongator tRNA^Met. The i-tRNA mutant which was most adept at both the initiation and elongation functions was the one that was also the most similar to mitochondrial tRNA^Met, thus implying that bacterial elongator and initiator tRNA^Met may have originated from a single dual function tRNA ⁴⁵.  

4. Events in mitochondrial translation initiation

Although the 28S mitoribosomal subunit can interact with the mRNA independent of its sequence, the mRNA needs to be at least 350 nucleotides for suitable binding²³. It has also been shown by in vitro studies that the mRNA may bind to the 28S subunit in the absence of any initiation factors ⁴⁶. Mitochondria do not have polysomes and only a single 28S subunit binds to an mRNA at a time²³. It is worth noting that in yeast mitochondria, translational activators such as Pet309 for COX1 mRNA translation or Nca2 for ATP8 mRNA translation are utilised to position mitoribosomes containing the specific mRNAs to the mitochondrial membrane and allow insertion of the nascent polypeptides into the membrane ⁴⁷. 

The initial model for mitochondrial translation postulated by the Spremulli lab proposed that binding of fMet-tRNA^Met by IF2_mt in the absence of mRNA is destabilised by IF3_mt leading to the formation of a nonproductive complex³⁷. In the productive pathway, the 28S subunit may bind the mRNA first but its position on the mRNA would be uncertain due to the absence of an SD-aSD interaction. IF3_mt might alter the position of the mRNA such that the AUG (or an alternate initiation codon) is positioned at the P site (Fig. 5).  Subsequently, IF2_mt might promote fMet-tRNA^Met binding to the P site. This would be followed by 39S binding and subsequent dissociation of the initiation factors and 55S IC formation. In the absence of the C_ext, IF3_mt is not able to dissociate a nonproductive complex. Therefore, the C_ext may have evolved to create an ordered translation initiation pathway. Further, in the absence of extensions, IF3_mt has an enhanced affinity for the 39S subunit and it may allow 39S docking prematurely³⁸. Therefore, another reason for the occurrence of the terminal extensions is to reduce the affinity of IF3_mt to the 39S subunit in order to prevent the formation of incorrect initiation complexes. Ensuing studies by the Spremulli group have allowed the authors to propose that IF3_mt is a dissociation factor and not an anti-association factor for the ribosomal subunits³¹. While studying the preference of leaderless mRNAs in mitochondrial translation ³⁴, the Spremulli group proposed that after dissociation of 55S complexes by IF3_mt, an mRNA enters the ribosome and the ribosome pauses when the first 17 nucleotides enter to inspect the codon at the 5’ end of the mRNA, even if it is not an AUG codon. During this step, IF2_mt allows fMet-tRNA^Met binding at the P site. In the event the start codon or the tRNA at the P site are incorrect, the mRNA moves through the ribosome and the monosome eventually dissociates. If the start codon and the tRNA are canonical, IF2_mt hydrolyzes GTP, initiation factors are released and the 39S subunit associates to form the 55S IC.

Work from our lab has shown that both IF3_mt and EcoIF3 are capable of examining the 3GC base pairs in the anticodon of the tRNA^{Met 39}.However, when both the N_ext and C_ext [IF3_mtΔ(N_extC_ext)] are absent, there is an improvement in the fidelity of i-tRNA selection. Based on the in vitro data, the absence of the extensionsmay facilitate i-tRNA accommodation at the P-site even in the absence of mRNA ³⁷. Thus, any scrutiny of i-tRNA binding would  occur only through the anticodon stem. The absence of the stabilising effect of a cognate codon-anticodon interaction would increase the likelihood of ejection of an incorrect tRNA from the ribosome. The, i-tRNA binding would be stabilised only after mRNA binding ³⁷ in the presence of IF3_mt. If an anticodon stem mutant i-tRNA were to bind to the P site, the presence of a complementary codon-anticodon interaction would be required to decrease the probability of i-tRNA dissociation. Since non-AUG initiation codons are present in the mitochondrial system, such an ejection of i-tRNA would slow down mitochondrial protein synthesis. Thus, the extensions of IF3_mt may have evolved to somewhat lessen anticodon stem based discrimination to allow initiation with non-canonical initiation codons such as AUU and AUA.

Recent in vivo studies ⁴⁰ and structural studies ⁴⁸ have suggested that prokaryotic IF3 may remain bound to the 70S initiation complex due to a recently discovered binding site on the 50S ribosome. These findings are crucial in explaining the occurrence of the 70S mode of initiation for translation of leaderless mRNAs. The Spremullli group has seen that IF3_mt can bind to the 55S ribosome, for the purpose of ribosome dissociation. Further IF3_mt only permits initiation with leaderless mRNAs³⁴. Since the mammalian mitochondrial system utilizes leaderless mRNAs, we propose that it is entirely possible that the mammalian mitochondrial system may also be capable of initiating with monosomes under certain circumstances. It would be interesting to investigate if 55S initiation does indeed occur in mammalian mitochondria and what factors might influence such non-canonical modes of initiation. 

In vivo studies of IF2_mt have been crucial in deciphering the order of the departure of prokaryotic initiation factors from the initiation complex²⁶. Earlier studies in prokaryotes have indicated that the affinity of IF2 for the 30S subunit increases while IF1 is present, whereas the release of IF1 from the ribosomal subunit leads to ejection of IF2 ⁴⁹. Since IF2_mt is a protein that is capable of complementing E. coli for the functions of eubacterial IF1 and IF2, it has been proposed that in the bacterial system also, IF1 and IF2 may depart from the 70S complex simultaneously.  

5. Diseases of mitochondrial translation initiation

Mitochondrial pathologies which can be caused by either nuclear or mitochondrial DNA mutations affect 1 in 5000 people ^{50, 51}. These diseases cause OXPHOS dysfunction that can lead to a variety of diseases, which may be multi-systemic or tissue-specific with varying degrees of severity. Defects in mitochondrial translation are responsible for a large number of these pathologies. Mutations in elongation factors have been known to cause encephalopathy with liver or heart involvement ⁵² while mutation in the mitochondrial termination factor C12orf65 can cause Leigh Syndrome⁵³. Mutations in mitoribosomal proteins such as bS16m may cause neonatal lactic acidosis ⁵⁴ while an uL3m mutation causes cardiomyopathy ⁵⁵. Mitochondrial DNA rearrangements as seen in Kearns Sayre Syndrome can lead to mitochondrial tRNA or rRNA defects ⁵⁰. A large number of mitochondrial DNA mutations affect tRNA genes such as tRNA^Lys (MERRF) and tRNA^Leu (MELAS). Additionally, mutations of factors involved in mitochondrial mRNA maturation, and mitochondrial tRNA processing and aminoacylation have also been implicated in a wide variety of mitochondrial pathologies⁵¹. In this review, we will focus on diseases of mitochondrial translation initiation.    

IF3_mt has been implicated in the pathogenesis of various disorders like Parkinson’s disease (PD), obesity and diabetes.  In PD, a mutation in mitochondrial IF3 has been tentatively implicated in the pathogenesis ⁵⁶. However, a synonymous polymorphism (D266D, resulting from C798T nucleotide change) has been associated with sporadic PD. IF3_mt mutations have also been implicated in obesity, where an SNP in IF3_mt (rs4771122) in  Mexican children was associated with increased body mass index (BMI) ⁵⁷. Interestingly, the same SNP was associated with long term weight loss after bariatric surgery⁵⁸. Each copy of the minor G allele of IF3_mt (rs1885988) was associated with greater weight loss following lifestyle intervention of the Diabetes Prevention Program⁵⁹. Additionally, autoantibodies to IF3_mt were identified in type I diabetes patients ⁶⁰, further implicating the role of IF3_mt in diabetes. Disruptions in modifications of the i-tRNA have been implicated in various disease phenotypes. Mutations in MTFMT (encoding mitochondrial methionyl-tRNA formyltransferase which formylates tRNA^Met) have been isolated in patients with Leigh syndrome and combined OXPHOS deficiency⁶¹. Patient fibroblasts had very low levels of fMet-tRNA^Met that was rescued by overexpression of wild type MTFMT. There are numerous documented cases of mitochondrial disease caused by mitochondrial tRNA^Met mutations ^62-69, all of which are characterized by either mitochondrial myopathy or hypertension. In addition, pathogenic mutations in the anticodon stem loop region of mitochondrial tRNA^Met have been isolated which lead to impaired NSUN3 binding causing hypomodification of ^f5C34 ⁴⁴. Similarly, a compound heterozygous mutation in NSUN3 led to severe mitochondrial disease characterised by numerous clinical presentations including combined OXPHOS deficiency in skeletal muscle, microcephaly and failure to thrive ⁷⁰. Mutations of the AUA initiation codon of the ND1 gene and the AUG initiation codon of the gene encoding subunit II of cytochrome c oxidase to ACG were also shown to be pathogenic mutations ^{71, 72}.

6. Available methods of studying mitochondrial translation in vivo

In vitro mitochondrial translation studies have been limited due to technical difficulties in reconstituting the entire translational machinery ⁷³, and in vivo studies have not been feasible due to inadequate methodologies to manipulate mitochondria ³. Mitochondria are incapable of propagation and survival outside eukaryotic cells. Therefore, a robust in vivo system to study mitochondrial translation is crucial. The resemblances between the eubacterial and mitochondrial protein synthesis have allowed us to establish that IF2_mt can complement E. coli for the essential roles of EcoIF1 and EcoIF2 in E. coli ²⁶. It was shown that the 37 amino acid protrusion of IF2_mt was functionally equivalent to EcoIF1 and this in vivo data was further validated by structural studies ²⁷. Thus, E. coli was found to be an able ‘substitute in vivo system’ to characterise proteins involved in mitochondrial translation. Such a system can be exploited to a greater extent by using reporter genes to functionally characterise a mitochondrial protein. Our lab has recently developed an E. coli strain (infCΔ55- lacking 55 amino acids from the N-terminus of IF3), with compromised IF3 activity⁴⁰. This strain, in conjunction with the chloramphenicol acetyltransferase reporter system has provided a useful heterologous system for characterization of IF3_mt ³⁹. Our group was also able to use a system of tRNA gene deletions in E. coli to investigate how initiator and elongator tRNAs may have evolved from a single bifunctional tRNA^Met45. 

The ideal in vivo method of studying mitochondrial translation would be to carry out reporter-based experiments in human cell lines or whole organisms. Unfortunately, both DNA and RNA import are difficult in mammalian mitochondria ³. To recapitulate, mammalian mitochondria only utilise mRNAs transcribed from their own genomes for translation. Unlike yeast and kinetoplastid protozoa, mammalian mitochondria are self-sufficient for all the tRNAs required during translation and they do not need to import tRNAs from the cytosol. Similarly, the mammalian mitochondrial genome also encodes its own rRNAs (12S and 16S) and mitochondrially encoded tRNA^Val is found in the position of bacterial 5S rRNA ^{8, 74}. Mitochondrial RNaseP, which is an endonuclease that plays a role in the maturation of tRNAs was earlier thought to have a nuclear encoded RNA and multiple protein components like its bacterial and cytosolic counterparts ⁷⁵. However, it was subsequently shown that human mitochondrial RNase P is a protein enzyme without a mitochondrially imported RNA component ⁷⁶. Therefore, it is important to note that there seems to be no requirement for RNA import in mammalian mitochondria and the broader impact of RNA import is still to be shown.

Although reporter studies in mammalian mitochondria appear difficult, work from the Tarassov and Adhya groups have shown that the yeast and Leishmania model systems function for mRNA import. Kinetoplastid protozoa such as the Leishmania species import all the required tRNAs from the cytosol into mitochondria as their mitochondrial genomes do not encode any tRNAs. Work from the Adhya group has shown that tRNA import is mediated by a large multiprotein complex (RNA import complex or RIC) and it is dependent on a tRNA motif (similar to the D arm of tRNA^Tyr) which is present in a majority of Leishmania tRNAs ^{77, 78}. Mitochondrial translation can potentially be studied in the Leishmania system, with the help of reporter mRNAs tagged with the D arm of tRNA^Tyr. In S. cerevisiae, two species of tRNA^Lys have been found in mitochondria ⁷⁹. Since tRNA^LysUUU is fully capable of decoding AAA and AAG codons in the mitochondrial genome, it was surprising that the nuclear encoded tRNA^LysCUU was also found in mitochondria. Subsequent studies revealed that at a high temperature (37 ^◦C), tRNA^LysUUU is undermodified and hence unable to fulfil its functions⁸⁰. At this temperature, tRNA^LysCUU is recruited to decode the AAG codon. Recruitment of tRNA^LysCUU is a two-step process. On aminoacylation by the cognate cytoplasmic tRNA synthetase, tRNA^LysCUU can either enter the cytoplasmic translation system or bind to enolase-2 which escorts the tRNA to the precursor of the mitochondrial lysyl tRNA synthetase on the outer mitochondrial membrane^{81, 82}. With the help of the Tim/Tom protein import machinery, tRNA^LysCUU is finally able to enter the mitochondrion ⁸³. It has been shown that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNA^LysCUU and facilitates internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be a member of the yeast RNA import machinery. The Tarassov lab has shown that short synthetic RNAs (referred to as FD-RNA) comprising two domains of the tRNA^Lys were individually sufficient for highly efficient to deliver RNAs into mitochondria. This technology could be adopted to send reporter mRNAs into yeast mitochondria in order to characterise mitochondrial translation in vivo.Such systems would help us study initiation codon preference, codon usage and the influence of a 5’ UTR on mitochondrial translation in vivo. In addition, there are a number of untapped resources to potentially target nucleic acids to mitochondria. For instance, triphenylphosphine-coated nanoparticles have been used to target cancer drugs to the mitochondria of breast cancer cells ⁸⁴. This technology could be engineered to target reporter mRNAs to human cells.  

In the absence of reporter genes, another method of studying mitochondrial translation initiation is by means of gene deletions and knockdowns. Currently, our group is in the process of obtaining and analysing mitochondrial initiation factor knockdowns and knockouts. Such a system can also be used to study the functions of individual domains of translational factors as well as their disease-causing mutants.

7. Concluding remarks

Considerable progress has been made in the last decade to decipher the cryptic process of mitochondrial translation initiation: from superior structural analyses of the human mitoribosome to clearer biochemical characterisations of mitochondrial i-tRNA and initiation factors, and studies of naturally occurring pathogenic mutations. However, the need of the hour is to develop suitable RNA or DNA import mechanisms for efficient reporter studies to comprehensively characterise translation in mammalian mitochondria.

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Table 1: Human diseases due to defects in mitochondrial translation initiation.

Figure legends

Fig 1. Structures of (A) human mitochondrial (PDB ID: 3J9M) and (B) E. coli ribosomes (PDB ID: 6O9J) bound to tRNA^Val and 5S rRNA, respectively.

Fig 2. Multiple sequence alignment of uS9m C-terminal tail (in the red box) from mammalian mitochondria and uS9 from E. coli.

Fig 3. Initiation factor 2. (A) Multiple sequence alignment of IF2 from E. coli, T. thermophilus and human mitochondria. Structures of (B) Human IF2_mt (PDB ID: 6GAZ) (C) T. thermophilus IF2 (PDB ID: 3J4J). The region in pink in (B) and the red box in (A) denote the extension of human IF2_mt that discharges the functions of bacterial IF1.

Fig 4. Initiation factor 3. (A) Multiple sequence alignment of IF3 from E. coli, T. thermophilus and human mitochondria. Structure of (B) Human IF3_mt (PDB ID: 6NEQ) (C) T. thermophilus IF3 (PDB ID: 5LMN). The colours in the structures represent the domains shown by arrows in (A).

Fig 5.  Mitochondrial translation initiation pathway.