Mitochondrial transcription and translation: overview

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

Received: 26 March 2018

Revised: 14 May 2018

Accepted: 14 May 2018

Version of Record published:

20 July 2018

Review Article

Mitochondrial transcription and translation:

overview

Aaron R. D’Souza and Michal Minczuk

MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, U.K.

Correspondence: Michal Minczuk ([email protected])

Mitochondria are the major source of ATP in the cell. Five multi-subunit complexes in the

inner membrane of the organelle are involved in the oxidative phosphorylation required for

ATP production. Thirteen subunits of these complexes are encoded by the mitochondrial

genome often referred to as mtDNA. For this reason, the expression of mtDNA is vital for

the assembly and functioning of the oxidative phosphorylation complexes. Defects of the

mechanisms regulating mtDNA gene expression have been associated with deciencies

in assembly of these complexes, resulting in mitochondrial diseases. Recently, numerous

factors involved in these processes have been identied and characterized leading to a

deeper understanding of the mechanisms that underlie mitochondrial diseases.

Introduction

Mitochondrial gene expression is central to maintaining cellular homoeostasis. The control of mitochon-

drial gene expression is unique in that its components have dual origins in the mitochondria (all RNAs)

and nucleus (all protein factors). The regulation of the synthesis anddegradation of mitochondrial (mt-)

RNAs determines steady-state levelsofmitochondriallyencoded proteins allowing fine control of the

mitochondrial energy metabolism. Therefore, the cell can adapttochangingenvironmental stresses and

satisfy changing cellular energy demands. Defects in the mitochondrial gene expression can lead

to respi-

ratory chain dysfunction resulting in a multi-system disease phenotype, predominantlyaffectingmuscular

and neuronal tissues.

The mammalian mitochondrial genome is highlycondensed as far as genetic information is concerned.

The mitochondrial genome encodes 2 rRNAs, 22 tRNAs and mRNAs for 13polypeptides of the oxidative

phosphorylation (OxPhos) system. Insomecases,thereduction of the mitochondrial genome has led to

overlapping genes (MT-ATP8/6, MT-N D4/4L and mt-tRNA

Tyr

/mt-tRNA

Cys

). The entire mitochondrial

genome is transcribed from both the strandsaslong polycistronic transcripts. These strandsarenamed

heavy (H)orlight (L) based on their buoyancy in caesium chloride density gradients. The long poly-

cistronic transcripts require multiple processing steps before individual RNA species become functional.

After endonucleolytic cleavage of the primary transcript, the ribosomal RNAs undergo chemical modifi-

cations before it can function correctly within the mitoribosome, the tRNA s also undergo a large number

of chemical modifications, in addition to further polymerization and aminoacylation, and the mRNAs are

differentiallypolyadenylate

d.Finally, the mRNAs, tRNAs and the assembled mitoribosome come together

in the translation apparatus where translational factors direct the progression of translation (Figure 1).

Inthisarticle, we briefly overview the key stages of mitochondrial gene expression in humans, pro-

viding a useful basis for the articlebyBoczonadietal.thatdealswithhumandiseases resulting from the

defects in this pathway [1].Themaingoal of our articleistopresentthebasicmitochondrial function

of protein factors that have been associated with mitochondrialdisease, therefore, some proteins known

to be involved in mitochondrial transcription and translation might have not been described in this brief

overview.

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution

License 4.0 (CC BY).

309

brought to you by COREView metadata, citation and similar papers at core.ac.uk

provided by Apollo

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

Figure 1. Overview of human mitochondrial transcription, RNA processing and translation

The list of proteins mentioned in the gure are biased towards those that are associated with mitochondrial diseases, as explained

in the article by Boczonadi et al. [1]. Aminoacyl-tRNA synthetases are abbreviated as xARS and xARS2.

Mitochondrial transcription

Transcription of the mitochondrial geno me originates in the major non-coding region containing the L-strand (LSP)

andH-strand (HSP) promoters. The light strand promoter controls the transcription of eight of the tRNAs and the

MT-ND6 gene. On the heavy strand,twoH-strand two-promoter systems have historicallybeenproposed,where

HSP1 transcription produces a transcript containing tRNA

Phe

,tRNA

Val

and the two rRNAs (12S and16S), while

transcription from HSP2 generates a transcript that spans almost the entire genome [2-4].Thistwo-promoter model

of H-strand transcription was proposed to explain the high abundance of the two rRNAs. However, more recent

animal models[5] and in vitro [6] experiments suggest that heavy strand transcription is under the control of a

singlepromoterand that the difference in rRNA abundance may be a consequence of dif ferential turnover .

Transcription initiation

Transcription in human mitochondria is driven by a DNA-dependant RNA polymerase called POLRMT, which is

structurallysimilar to RNA polymerases in T3 and T7 bacteriophages [7,8].Thisincludes high sequence homology

to the C-terminal catalytic core of the enzyme [9].AttheN-terminaldomain, POLRMTalso contains two p en-

tatricopeptide repeat (PPR) domains, commonlyfound in RNA-associated proteins, where they are required for

site-specific interactions [7,10]. Incontrastwithbacteriophagepolymerases, which can recognize promoter regions

without auxiliary proteins, additional factors are required to perform this function by POLRMT. The initiation of

310

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons

Attribution License 4.0 (CC BY).

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

transcription requires the association of POLRMT with mitochondrial transcription factor A (TFAM)and mito-

chondrial transcription factor B2 (TFB2M). TFAM is a DNA-binding protein, w hich, in addition to transcription

activation, also packages DNA in the nucleoid [11].TFB2M was produced as a r esultofageneduplication event.

TFB1M, the other product of this duplication event, is a ribosomal RNA methyltransferase (see below). Although

TFB2M alsocontainsarRNAmethyltransferase domain, the key function of this protein is DNA melting during the

initiation of transcription [12-16].Recentevidence shows that in the transcription initiation complex, at both the HSP

and

LSP, TFAM,bound to DNA, recruits POLRMTtothepromoterviaitsN-terminal extension. TFB2M modifies

the structure of POLRMTtoinduce opening of the promoter [14,17,18].

Transcription elongation

POLRMTrequiresanadditional transcription elongation factor (TEFM)fortheelongation stage [19].Recombinant

TEFM stronglypromotesPOLRMTprocessivityasitstimulates the formation of longer transcripts in vitro [20].

Also, depletion of TEFM in living cells leadstoareduction in promoter–distal transcription elongation products

[19]. Transcription from LSP is often prematurelyterminated around the conserved sequence block 2 (CSB2) of

the major non-coding region of the mitochondrial genome. The short RNA moleculeproduced has been suggested

to p

lay a key roleinprimingDNA replication as multipleRNAtoDNA transition sites clustering aroundCSB2

[21,22] (see also the articlebyMaria Falkenberg [23]). Stimulation of POLRMTprocessivitybyTEFM prevents the

formation of G-quadraplexes that inhibit the progression of the elongation complex at CSB2 [20,24].Thecapability of

TEFM to abolish premature transcription termination has been proposed to function as the switch from replication

to the transcription of the LSP-derived primary transcript [24].Recentstructural work showed that TEFM contains

apseudonuclease core that forms a ‘sliding clamp’ around the mtDNA

downstream of the transcribing POLRMT,

interacting with POLRMTviaitsC-terminaldomain [25].

Transcription termination

The mechanism of termination of HSP transcription is still unclear. Itwaspreviouslysuggested that mitochondrial

termination factor 1 (MTERF)bendsthemtDNA connecting the HSP1 promoter site and its apparent tRNA

Leu(UUR)

termination site. MTERF1 would then induce transcription termination through base flipping andDNA unwinding

[26-28].Thismodel was originallyproposed to explain the 50-fold higher abundance of mitochondrial rRNAs [27].

However, more recent e vidence contradicts this hypothesis. Studies in MTERF 1 knockout mice donotshowaneffect

on rRNA steady-state levels[5]. Their increase in abundance is probablyaproduct of increased stability rather than

due to the presence of a different promoter. Moreover, it was also recently sho wn that transcription from the LSP is

prematurelyterminated by MTERF1 at the 3



-end of the mt-rRNA coding sequence. Binding of MTERF1 to this site

prevents the replication fork f rom progressing into the mt-rRNA genes while they are being transcribed,whilst also

preventing transcription of the antisense sequence of the rRNA [5,29,30].

Maturation of the primary transcript

Transcription from the heavy andlight strand promoters produces long polycistronic transcripts. The mt-rRNA cod-

ing sequences and most of the protein coding sequences are separated by mt-tRNAs. Endonucleolytic excision of

these mt-tRNAs releases the mRNAs and rRNAs – aconceptknownasthe‘tRNA Punctuation Model’ [31,32].The

processing of mt-tRNAs from the primary transcript is performed by RNase P and RNase Z at the 5



- and 3



-end

respectively. Unlike previously characterized cytoplasmic and bacterial RNase P enzymes, which contain a catalytic

RNA subunit, mammalian mitochondrial RNase P is an entirely proteinaceous heterotrimeric endonuclease. This

enzyme is composed of a tRNA m

R9 methyltransferase, TRMT10C (MRPP1), a member of the short-chain dehy-

drogenase/reductase (SDR) family, SDR5C1 (HSD17B10, MRPP2), and aproteinwithhomology to PiITN-terminus

(PIN) domain-like metallon ucleases, PRORP (MRPP3) and cleaves the primary transcript at the 5



-end of tRNAs [33].

ELAC2isanendonuclease that executes 3



-end maturation of both mitochondrial and nuclear pre-tRNAs [34-36].

The ‘tRNA punctuation model’ does not explain all the primary transcript cleavage events, as not all mRNAs are

immediatelyflanked by tRNAs. Various Fas-activated serine/threonine kinase (FASTK) proteins have been shown to

be required for mtRNA stability and the processing of precursors, especiallythenon-canonical cleavage sites. They all

contain a conserved nuclease fold (RAP domain); however, endonucleolytic activity has not been shown for any of the

FASTK proteins [37]. For example, depletionorknockoutofFASTKD2 leadstotheaccumulation of various cl

eav-

age precursors, especially 16rRNAand ND6mRNA[38,39].Additionally, FASTK has been implicated in MT-ND6

maturation and stability, and FASTKD5,similar to FASTKD4,regulates the maturation of those precursor RNAs that

cannot be processed by RNase P and ELAC2 [39,40]. Furthermore, cross-linking immunoprecipitation (CLIP)-based

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution

License 4.0 (CC BY).

311

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

analysis o f FADTKD2binding sites identified16S rRNA and ND6asitstargets[38].Recently, FASTKD4 was shown to

be required for the stable expression of several mt-mRNAs, whereas FASTKD1 had the opposite effect on the stability

of the MT-ND3mt-mRNA. Interestingly, depletion of both FASTKD1 and FASTKD4 also caused a loss of MT-ND3,

suggesting that the loss of FASTKD4 is epistatic [37]. Moreover, FASTKD4 has been suggested to be responsiblefor

the cleavage of the MT-ND5-CYBprecursor.Adetaile

d characterization of how the FASTK proteins regulate the

mitochondrial transcriptome is likelytobeasubject of intense studyinthenearfuture.

mRNA maturation and stability

After excision from the primary transcript, all mRNAs, except MT-ND6, undergo 3



polyadenylation. Polyadeny lation

in mitochondria is performed by a homodimeric polyadenylic acid RNA polymerase (mtPAP) [41-43].Sevenofthir-

teen mt-mRNAs donotcontaina3



stop codon. In these cases, 3



adenylation completes these stop codons and thus,

the open reading frame. Polyadenylation of bacterial transcripts generallymarkthemfordegradation, whereas addi-

tion of poly(A) tailstoeukaryotic,nuclear-encoded mRNA is necessary for their stability. However , in mammalian

mitochondria the effect of polyadenylation on steady-state levelsismRNA-sp ecific. For example, deadenylation de-

creases complex IV mt-mRNA and increases complex I mt-mRNA levels[41,

44-46].

The stability of HSP -derived mitochondrial transcripts is regulated by leucine-rich penticopeptiderichdomain

containing protein (LRPPRC)[47]. Loss of LRPPRC reduces the steady-state levelsofmRNAswhilst not affecting

rRNAs and tRNAs, consequently leading to a t ranslation defect andloss of respiratory complexes [48-51].Thepres-

ence or absence of LRPPRC in the mitochondria correlates with the level of mt-mRNA polyadenylation [52,53].As

such, LRPPRC mouse knockout models d

isplay a loss in HSP-derived transcripts, loss of poly(A) tailsand asevere

translationaldefect [50]. More recent data show that LRPPRC is a mt-mRNA chaperone that relaxes secondary struc-

tures, therefore, facilitating RNA polyadenylation and coordinated mitochondrial translation [54].Following translo-

cation into mitochondria, LRPPRC forms a complex with a stem–loop interacting RNA-binding protein (SLIRP) [55].

Within this complex, SLIRP stabilizes LRPPRC by protecting it from degradation [56]

,whilst being dispensablefor

polyadenylation of mtDNA-encoded mRNAs [56]. The LRPPRC–SLIRP complex has also been shown to suppress

their degradation of mt-mRNAs [57].

Human mitochondrial RNA decay is mediated by a complex of p olynucleotide phospho rylase (PNPase) and human

Suv3 protein (hSuv3) [58].PNPaseisa3



–5



exoribonuclease which has been shown to localize to the intermembrane

space [59],and also in distinct foci with hSuv3p and mitochondrial RNA [58].Knockdown of PNPase leadstothe

increase in the half-life of mitochondrial transcripts and the accumulation of RNA decay intermediates [58].hSuv3p

is an NTP-dependent helicase. The lack of a functioning hSuv3 helicase leadstotheaccumulation of aberrant RNA

species, polyadenylated molecules anddegradation intermediates [60].Recentevidence shows that exposure to the

intercalating agent ethidium bromide (EtBr), which disrupts tRNA second

ary structure, causes them to be polyadeny-

lated.Subsequentwithdrawal of EtBr causes the polyadenylated tRNAs to be rapidly degraded by the PNPase–hSuv3

degradosome [61].Knockdown of PNPase leadstolengthening of the poly(A) tails due to inhibited tRNA turnover

[61]. Controversially, the localization of PNPase in the intermembrane space has led to implications that it plays a

roleintheimportofendogenous RNA into mitochondria. However, various pieces of evidence suggest that this is

not the case and that it primarily functions in the RNA degradosome [62].

tRNA maturation

The mt-tRNAs undergo extensive post-transcriptional maturation including chemical nucleotidemodifications and

CCAaddition at the 3



-enddeadenylation. One of the key tRNA positions of chemical modification is the ‘wob-

ble’ base (p osition 34)attheanticodon of the tRNAs. During translation, the appropriate amino acyl-tRNA is po-

sitioned in the mitoribosome through the accurate recognition of a cognate mRNA codon. However, since, many

codons codeforthesameaminoacid,thefirstpositionofthetRNAanticodon is chemicallymodified to facilitate

non-Watson–Crick bas e pairing, therefore expanding codon recognition during mitochondrial translation. Some of

the enzymes involved in modifying this position include: NSUN3 and ABH1 which are required for the introduc-

tion of 5-formy

lcytosine at the wobbleposition(f

C34)ofmt-tRNA

Met

[63-66], MTO1 andGTPBP3 are required for

the biogenesis of 5-taurinomethyluridine (τm

U)[67,68],andMTU1 (TRMU)whichcatalyses the 2-thiolation of

5-taurinomethylridine to form τm

U of a subset of mt-tRNAs [69,70].

Inaddition to the modification of the wobbleposition,position37downstream of the anticodon is also frequently

chemicallymodified in order to facilitate stablecodon–anticodon interactions and, therefore, increasing accuracy

and fidelity of mitochondrial translation. Examples of enzymes that are responsibleformodifying mt-tRNA position

312

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons

Attribution License 4.0 (CC BY).

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

37 includeTRIT1 responsiblefortheintroduction of an isopentenyl group onto N

of 37 adenine (i

A37)inasmall

subset of mt-tRNAs [71] or TRMT5 which introduces N

-methylation of the 37 guanosine (m

G37)[72].

Pseudouridine (Psi), the most common RN A modification, is often referred to as the fifth nucleotide. Itisastruc-

tural isomer of uridine produced by a rotation around the N3–C6 axis. Psi is generallyassociated with a stabilizing

role, by providing structural rigidity to RNA molecules regardless of sequence or str ucture, and has been detected in

several mt-tRNAs. PUS1 is a pseudouridinesynthetasewhichmodifies U27 andU28 on mt-tRNAs [73,74].Recently,

apseudouridine synthetase, RPU

SD4,wascharacterized as introducing pseudouridine at position 39 of tRNA

Phe

[75].

Other putative PUSs have been identified as necessary for mitochondrial translation, including RPUSD3and TRUB2;

however, their exact mtRNA targets remain to be further characterized [76,77].

The CCAfound at the 3



-end of all mature mt-tRNAs is not encoded by mtDNA and is instead

post-transcriptionallysynthesized by tRNA -nucleotidyltransferase 1 (TRNT1): TRNT1does not require a template

sequence, instead preferentiallyselecting CTP and ATP for polymerization [78].Similarly, a non-encoded5



guanine

on mt-tRNA

His

is post-transcriptionallyadded by 3



–5



polymerase activity probablyprovided by THG1L[79].The3



endsofseveral mt-tRNAs undergo spurious, mistargeted adenylation precluding correct aminoacylation at the 3



-end

(see below). A 3



–5



exonuclease, PDE12, is required for the removal of these spurious poly(A) tails[45,80].

tRNA aminoacylation

Mitochondrial translationrequiresthateachtRNAischarged with the cognate amino acid.Thisprocessismediated by

the mitochondrial aminoacyl-tRNA synthetases (ARS2s), which are encoded by nuclear genes. Ofthese,17 ARS2s are

unique to the mitochondria, while GARS (Glycyl-tRNA synthetase) and KARS (Lysyl-tRNA synthetase) are encoded

by the same loci as the c ytoplasmic enzymes, with the mitochondrial isoforms being generated by alternative transla-

tion initiation (GARS) [81] or alternative splicing (KARS) [82]. Interestingly, glutaminyl mt-tRNA (mt-tRNA

Gln

)

is aminoacylated by an indirect pathway, in which it is first charged with glutamic acid (Glu) by mitochondrial

glutamyl-tRNA synthetase (EARS2), after which the Glu-mt-tRNA

Gln

is transamidated into Gln-mt-tRNA

Gln

,us-

ing free glutamine as an amide donor [83].Thislatter conversion is performed by GatCAB, the glutamyl-tRNA

Gln

amidotransferase protein complex, which consists of three subunits:GatA (QRSL1), GatB (GATB) andGatC (GATC)

[84].

Mitochondrial ribosome: structure and assembly

The mitoribosome consists of a large 39S subunit (mtLSU)and asmall 28S (mtSSU)subunit.Compared with

the bacterial ribosome, the mammalian mitoribosome has reduced rRNA components. To compensate for this, 36

mitochondria-specific proteins have been recruited to the ribosome, primarilyfound at the periphery of the com-

plex surrounding a highlyconserved catalytic core [85-89]. Inthebacterial ribosome, a 5SrRNAactsasascaffold

interconnecting the LSU,SSU and the tRNAs in the intersubunit space. However, recent structures of the mitoribo-

some instead ident i fied the recruitment of a mitochondriallyencoded tRNA to this site (tRNA

Val

in humans and rat,

tRNA

Phe

in porcine and bovine mitochondr ia) [88,90,91].

The maturation of the mitoribosome requires the post-transcriptional processing of the catalytic rRNA in addition

to the import and assemblyofabout80 nuclear-encoded proteins (MRPL andMRPS proteins). As for other ribosomes,

both the small subunit and the large subunit rRNA undergo chemical nucleotidemodifications. These modifications

includebasemethylations, 2



-O-ribose methylations and pseudouridylation, with several enzymes responsiblefor

these modifications having been identified (TRMT61B[92]; TFB1M [93]; NSUN4 [94]; MRMs[95]; RPUSD4 [76];

reviewed in [96]). For example, the A -loop region of the 16S rRNA is methylated at position U1369 andG1370

(human mtDNA numbering). This site directly interacts with the aminoacyl-tRNA [88,97]. U1369 is methylated by

MRM2, which has been shown to interact with the mtLSU. Depletion of the protein

leadstodefective biogenesis of the

mtLSU and consequently, a deficiency in translation [97].Also, several p r otein factors not directlyinvolved in rRNA

modification have been identified to coordinate the assemblyofthemitoribosomereviewed in [96]. For example,

ERAL1,ahomolog of the bacterial Era protein that belongs to the conserved familyofGTP-binding proteins, has

been proposed toactasanRNAchaperonethatstabilizes 12S mt-rRNA during mtSSU assembly[98].

Man y of the proteins involved in mitoribosome assemblyand the post-transcriptional processing of the nascent

transcript, inclu

ding FASTK proteins, ELAC2 or RNase P (see above) are found in distinct foci called mitochondrial

RNA g ranules (MRG). This compartmentalization has been proposed to facilitatemoreefficientand accurate gene

expression [40,99,100]. Ithasbeensuggested that an integral inner membrane protein, RMND1,stabilizes and an-

chors the mitochondrial ribosome at the inner membrane, adjacent to MRGswherethemRNAsareproduced and

processed [101]. How ever, the exact function and mechanism of this protein is still unclear.

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution

License 4.0 (CC BY).

313

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

Translation

Mitochondrial translation is fully dependent on various nuclear-encoded regulatory proteins. Inthemammalian mi-

tochondria, the mitochondrial initiation factors, mtIF2 and mtIF3, control the initiation of translation [102]. During

initiation, mtIF3 positions the AUG or AUAinitiationcodons of the mRNA at the peptidyl (P) site in the mtSSU and

prevents the premature association of the mtLSU and mtSSU [103-105].Asinall protein synthesis systems, translation

in mitochondria is initiated with a methionine residue. However , mitochondria differ in that onlyasingletRNA

Met

is used for both initiation and elongation. Discrimination, instead,isachieved through a post-transcriptional modi-

fication, with the aminoacylated initiator mt-tRNA

Met

being subjected to formylation of methionine (fMet), thereby

increasing its affinity for mtIF2 [106].mtIF2 directs the association of t he fMet-tRNA

Met

with the mRNA, and guides

the assemblyofthemitochondrial monosome and the initiation of t ranslation [107,108].

Translationinmammalian mitochondria differs from that of the cytoplasmorthatoftheyeastmitochondria in part

due to the general absence of 5



-untranslated regions (UTRs) on mRNAs, gene-specific RNA cis-acting regulatory

elements and introns. Inyeast,a5



-UTRs allow mRNA -specific, translational activators to bind anddirect the mRNA

into the mitoribosome for translation. However , in mammalian mitochondria, such mRNA regulatory elements have

not been identified. Hence, alternative mechanisms are in place for the regulation of translation. Unlike UTR-based

regulation, these protein factors have to binddirectlytothemitochondrial transcript and affect gene expression.

For example,variousproteinfactorssuchasTACO1, MITRAC or C12orf62 have been recruited to modulate the

translation of complex IV subunit CO1. Absence of any of these protein leadstoacomplex IV deficiency [109-111]

Elongation of translation is mediated by mitochondrial elongation factors, EFTu (TUFM), EFTs (TSFM)and EFGM

(GFM1)[112,113]. Inelongation, EFTu forms a complex with GTP and aminoacyl tRNA. It directs the tRNA to

the acceptor (A) site where the tRNA base pairs with the mRNA at the codon–anticodon site. The hydrolysis of

GTP catalyses peptidebond formation. EFTu is released and the GTP:EFTu complex is re-established by EFTs [114].

EFG1-mt causes the release of the deacetylated

tRNA from the P-site, trans locates the peptidyl-tRNAs from the A

and PsitetothePand exit (E) site, also causing the mRNA to move along by one codon.

Termination of mitochondrial translation is finallytriggered by the presence of a stop codon at the A-site. Four

mitochondrial proteins with homology to ribosome release factors have been identified in humans, including mtRF1,

mtRF1a, C12orf65 andICT1.Thesefactorsarecharacterized bythepresenceofatripeptide GGQ motif that confers

peptidyl-tRNA hydrolase activity [115,116].Structural analysis of mtRF1 suggested that it is capableofrecognizingthe

UAA andUAG

stop codons, targeting ribosomes with a vacant A-site [117,118]. However, it does not exhibit release

activity in vitro [119].mtRF1acatalyses the hydrolysis of peptidyl tRNA at the UAA andUAG stop codons [120].

mtRF1ahasbeenproposed to be sufficient for the termination of translation of all 13mtDNA-encoded polypeptides,

despite the mRNAs for MT-CO1 andMT-ND6 lacking the UAA andUAG stop codons at the end oftheopenreading

frame (ORF). Instead,theseO

RFs contain in-frame AGAand AGG as the last codons respectively. AGAand AGG

are used to encode Arg according to the universal genetic code; however, they are not used for this purpose in any

of the mitochondria ORFs. Fine mapping of the termination codons of the mRN As sho wed that these two mRNAs

terminate at the UAG stop codon possiblycreated as a resultofa−1 frameshift of the mitoribosome [121,122].

Initially, ICT1 was suggested as the protein that performed the termination function at the AGAand AGG codons.

However, neither ICT1 nor C12orf65 release factor homol

ogues containing the specific domains required for UAA

andUAG stop codon recognition [116].Recentevidence also suggests that ICT1 is capableofinducing hydrolysis of

the peptidyl tRNAs in stalled mitoribosomes [119,123].SinceICT1 is incapableofperformingthepeptidyl hydrolase

activity, where the RNA template extends 14 nucleotides beyond the A-site,asisthecaseinMT-CO1 andMT-ND6,

ICT1 may not be directlyinvolved in the termination of transl

ation of these two mRNAs [124,125].

Finally, after the release of the polypeptide, mitochondrial ribosomal recycling factors, mtRRF and EFG2(also

known as RRF2M,ahomologue of EFGM)catalyse the release of the mRNAs, deacetylated tRNAs and the ribosomal

subunits [126,127].

Concluding remarks

Diseases affecting mitochondrial transcription and translation, as described in the articlebyBoczonadietal.[1],can

have multi-systemic and severe manifestation. The develop ment of novel,treatmentsofthesemitochondrialdiseases

canbemademoreeffectivethroughadeeper understanding of the underlying mechanisms that cause them.

314

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons

Attribution License 4.0 (CC BY).

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

Summary

• Progression of mitochondrial transcription and translation requires the sequential recruitment of

different, nuclear-encoded initiation, elongation and termination factors.

• Almost the entire mitochondrial genome is transcribed as long polycistronic transcripts.

• Maturation of the transcripts requires endonucleolytic cleavage, but not all mRNAs are produced

through RNase P and RNase Z function.

• Mitochondrial mRNA steady-state levels are mainly controlled post-transcriptionally.

• The role mitochondrial mRNA polyadenylation is not fully understood.

• Mitochondrial tRNAs undergo extensive chemical modications, including the addition and re-

moval of nucleotides during their maturation.

• Aminoacyl tRNA-synthetases charge tRNAs with their cognate amino acid, many of which are

unique to the mitochondria.

• Mammalian mitoribosomes differ considerably from other ribosomes as far as architecture and

composition are concerned, with the key differences being the reversed protein:RNA mass ratio,

incorporation of mtDNA-encoded structural tRNA and many novel, mitochondria-specic protein

components.

• The assembly of the mitoribosome assembly pathway is likely to be considerably different from

its bacterial counterpart, implying the presence of mitochondria-specic regulatory factors.

Acknowledgements

We thank Dr Christopher Powell and other members of the Mitochondrial Genetics group at the MRC MBU, University of Cam-

bridge for stimulating discussion during the course of this work.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the Medical Research Council, U.K. [MC U105697135 and MC UU 00015/4].

Author Contribution

A.R.D. contributed to drafting this review. A.R.D. and M.M. contributed to revising it and approved the nal version. A.R.D. pre-

pared the gure.

Abbreviations

ARS2, aminoacyl-tRNA synthetase; CSB2, conserved sequence block 2; FASTK, Fas-activated serine/threonine kinase; HSP,

H-strand promoter; hSuv3, human Suv3 protein; LRPPRC, leucine-rich penticopeptide rich domain containing protein; LSP,

L-strand promoter; MTERF1, mitochondrial termination factor 1; PNPase, polynucleotide phosphorylase; SLIRP, stem–loop

interacting RNA-binding protein; TRNT1, tRNA-nucleotidyltransferase 1.

References

1 Boczonadi, V., Ricci, G. and Horvath, R. (2018) Mitochondrial DNA transcription and translation: clinical syndromes. Essays Biochem. 62, 321–340,

https://doi.org/10.1042/EBC20170103

2 Chang, D.D. and Clayton, D.A. (1984) Precise identiﬁcation of individual promoters for transcription of each strand of human mitochondrial DNA. Cell

36, 635–643, https://doi.org/10.1016/0092-8674(84)90343-X

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons

Attribution License 4.0 (CC BY).

315

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

3 Montoya, J., Christianson, T., Levens, D., Rabinowitz, M. and Attardi, G.(1982) Identiﬁcation of initiation sites for heavy-strand and light-strand

transcription in human mitochondrial DNA. Proc. Natl. Acad. Sci. U.S.A. 79,7195–7199, https://doi.org/10.1073/pnas.79.23.7195

4Montoya,J.,Gaines, G.L. and Attardi, G.(1983) The pattern of transcription of the human mitochondrial rRNA genes reveals two overlapping

transcription units. Cell 34, 151–159, https://doi.org/10.1016/0092-8674(83)90145-9

5 Terzioglu, M., Ruzzenente, B., Harmel, J., Mourier, A., Jemt, E., Lopez, M.D. et al. (2013) MTERF1 binds mtDNA to prevent transcriptional interference

at the light-strand promoter but is dispensable for rRNA gene transcription regulation. Cell Metab. 17, 618–626,

https://doi.org/10.1016/j.cmet.2013.03.006

6 Litonin, D., Sologub, M., Shi, Y., Savkina, M., Anikin, M., Falkenberg, M. et al. (2010) Human mitochondrial transcription revisited: only TFAM and

TFB2M are required for transcription of the mitochondrial genes in vitro. J. Biol. Chem. 285, 18129–18133, https://doi.org/10.1074/jbc.C110.128918

7 Ringel, R., Sologub, M., Morozov, Y.I., Litonin, D., Cramer, P. and Temiakov, D. (2011) Structure of human mitochondrial RNA polymerase. Nature 478,

269–273, https://doi.org/10.1038/nature10435

8Tiranti,V.,Savoia, A., Forti, F., D’Apolito, M.F., Centra, M., Rocchi, M. et al. (1997) Identiﬁcation of the gene encoding the human mitochondrial RNA

polymerase (h-mtRPOL) by cyberscreening of the expressed sequence tags database. Hum. Mol. Genet. 6, 615–625,

https://doi.org/10.1093/hmg/6.4.615

9 Jeruzalmi, D. and S teitz, T.A. (1998) Structure of T7 RNA polymerase complexed to the transcriptional inhibitor T7 lysozyme. EMBO J. 17,

4101–4113, https://doi.org/10.1093/emboj/17.14.4101

10 Barkan, A., Rojas, M., Fujii, S., Yap, A., Chong, Y.S., Bond, C.S. et al. (2012) A combinatorial amino acid code for RNA recognition by pentatricopeptide

repeat proteins. PLoS Genet. 8, e1002910, https://doi.org/10.1371/journal.pgen.1002910

11 Kanki, T., Nakayama, H., Sasaki, N., Takio, K., Alam, T.I., Hamasaki, N. et al. (2004) Mitochondrial nucleoid and transcription factor A. Ann. N. Y. Acad.

Sci. 1011, 61–68, https://doi.org/10.1196/annals.1293.007

12 Falkenberg, M., Gaspari, M., Rantanen, A., Trifunovic, A., Larsson, N.G.andGustafsson, C.M. (2002) Mitochondrial transcription factors B1 and B2

activate transcription of human mtDNA. Nat. Genet. 31,289–294, https://doi.org/10.1038/ng909

13 McCulloch, V., Seidel-Rogol, B.L. and Shadel, G.S. (2002) A human mitochondrial transcription factor is related to RNA adenine methyltransferases and

binds S-adenosylmethionine. Mol. Cell. Biol. 22, 1116–1125, https://doi.org/10.1128/MCB.22.4.1116-1125.2002

14 Posse, V. and Gustafsson, C.M. (2017) Human mitochondrial transcription factor B2 is required for promoter melting during initiation of transcription. J.

Biol. Chem. 292, 2637–2645, https://doi.org/10.1074/jbc.M116.751008

15 Seidel-Rogol, B.L., McCulloch, V. and Shadel, G.S. (2003) Human mitochondrial transcription factor B1 methylates ribosomal RNA at a conserved

stem-loop. Nat. Genet. 33, 23–24, https://doi.org/10.1038/ng1064

16 Sologub, M., Litonin, D., Anikin, M., Mustaev, A. and Temiakov, D. (2009) TFB2 is a transient component of the catalytic site of the human

mitochondrial RNA polymerase. Cell 139, 934–944, https://doi.org/10.1016/j.cell.2009.10.031

17 Hillen, H.S., Morozov, Y.I., Sarfallah, A., Temiakov, D. and Cramer, P. (2017) Structural Basis of Mitochondrial Transcription Initiation. Cell 171,

1072e10–1081e10, https://doi.org/10.1016/j.cell.2017.10.036

18 Ramachandran, A., Basu, U., Sultana, S., Nandakumar, D. and Patel, S.S. (2017) Human mitochondrial transcription factors TFAM and TFB2M work

synergistically in promoter melting during transcription initiation. Nucleic Acids Res. 45, 861–874, https://doi.org/10.1093/nar/gkw1157

19 Minczuk, M., He, J., Duch, A.M., Ettema, T.J., Chlebowski, A., Dzionek, K. et al. (2011) TEFM (c17orf42) is necessary for transcription of human

mtDNA. Nucleic Acids Res. 39, 4284–4299, https://doi.org/10.1093/nar/gkq1224

20 Posse, V., Shahzad, S., Falkenberg, M., Hallberg, B.M. and Gustafsson, C.M. (2015) TEFM is a potent stimulator of mitochondrial transcription

elongation in vitro. Nucleic Acids Res. 43, 2615–2624, https://doi.org/10.1093/nar/gkv105

21 Pham, X.H., Farge, G., Shi, Y., Gaspari, M., Gustafsson, C.M. and Falkenberg, M. (2006) Conserved sequence box II directs transcription termination

and primer formation in mitochondria. J. Biol. Chem. 281, 24647–24652, https://doi.org/10.1074/jbc.M602429200

22 Wanrooij, P.H., Uhler, J.P., Simonsson, T., Falkenberg, M. and Gustafsson, C.M. (2010) G-quadruplex structures in RNA stimulate mitochondrial

transcription termination and primer formation. Proc.Natl.Acad.Sci.U.S.A.107, 16072–16077, https://doi.org/10.1073/pnas.1006026107

23 Falkenberg, M. (2018) Mitochondrial DNA replication in mammalian cells: overview of the pathway. Essays Biochem. 62,287–297,

https://doi.org/10.1042/EBC20170100

24 Agaronyan, K., Morozov, Y.I., Anikin, M. and Temiakov, D. (2015) Mitochondrial biology. Replication-transcription switch in human mitochondria.

Science 347, 548–551, https://doi.org/10.1126/science.aaa0986

25 Hillen, H.S., Parshin, A.V., Agaronyan, K., Morozov, Y.I., Graber, J.J., Chernev, A. et al. (2017) Mechanism of transcription anti-termination in human

mitochondria. Cell 171, 1082e13–1093e13, https://doi.org/10.1016/j.cell.2017.09.035

26 Jimenez-Menendez, N., Fernandez-Millan, P., Rubio-Cosials, A., Arnan, C., Montoya, J., Jacobs, H.T. et al. (2010) Human mitochondrial mTERF wraps

around DNA through a left-handed superhelical tandem repeat. Nat. Struct. Mol. Biol. 17,891–893, https://doi.org/10.1038/nsmb.1859

27 Martin, M., Cho, J., Cesare, A.J., Grifﬁth, J.D. and Attardi, G. (2005) Termination factor-mediated DNA loop between termination and initiation sites

drives mitochondrial rRNA synthesis. Cell 123, 1227–1240, https://doi.org/10.1016/j.cell.2005.09.040

28 Yakubovskaya, E., Mejia, E., Byrnes, J., Hambardjieva, E. and Garcia-Diaz, M. (2010) Helix unwinding and base ﬂipping enable human MTERF1 to

terminate mitochondrial transcription. Cell 141, 982–993, https://doi.org/10.1016/j.cell.2010.05.018

29Shi, Y., Posse, V., Zhu, X., Hyvarinen, A.K., Jacobs, H.T., Falkenberg, M. et al. (2016) Mitochondrial transcription termination factor 1 directs polar

replication fork pausing. Nucleic Acids Res. 44, 5732–5742, https://doi.org/10.1093/nar/gkw302

30 Hyvarinen, A.K., Kumanto, M.K., Marjavaara, S.K. and Jacobs, H.T. (2010) Effects on mitochondrial transcription of manipulating mTERF protein levels

in cultured human HEK293 cells. BMC Mol. Biol. 11, 72, https://doi.org/10.1186/1471-2199-11-72

31 Ojala, D., Montoya, J. and Attardi, G .(1981) tRNA punctuation model of RNA processing in human mitochondria. Nature 290, 470–474,

https://doi.org/10.1038/290470a0

316

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons

Attribution License 4.0 (CC BY).

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

32 Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H., Coulson, A.R., Drouin, J. et al. (1981) Sequence and organization of the human mitochondrial

genome. Nature 290, 457–465, https://doi.org/10.1038/290457a0

33 Holzmann, J., Frank, P., Lofﬂer, E., Bennett, K.L., Gerner, C. and Rossmanith, W. (2008) RNase P without RNA: identiﬁcation and functional

reconstitution of the human mitochondrial tRNA processing enzyme. Cell 135, 462–474, https://doi.org/10.1016/j.cell.2008.09.013

34 Brzezniak, L.K., Bijata, M., Szczesny, R.J. and Stepien, P.P. (2011) Involvement of human ELAC2 gene product in 3



end processing of mitochondrial

tRNAs. RNA Biol. 8, 616–626, https://doi.org/10.4161/rna.8.4.15393

35 Rossmanith, W. (2011) Localization of human RNase Z isoforms: dual nuclear/mitochondrial targeting of the ELAC2 gene product by alternative

translation initiation. PLoS One 6,e19152, https://doi.org/10.1371/journal.pone.0019152

36 Sanchez, M.I., Mercer, T.R., Davies, S.M., Shearwood, A.M., Nygard, K.K., Richman, T.R. et al. (2011) RNA processing in human mitochondria. Cell

Cycle 10,2904–2916, https://doi.org/10.4161/cc.10.17.17060

37 Boehm, E., Zaganelli, S., Maundrell, K., Jourdain, A.A., Thore, S. and Martinou, J.C. (2017) FASTKD1 and FASTKD4 have opposite effects on

expression of speciﬁc mitochondrial RNAs, depending upon their endonuclease-like RAP domain. Nucleic Acids Res. 45, 6135–6146,

https://doi.org/10.1093/nar/gkx164

38 Popow, J., Alleaume, A.M., Curk, T., Schwarzl, T., Sauer, S. and Hentze, M.W. (2015) FASTKD2 is an RNA-binding protein required for mitochondrial

RNA processing and translation. RNA 21, 1873–1884, https://doi.org/10.1261/rna.052365.115

39 Antonicka, H. and Shoubridge, E.A. (2015) Mitochondrial RNA granules are centers for posttranscriptional RNA processing and ribosome biogenesis.

Cell Rep., https://doi.org/10.1016/j.celrep.2015.01.030

40 Jourdain, A.A., Koppen, M., Rodley, C.D., Maundrell, K., Gueguen, N., Reynier, P et al. (2015) A mitochondria-speciﬁc isoform of FASTK is present in

mitochondrial RNA granules and regulates gene expression and function. Cell Rep. 10, 1110–1121, https://doi.org/10.1016/j.celrep.2015.01.063

41 Tomecki, R., Dmochowska, A., Gewartowski, K., Dziembowski, A. and Stepien, P.P. (2004) Identiﬁcation of a novel human nuclear-encoded

mitochondrial poly(A) polymerase. Nucleic Acids Res. 32, 6001–6014, https://doi.org/10.1093/nar/gkh923

42 Bai, Y., Srivastava, S.K., Chang, J.H., Manley, J.L. and Tong, L. (2011) Structural basis for dimerization and activity of human PAPD1, a noncanonical

poly(A) polymerase. Mol. Cell 41, 311–320, https://doi.org/10.1016/j.molcel.2011.01.013

43 Lapkouski, M. and Hallberg, B.M. (2015) Structure of mitochondrial poly(A) RNA polymerase reveals the structural basis for dimerization, ATP

selectivity and the SPAX4 disease phenotype. Nucleic Acids Res. 43, 9065–9075, https://doi.org/10.1093/nar/gkv861

44 Nagaike, T., Suzuki, T., Katoh, T. and Ueda, T. (2005) Human mitochondrial mRNAs are stabilized with polyadenylation regulated by

mitochondria-speciﬁc poly(A) polymerase and polynucleotide phosphorylase. J. Biol. Chem. 280,19721–19727,

https://doi.org/10.1074/jbc.M500804200

45 Rorbach, J., Nicholls, T.J. and Minczuk, M. (2011) PDE12 removes mitochondrial RNA poly(A) tails and controls translation in human mitochondria.

Nucleic Acids Res. 39, 7750–7763, https://doi.org/10.1093/nar/gkr470

46 Wydro, M., Bobrowicz, A., Temperley, R.J., Lightowlers, R.N. and Chrzanowska-Lightowlers, Z.M. (2010) Targeting of the cytosolic poly(A) binding

protein PABPC1 to mitochondria causes mitochondrial translation inhibition. Nucleic Acids Res. 38, 3732–3742, https://doi.org/10.1093/nar/gkq068

47 Sterky, F.H., Ruzzenente, B., Gustafsson, C.M., Samuelsson, T. and Larsson, N.-G. (2010) LRPPRC is a mitochondrial matrix protein that is conserved

in metazoans. Biochem. Biophys. Res. Commun. 398,759–764, https://doi.org/10.1016/j.bbrc.2010.07.019

48 Sasarman, F., Brunel-Guitton, C., Antonicka, H., Wai, T., Shoubridge, E.A. and Consortium, L. (2010) LRPPRC and SLIRP interact in a ribonucleoprotein

complex that regulates posttranscriptional gene expression in mitochondria. Mol. Biol. Cell 21, 1315–1323,

https://doi.org/10.1091/mbc.e10-01-0047

49Gohil, V.M., Nilsson, R., Belcher-Timme, C.a., Luo, B., Root, D.E. and Mootha, V.K. (2010) Mitochondrial and nuclear genomic responses to loss of

LRPPRC expression. J. Biol. Chem. 285, 13742–13747, https://doi.org/10.1074/jbc.M109.098400

50 Ruzzenente, B., Metodiev, M.D., Wredenberg, A., Bratic, A., Park, C.B., C

amara, Y. et al. (2012) LRPPRC is necessary for polyadenylation and

coordination of translation of mitochondrial mRNAs. EMBO J. 31, 443–456, https://doi.org/10.1038/emboj.2011.392

51 Mourier, A., Ruzzenente, B., Brandt, T., K

uhlbrandt, W. and Larsson, N.-G. (2014) Loss of LRPPRC causes ATP synthase deﬁciency. Hum. Mol. Genet.

23, 2580–2592, https://doi.org/10.1093/hmg/ddt652

52 Wilson, W.C., Hornig-Do, H.T., Bruni, F., Chang, J.H., Jourdain, A.A., Martinou, J.C. et al. (2014) A human mitochondrial poly(A) polymerase mutation

reveals the complexities of post-transcriptional mitochondrial gene expression. Hum. Mol. Genet. 23, 6345–6355,

https://doi.org/10.1093/hmg/ddu352

53 Chujo, T., Ohira, T., Sakaguchi, Y., Goshima, N., Nomura, N., Nagao, A. et al. (2012) LRPPRC/SLIRP suppresses PNPase-mediated mRNA decay and

promotes polyadenylation in human mitochondria. Nucleic Acids Res. 40, 8033–8047, https://doi.org/10.1093/nar/gks506

54 Siira, S .J., Spahr, H., Shearwood, A.J., Ruzzenente, B., Larsson, N.G., Rackham, O. et al. (2017) LRPPRC-mediated folding of the mitochondrial

transcriptome. Nat. Commun. 8, 1532, https://doi.org/10.1038/s41467-017-01221-z

55 Baughman, J.M., Nilsson, R., Gohil, V.M., Arlow, D.H., Gauhar, Z. and Mootha, V.K. (2009) A computational screen for regulators of oxidative

phosphorylation implicates SLIRP in mitochondrial RNA homeostasis. PLoS Genet. 5, e1000590, https://doi.org/10.1371/journal.pgen.1000590

56 Lagouge, M., Mourier, A., Lee, H.J., Spahr, H., Wai, T., Kukat, C. et al. (2015) SLIRP Regulates the Rate of Mitochondrial Protein Synthesis and Protects

LRPPRC from Degradation. PLoS Genet. 11, e1005423, https://doi.org/10.1371/journal.pgen.1005423

57 Chujo, T. and Suzuki, T. (2012) Trmt61B is a methyltransferase responsible for 1-methyladenosine at position 58 of human mitochondrial tRNAs. RNA

18, 2269–2276, https://doi.org/10.1261/rna.035600.112

58 Borowski, L.S., Dziembowski, A., Hejnowicz, M.S., Stepien, P.P. and Szczesny, R.J. (2013) Human mitochondrial RNA decay mediated by

PNPase-hSuv3 complex takes place in distinct foci. Nucleic Acids Res. 41, 1223–1240, https://doi.org/10.1093/nar/gks1130

59 Chen, H.W., Rainey, R.N., Balatoni, C.E., Dawson, D.W., Troke, J.J., Wasiak, S. et al. (2006) Mammalian polynucleotide phosphorylase is an

intermembrane space RNase that maintains mitochondrial homeostasis. Mol. Cell. Biol. 26, 8475–8487, https://doi.org/10.1128/MCB.01002-06

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons

Attribution License 4.0 (CC BY).

317

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

60 Szczesny, R.J., Borowski, L.S., Brzezniak, L.K., Dmochowska, A., Gewartowski, K., Bartnik, E. et al. (2010) Human mitochondrial RNA turnover caught

in ﬂagranti: involvement of hSuv3p helicase in RNA surveillance. Nucleic Acids Res. 38,279–298, https://doi.org/10.1093/nar/gkp903

61 Toompuu, M., Tuomela, T., Laine, P., Paulin, L., Dufour, E. and Jacobs, H.T. (2018) Polyadenylation and degradation of structurally abnormal

mitochondrial tRNAs in human cells. Nucleic Acids Res., https://doi.org/10.1093/nar/gky159

62 Gammage, P.A., Moraes, C.T. and Minczuk, M. (2017) Mitochondrial genome engineering: the revolution may not be CRISPR-Ized. Trends Genet. 34,

101–110

63 Haag, S., Sloan, K.E., Ranjan, N., Warda, A.S., Kretschmer, J., Blessing, C. et al. (2016) NSUN3 and ABH1 modify the wobble position of mt-tRNAMet

to expand codon recognition in mitochondrial translation. EMBO J. 35, 2104–2119, https://doi.org/10.15252/embj.201694885

64 Nakano, S., Suzuki, T., Kawarada, L., Iwata, H., Asano, K. and Suzuki, T. (2016) NSUN3 methylase initiates 5-formylcytidine biogenesis in human

mitochondrial tRNA(Met). Nat. Chem. Biol. 12, 546–551, https://doi.org/10.1038/nchembio.2099

65 Van Haute, L., Dietmann, S., Kremer, L., Hussain, S., Pearce, S.F., Powell, C.A. et al. (2016) Deﬁcient methylation and formylation of mt-tRNA(Met)

wobble cytosine in a patient carrying mutations in NSUN3. Nat. Commun. 7, 12039, https://doi.org/10.1038/ncomms12039

66 Van Haute, L., Powell, C.A. and Minczuk, M. (2017) Dealing with an unconventional genetic code in mitochondria: the biogenesis and pathogenic

defects of the 5-formylcytosine modiﬁcation in mitochondrial tRNAMet. Biomolecules 7,e24

67 Asano, K., Suzuki, T., Saito, A., Wei, F.Y., Ikeuchi, Y., Numata, T. et al. (2018) Metabolic and chemical regulation of tRNA modiﬁcation associated with

taurine deﬁciency and human disease. Nucleic Acids Res. 46, 1565–1583, https://doi.org/10.1093/nar/gky068

68 Villarroya, M., Prado, S., Esteve, J.M., Soriano, M.A., Aguado, C., Perez-Martinez, D. et al. (2008) Characterization of human GTPBP3, a GTP-binding

protein involved in mitochondrial tRNA modiﬁcation. Mol. Cell. Biol. 28, 7514–7531, https://doi.org/10.1128/MCB.00946-08

69Sasarman, F., Antonicka, H., Horvath, R. and Shoubridge, E.A. (2011) The 2-thiouridylase function of the human MTU1 (TRMU) enzyme is dispensable

for mitochondrial translation. Hum. Mol. Genet. 20, 4634–4643, https://doi.org/10.1093/hmg/ddr397

70 Wu, Y., Wei, F.Y., Kawarada, L., Suzuki, T., Araki, K., Komohara, Y. et al. (2016) Mtu1-mediated thiouridine formation of mitochondrial tRNAs is required

for mitochondrial translation and is involved in reversible infantile liver injury. PLoS Genet. 12, e1006355,

https://doi.org/10.1371/journal.pgen.1006355

71 Yarham, J.W., Lamichhane, T.N., Pyle, A., Mattijssen, S., Barufﬁni, E., Bruni, F. et al. (2014) Defective i6A37 modiﬁcation of mitochondrial and cytosolic

tRNAs results from pathogenic mutations in TRIT1 and its substrate tRNA. PLoS Genet. 10, e1004424, https://doi.org/10.1371/journal.pgen.1004424

72 Powell, C.A., Kopajtich, R., D’Souza, A.R., Rorbach, J., Kremer, L.S., Husain, R.A. et al. (2015) TRMT5 mutations cause a defect in post-transcriptional

modiﬁcation of mitochondrial tRNA associated with multiple respiratory-chain deﬁciencies. Am.J.Hum.Genet.97,319–328,

https://doi.org/10.1016/j.ajhg.2015.06.011

73 Zhao, X., Patton, J.R., Davis, S.L., Florence, B., Ames, S.J. and Spanjaard, R.A. (2004) Regulation of nuclear receptor activity by a pseudouridine

synthase through posttranscriptional modiﬁcation of steroid receptor RNA activator. Mol. Cell 15,549–558,

https://doi.org/10.1016/j.molcel.2004.06.044

74 Patton, J.R., Bykhovskaya, Y., Mengesha, E., Bertolotto, C. and Fischel-Ghodsian, N. (2005) Mitochondrial myopathy and sideroblastic anemia

(MLASA): missense mutation in the pseudouridine synthase 1 (PUS1) gene is associated with the loss of tRNA pseudouridylation. J. Biol. Chem. 280,

19823–19828, https://doi.org/10.1074/jbc.M500216200

75 Zaganelli, S., Rebelo-Guiomar, P., Maundrell, K., Rozanska, A., Pierredon, S., Powell, C.A. et al. (2017) The pseudouridine synthase RPUSD4 is an

essential component of mitochondrial RNA granules. J. Biol. Chem. 292, 4519–4532, https://doi.org/10.1074/jbc.M116.771105

76 Antonicka, H., Choquet, K., Lin, Z.Y., Gingras, A.C., Kleinman, C.L. and Shoubridge, E.A. (2017) A pseudouridine synthase module is essential for

mitochondrial protein synthesis and cell viability. EMBO Rep. 18, 28–38, https://doi.org/10.15252/embr.201643391

77 Arroyo, J.D., Jourdain, A.A., Calvo, S.E., Ballarano, C.A., Doench, J.G., Root, D.E. et al. (2016) A genome-wide CRISPR death screen identiﬁes genes

essential for oxidative phosphorylation. Cell Metab., https://doi.org/10.1016/j.cmet.2016.08.017

78 Nagaike, T., Suzuki, T., Tomari, Y., Takemoto-Hori, C., Negayama, F., Watanabe, K. et al. (2001) Identiﬁcation and characterization of mammalian

mitochondrial tRNA nucleotidyltransferases. J. Biol. Chem. 276, 40041–40049, https://doi.org/10.1074/jbc.M106202200

79 Nakamura, A., Nemoto, T., Heinemann, I.U., Yamashita, K., Sonoda, T., Komoda, K. et al. (2013) S tructural basis of reverse nucleotide polymerization.

Proc. Natl. Acad. Sci. U.S.A. 110,20970–20975, https://doi.org/10.1073/pnas.1321312111

80 Pearce, S.F., Rorbach, J., Van Haute, L., D’Souza, A.R., Rebelo-Guiomar, P., Powell, C.A. et al. (2017) Maturation of selected human mitochondrial

tRNAs requires deadenylation. Elife 6, e27596, https://doi.org/10.7554/eLife.27596

81 Chihara, T., Luginbuhl, D. and Luo, L. (2007) Cytoplasmic and mitochondrial protein translation in axonal and dendritic terminal arborization. Nat.

Neurosci. 10, 828–837, https://doi.org/10.1038/nn1910

82 Tolkunova, E., Park, H., Xia, J., King, M.P. and Davidson, E. (2000) The human lysyl-tRNA synthetase gene encodes both the cytoplasmic and

mitochondrial enzymes by means of an unusual alternative splicing of the primary transcript. J. Biol. Chem. 275, 35063–35069,

https://doi.org/10.1074/jbc.M006265200

83 Nagao, A., Suzuki, T., Katoh, T., Sakaguchi, Y. and Suzuki, T. (2009) Biogenesis of glutaminyl-mt tRNAGln in human mitochondria. Proc. Natl. Acad.

Sci. U.S.A. 106, 16209–16214, https://doi.org/10.1073/pnas.0907602106

84 Echevarria, L., Clemente, P., Hernandez-Sierra, R., Gallardo, M.E., Fernandez-Moreno, M.A. and Garesse, R. (2014) Glutamyl-tRNAGln

amidotransferase is essential for mammalian mitochondrial translation in vivo. Biochem. J. 460, 91–101, https://doi.org/10.1042/BJ20131107

85 Sharma, M.R., Koc, E.C., Datta, P.P., Booth, T.M., Spremulli, L.L. and Agrawal, R.K. (2003) Structure of the mammalian mitochondrial ribosome reveals

an expanded functional role for its component proteins. Cell 115, 97–108, https://doi.org/10.1016/S0092-8674(03)00762-1

86 Kaushal, P.S., Sharma, M.R., Booth, T.M., Haque, E.M., Tung, C.S., Sanbonmatsu, K.Y. et al. (2014) Cryo-EM structure of the small subunit of the

mammalian mitochondrial ribosome. Proc. Natl. Acad. Sci. U.S.A. 111, 7284–7289, https://doi.org/10.1073/pnas.1401657111

318

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons

Attribution License 4.0 (CC BY).

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

87 Greber, B.J., Boehringer, D., Leibundgut, M., Bieri, P., Leitner, A., Schmitz, N. et al. (2014) The complete structure of the large subunit of the

mammalian mitochondrial ribosome. Nature 515, 283–286, https://doi.org/10.1038/nature13895

88 Greber, B.J., Bieri, P., Leibundgut, M., Leitner, A., Aebersold, R., Boehringer, D. et al. (2015) Ribosome. The complete structure of the 55S mammalian

mitochondrial ribosome. Science 348, 303–308, https://doi.org/10.1126/science.aaa3872

89 Brown, A., Amunts, A., Bai, X.C., Sugimoto, Y., Edwards, P.C., Murshudov, G. et al. (2014) Structure of the large ribosomal subunit from human

mitochondria. Science 346, 718–722, https://doi.org/10.1126/science.1258026

90 Amunts, A., Brown, A., Toots, J., Scheres, S.H. and Ramakrishnan, V. (2015) Ribosome. The structure of the human mitochondrial ribosome. Science

348, 95–98, https://doi.org/10.1126/science.aaa1193

91Rorbach,J.,Gao, F., Powell, C.A., D’Souza, A., Lightowlers, R.N., Minczuk, M. et al. (2016) Human mitochondrial ribosomes can switch their structural

RNA composition. Proc. Natl. Acad. Sci. U.S.A., https://doi.org/10.1073/pnas.1609338113

92 Bar-Yaacov, D., Frumkin, I., Yashiro, Y., Chujo, T., Ishigami, Y., Chemla, Y. et al. (2016) Mitochondrial 16S rRNA Is Methylated by tRNA

Methyltransferase TRMT61B in All Vertebrates. PLoS Biol. 14, e1002557, https://doi.org/10.1371/journal.pbio.1002557

93 Metodiev, M.D., Lesko, N., Park, C.B., Camara, Y., Shi, Y., Wibom, R. et al. (2009) Methylation of 12S rRNA is necessary for in vivo stability of the small

subunit of the mammalian mitochondrial ribosome. Cell Metab. 9, 386–397, https://doi.org/10.1016/j.cmet.2009.03.001

94 Metodiev, M.D., Spahr, H., Loguercio Polosa, P., Meharg, C., Becker, C., Altmueller, J. et al. (2014) NSUN4 is a dual function mitochondrial protein

required for both methylation of 12S rRNA and coordination of mitoribosomal assembly. PLoS Genet. 10, e1004110,

https://doi.org/10.1371/journal.pgen.1004110

95 Lee, K.W. and Bogenhagen, D.F. (2014) Assignment of 2



-O-methyltransferases to modiﬁcation sites on the mammalian mitochondrial large subunit 16

S ribosomal RNA (rRNA). J. Biol. Chem. 289,24936–24942, https://doi.org/10.1074/jbc.C114.581868

96 Pearce, S.F., Rebelo-Guiomar, P., D’Souza, A.R., Powell, C.A., Van Haute, L. and Minczuk, M. (2017) Regulation of mammalian mitochondrial gene

expression: recent advances. Trends Biochem. Sci. 42, 625–639, https://doi.org/10.1016/j.tibs.2017.02.003

97 Rorbach, J., Boesch, P., Gammage, P.A., Nicholls, T.J., Pearce, S.F., Patel, D. et al. (2014) MRM2 and MRM3 are involved in biogenesis of the large

subunit of the mitochondrial ribosome. Mol. Biol. Cell 25, 2542–2555, https://doi.org/10.1091/mbc.e14-01-0014

98 Dennerlein, S., Rozanska, A., Wydro, M., Chrzanowska-Lightowlers, Z.M. and Lightowlers, R.N. (2010) Human ERAL1 is a mitochondrial RNA

chaperone involved in the assembly of the 28S small mitochondrial ribosomal subunit. Biochem. J. 430, 551–558,

https://doi.org/10.1042/BJ20100757

99 Antonicka, H., Sasarman, F., Nishimura, T., Paupe, V. and Shoubridge, E.A. (2013) The mitochondrial RNA-binding protein GRSF1 localizes to RNA

granules and is required for posttranscriptional mitochondrial gene expression. Cell Metab. 17, 386–398, https://doi.org/10.1016/j.cmet.2013.02.006

100 Jourdain, A.A., Koppen, M., Wydro, M., Rodley, C.D., Lightowlers, R.N., Chrzanowska-Lightowlers, Z.M. et al. (2013) GRSF1 regulates RNA processing

in mitochondrial RNA granules. Cell Metab. 17,399–410, https://doi.org/10.1016/j.cmet.2013.02.005

101 Janer, A., van Karnebeek, C.D., Sasarman, F., Antonicka, H., Al Ghamdi, M., Shyr, C. et al. (2015) RMND1 deﬁciency associated with neonatal lactic

acidosis, infantile onset renal failure, deafness, and multiorgan involvement. Eur. J. Hum. Genet. 23, 1301–1307,

https://doi.org/10.1038/ejhg.2014.293

102 Gaur, R., Grasso, D., Datta, P.P., Krishna, P.D., Das, G., Spencer, A. et al. (2008) A single mammalian mitochondrial translation initiation factor

functionally replaces two bacterial factors. Mol. Cell 29, 180–190, https://doi.org/10.1016/j.molcel.2007.11.021

103 Liao, H.X. and Spremulli, L.L. (1989) Interaction of bovine mitochondrial ribosomes with messenger RNA. J. Biol. Chem. 264, 7518–7522

104 Haque, M.E. and Spremulli, L.L. (2008) Roles of the N- and C-terminal domains of mammalian mitochondrial initiation factor 3 in protein biosynthesis.

J. Mol. Biol. 384, 929–940, https://doi.org/10.1016/j.jmb.2008.09.077

105 Bhargava, K. and Spremulli, L.L. (2005) Role of the N- and C-terminal extensions on the activity of mammalian mitochondrial translational initiation

factor 3. Nucleic Acids Res. 33, 7011–7018, https://doi.org/10.1093/nar/gki1007

106 Spencer, A.C. and Spremulli, L.L. (2004) Interaction of mitochondrial initiation factor 2 with mitochondrial fMet-tRNA. Nucleic Acids Res. 32,

5464–5470, https://doi.org/10.1093/nar/gkh886

107 Ma, J. and Spremulli, L.L. (1996) Expression, puriﬁcation, and mechanistic studies of bovine mitochondrial translational initiation factor 2. J. Biol.

Chem. 271, 5805–5811, https://doi.org/10.1074/jbc.271.10.5805

108 Liao, H.X. and Spremulli, L.L. (1990) Identiﬁcation and initial characterization of translational initiation factor 2 from bovine mitochondria. J. Biol.

Chem. 265, 13618–13622

109 Richter-Dennerlein, R., Oeljeklaus, S., Lorenzi, I., Ronsor, C., Bareth, B., Schendzielorz, A.B. et al. (2016) Mitochondrial protein synthesis adapts to

inﬂux of nuclear-encoded protein. Cell 167, 471e10–483e10, https://doi.org/10.1016/j.cell.2016.09.003

110 Szklarczyk, R., Wanschers, B.F., Cuypers, T.D., Esseling, J.J., Riemersma, M., van den Brand, M.A. et al. (2012) Iterative orthology prediction uncovers

new mitochondrial proteins and identiﬁes C12orf62 as the human ortholog of COX14, a protein involved in the assembly of cytochrome c oxidase.

Genome Biol. 13, R12, https://doi.org/10.1186/gb-2012-13-2-r12

111 Weraarpachai, W., Sasarman, F., Nishimura, T., Antonicka, H., Aure, K., Rotig, A. et al. (2012) Mutations in C12orf62, a factor that couples COX I

synthesis with cytochrome c oxidase assembly, cause fatal neonatal lactic acidosis. Am.J.Hum.Genet.90, 142–151,

https://doi.org/10.1016/j.ajhg.2011.11.027

112 Ling, M., Merante, F., Chen, H.S., Duff, C., Duncan, A.M. and Robinson, B.H. (1997) The human mitochondrial elongation factor tu (EF-Tu) gene: cDNA

sequence, genomic localization, genomic structure, and identiﬁcation of a pseudogene. Gene 197, 325–336,

https://doi.org/10.1016/S0378-1119(97)00279-5

113 Hammarsund, M., Wilson, W., Corcoran, M., Merup, M., Einhorn, S., Grander, D. et al. (2001) Identiﬁcation and characterization of two novel human

mitochondrial elongation factor genes, hEFG2 and hEFG1, phylogenetically conserved through evolution. Hum. Genet. 109, 542–550,

https://doi.org/10.1007/s00439-001-0610-5

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons

Attribution License 4.0 (CC BY).

319

Essays in Biochemistry (2018) 62 309–320

https://doi.org/10.1042/EBC20170102

114 Cai, Y.C., Bullard, J.M., Thompson, N.L. and Spremulli, L.L. (2000) Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNA and

elongation factor Ts. J. Biol. Chem. 275, 20308–20314, https://doi.org/10.1074/jbc.M001899200

115 Frolova, L.Y., Tsivkovskii, R.Y., Sivolobova, G.F., Oparina, N.Y., Serpinsky, O.I., Blinov, V.M. et al. (1999) Mutations in the highly conserved GGQ motif of

class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis. RNA 5, 1014–1020,

https://doi.org/10.1017/S135583829999043X

116 Richter, R., Rorbach, J., Pajak, A., Smith, P.M., Wessels, H.J., Huynen, M.A. et al. (2010) A functional peptidyl-tRNA hydrolase, ICT1, has been

recruited into the human mitochondrial ribosome. EMBO J. 29, 1116–1125, https://doi.org/10.1038/emboj.2010.14

117 Huynen, M.A., Duarte, I., Chrzanowska-Lightowlers, Z.M. and Nabuurs, S.B. (2012) Structure based hypothesis of a mitochondrial ribosome rescue

mechanism. Biol Direct 7, 14, https://doi.org/10.1186/1745-6150-7-14

118 Lind, C., Sund, J. and Aqvist, J. (2013) Codon-reading speciﬁcities of mitochondrial release factors and translation termination at non-standard stop

codons. Nat. Commun. 4,2940, https://doi.org/10.1038/ncomms3940

119 Akabane, S., Ueda, T., Nierhaus, K.H. and Takeuchi, N. (2014) Ribosome rescue and translation termination at non-standard stop codons by ICT1 in

mammalian mitochondria. PLoS Genet. 10, e1004616, https://doi.org/10.1371/journal.pgen.1004616

120 Soleimanpour-Lichaei, H.R., Kuhl, I., Gaisne, M., Passos, J.F., Wydro, M., Rorbach, J. et al. (2007) mtRF1a is a human mitochondrial translation

release factor decoding the major termination codons UAA and UAG. Mol. Cell 27, 745–757, https://doi.org/10.1016/j.molcel.2007.06.031

121 Temperley, R., Richter, R., Dennerlein, S., Lightowlers, R.N. and Chrzanowska-Lightowlers, Z.M. (2010) Hungry codons promote frameshifting in

human mitochondrial ribosomes. Science 327, 301, https://doi.org/10.1126/science.1180674

122 Temperley, R.J., Wydro, M., Lightowlers, R.N. and Chrzanowska-Lightowlers, Z.M. (2010) Human mitochondrial mRNAs–like members of all families,

similar but different. Biochim. Biophys. Acta 1797, 1081–1085, https://doi.org/10.1016/j.bbabio.2010.02.036

123 Feaga, H.A., Quickel, M.D., Hankey-Giblin, P.A. and Keiler, K.C. (2016) Human cells require non-stop ribosome rescue activity in mitochondria. PLoS

Genet. 12, e1005964, https://doi.org/10.1371/journal.pgen.1005964

124 Chrzanowska-Lightowlers, Z.M. and Lightowlers, R.N. (2015) Response to “Ribosome Rescue and Translation Termination at Non-standard Stop

Codons by ICT1 in Mammalian Mitochondria”. PLoS Genet. 11, e1005227, https://doi.org/10.1371/journal.pgen.1005227

125 Chrzanowska-Lightowlers, Z.M., Pajak, A. and Lightowlers, R.N. (2011) Termination of protein synthesis in mammalian mitochondria. J. Biol. Chem.

286, 34479–34485, https://doi.org/10.1074/jbc.R111.290585

126 Rorbach, J., Richter, R., Wessels, H.J., Wydro, M., Pekalski, M., Farhoud, M. et al. (2008) The human mitochondrial ribosome recycling factor is

essential for cell viability. Nucleic. Acids. Res 36, 5787–5799, https://doi.org/10.1093/nar/gkn576

127 Tsuboi, M., Morita, H., Nozaki, Y., Akama, K., Ueda, T., Ito, K. et al. (2009)EF-G2mt is an exclusive recycling factor in mammalian mitochondrial

protein synthesis. Mol. Cell 35, 502–510, https://doi.org/10.1016/j.molcel.2009.06.028

320

 2018 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons

Attribution License 4.0 (CC BY).