Schizosaccharomyces pombe as a fundamental model for research on mitochondrial gene expression: Progress, achievements and outlooks

Schizosaccharomyces pombe (fission yeast) is an attractive model for mitochondrial research. The organism resembles human cells in terms of mitochondrial inheritance, mitochondrial transport, sugar metabolism, mitogenome structure and dependence of viability on the mitogenome (the petite‐negative phenotype). Transcriptions of these genomes produce only a few polycistronic transcripts, which then undergo processing as per the tRNA punctuation model. In general, the machinery for mitochondrial gene expression is structurally and functionally conserved between fission yeast and humans. Furthermore, molecular research on S. pombe is supported by a considerable number of experimental techniques and database resources. Owing to these advantages, fission yeast has significantly contributed to biomedical and fundamental research. Here, we review the current state of knowledge regarding S. pombe mitochondrial gene expression, and emphasise the pertinence of fission yeast as both a model and tool, especially for studies on mitochondrial translation.


| INTRODUCTION
Over the past 40 years, mitochondrial research has often employed Schizosaccharomyces pombe (fission yeast) as a complementary model to Saccharomyces cerevisiae (budding yeast), owing to several advantages.Firstly, molecular studies of S. pombe are supported by a wellmaintained genetic and protein database (PomBase), 1 coupled with a single-gene deletion library (Bioneer), 2 a system to investigate bypassable gene essentiality, 3 and more recently, a genetic engineering toolkit (POMBOX). 4econdly, similar to budding yeast, fission yeast is experimentally tractable, possessing a short generation time ($3 h), and typically proliferating in a haploid state.However, S. pombe better resembles the mitochondrial physiology of Homo sapiens (Table 1). 5For example, mitochondria are uniparentally inherited, and moved along microtubules in both organisms. 6,7Regarding the fission yeast, mitochondrial transport utilises the tethering protein Mmb1 and the polymer dynamics, rather than the tubulin-associated protein motors as per human cells. 7Whilst, S. cerevisiae shows a biparental pattern of mitochondrial inheritance, and depends on actin filaments for mitochondrial trafficking. 6,7n general, neither S. pombe nor human cells could proliferate in culture efficiently by galactose fermentation (Table 1).The energy metabolism of S. pombe adheres to the Crabtree effect, which is the glucose-dependent suppression of respiration in favour of fermentation for rapid growth. 8When galactose is the sole carbon source, fission yeast could import the substrate using Ght2, and afterwards engage Gal1, Gal7 and Gal10 in the conversion of galactose to glucose-6-phosphate (the Leloir pathway). 9,10owever, the general sugar transporter shows a relatively low affinity for galactose, and the galactose-assimilating genes are constitutively repressed.As a result, glucose-6-phosphate is produced from galactose in limited quantity, and must be both fermented and respired to generate sufficient energy for cellular proliferation.[10] Fission yeast also parallels human cells concerning the mitogenome (mitochondrial DNA, mtDNA) size, structure and dependence (Table 1).Both S. pombe and human mtDNAs are compact, and possess short intergenic regions, and few or no introns. 11,12Transcription of these genomes produce only a few polycistronic transcripts, in which rRNAs and mRNAs are virtually always punctuated by tRNAs, and contain short to no untranslated regions (UTRs).4][15] In contrast, the S. cerevisiae mitogenome is four-to five-fold larger, riddled with non-coding sequences, and could be lost under certain conditions (the petite-positive phenotype). 16,17A recent transcriptomics study on this species revealed the presence of 14 active mitochondrial promoters, over 10 times the numbers recorded for the S. pombe and human mtDNAs. 18onsidering the aforementioned resemblance to human cells, fission yeast is an attractive model for studies on mitochondrial biogenesis and diseases.Here, we review the progress in establishing such model organism, by (i) summarising our current understanding of mitochondrial gene expression in S. pombe, (ii) comparing and contrasting it to the S. cerevisiae and human system and (iii) offering critical perspectives on ambiguous areas of knowledge.We also appreciate the contributions of S. pombe to, and further accentuate its potential in, fundamental and therapeutic research relating to mitochondria.The complete sequence of the S. pombe genome was published in 2002, encompassing three linear nuclear chromosomes and one circular mtDNA. 11Annotations of the mtDNA were first made available in 2005, and has since been refined (Figure 1). 19,20The mitogenome of fission yeast encodes seven subunits of the OXPHOS complexes (Cytb, Cox1, Cox2, Cox3, Atp6, Atp8 and Atp9), three components of the mitoribosome (Urfa, rnl and rns), 25 tRNAs and a ribonuclease P (RNase P) RNA (rnpB).Similar gene content is observed amongst the five Schizosaccharomyces species, with the absence of (i) rnpB in S. japonicus, and (ii) trnI(cau) in S. cryophilus, S. octosporus and S. osmophilus. 21However, the mitogenome of fission yeast shows only partial synteny with those of the other Schizosaccharomyces species, and is the most compact owing to short intergenic regions.Depending on the strain under study, the S. pombe mtDNA could contain up to eight introns, distributed among cytb, cox1 and cox2 (Table S1). 20For instance, the reference strain (972 h À ) possesses only cytb-I1, cox1-I1b and cox1-I2b (Figure 1), 19,20 whose successive deletions generated a mitochondrial intron-less strain. 22he mtDNA gene content of S. pombe resembles that of S. cerevisiae, though the prior contains one additional tRNA sequence (Figure 1). 11,16In contrast, the human mtDNA encodes 13 subunits of the OXPHOS complexes (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, CYTB, COX1, COX2, COX3, ATP6 and ATP8), only the rRNA F I G U R E 1 Reference mitogenomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe and Homo sapiens.Each mitogenome encodes a variable set of mt-tRNAs, and one or several components of the mitochondrial complexes shown beneath.The mitochondrialencoded RNA and/or proteins are coloured accordingly.Question mark (?), unresolved structure; mt-RNase P, mitochondrial ribonuclease P; CI-V, complexes I-V. S. cerevisiae S288C mtDNA (NC_001224), 16 mitoribosome (5MRC), 76 complex III (1KYO), 135 complex IV (6YMY) 136 and complex V (6CP6) 137 ; S. pombe 972 h À mtDNA (NC_001326), 19 complex III (8Q1B) 121 and complex IV (8C8Q) 122 ; H. sapiens mtDNA (NC_012920), 12 mitoribosome (3J9M), 67 complex I (5XTD), 138 complex III (5XTE), complex IV (5Z62) 139 and complex V (8H9T). 140omponents of the mitoribosome (RNR1 and RNR2), 22 tRNAs and no RNase P RNA. 12Distinctively, coding regions are localised to a single mtDNA strand in S. pombe, rather than both strands as per S. cerevisiae and humans. 12,16,20Moreover, although mitochondrial nucleoids have been observed in fission yeast, 23 no apparent mtDNA-packaging protein has been recognised (Table 1). 1 Whilst, S. cerevisiae and human nucleoids are respectively maintained by the high mobility group (HMG)-box proteins Abf2 and TFAM, the latter of which also functions in mtDNA transcription. 24,25

| S. pombe mtDNA replication abides by the rolling circle model
To initiate rolling circle replication, a single-stranded DNA (ssDNA) invades the circular mitogenome and commences leading-strand synthesis. 26,27Simultaneously, the lagging strand is generated as ligated Okazaki fragments.Repeated DNA synthesis produces a linear molecule of several genomic units fused head-to-tail, which may undergo homologous recombination to regenerate the circular genome.The initial ssDNA could result from a previous replication cycle, or from end resection after a double-stranded break.The rolling circle model was supported by the observation that upon pulsed field gel electrophoresis, the S. pombe mtDNA appeared primarily as linear molecules of $5 genome units in vivo. 26Moreover, via two-dimensional agarose gel analysis, the predominant replication intermediates were simple Y-shaped replication forks, contrary to bacterial replication bubbles and human mtDNA displacement loop (D-loop).The S. cerevisiae mitogenome is also replicated according to the rolling circle mechanism, while human mtDNA replication emulates the strand displacement model under normal conditions, and the rolling circle mode upon oxidative stress. 27No mitochondrial origins of replication have been published in S. pombe, in contrast to the extensively mapped sequences in the other two species (Figure 1).
However, the three organisms consistently recruit the cruciform-endonuclease Cce1, the ssDNA binding protein Rim1/SSBP1 and/or the DNA polymerase gamma (Polγ) for mtDNA replication.Firstly, both the fission and budding yeast require Cce1 for the resolution of Holliday junction during homologous recombination. 28,29he human homologue of these proteins has not been identified. 1,30Secondly, S. pombe Rim1 and its homologues (S. cerevisiae Rim1 and human SSBP1) have each been demonstrated to interact with ssDNA in vitro and form homotetramers in vivo. 25,29,31Thirdly, the incorporation of deoxynucleotide triphosphates (dNTPs) to the growing mtDNA requires Polγ, whose catalytic subunit is named Pog1, Mip1 and POLG in S. pombe, S. cerevisiae and humans, respectively. 25,29,32Surprisingly, the deletion of pog1 in haploid S. pombe was stably obtained by tetrad dissection of a Δpog1/pog1 hetero-diploid, that is, pog1 did not appear essential to viability. 32The haploid strain showed a near abolition of fermentative growth, abnormal cellular and mitochondrial morphology, and minimal mtDNA presence.The authors suggested that the basal mitogenome number could have been carried over from the pog1/Δpog1 mother cell.However, upon prolonged incubation, the Δpog1 strain continued to proliferate.We proposed that suppressor mutations might have occurred, and produced petite-positive revertants.Alternatively, mtDNA replication could be feebly maintained by other DNA polymerases.In S. cerevisiae, the DNA polymerase zeta (Polζ) contributes to both nuclear and mitochondrial genome maintenance. 33Each subunit of the Polζ heterotrimer (Rev3, Rev7 and Rev1) has an orthologue in S. pombe, 1 and such functional duality was also observed for one composition (REV3L) of the human Polζ. 33

| MAINTENANCE OF MITOCHONDRIAL DNA INTEGRITY
3.1 | Mitogenome integrity is vital to S. pombe, a petite-negative yeast Mitochondria are essential for various cellular processes other than respiration.To fulfil such functions, mitochondria must import nuclear-encoded proteins from the cytoplasm, which requires the electrochemical gradient across the inner mitochondrial membrane (IMM). 17,34In wild-type S. pombe and S. cerevisiae, the gradient is accomplished by the OXPHOS complexes III and IV, which pump protons from the mitochondrial matrix to the intermembrane space, and complex V, which utilises the electrochemical gradient to phosphorylate ADP.The yeast mtDNAs encode core components of the former two complexes, alongside three subunits in the transmembrane (F 0 ) domain of complex V (Figure 1).As a petite-negative species (briefly discussed in 'Introduction'), S. pombe must maintain the mitogenome integrity to ensure functional OXPHOS complexes, thereby sustaining the IMM potential and supporting life.On the contrary, in S. cerevisiae (a petite-positive yeast), the electrochemical gradient could also be achieved via the coupling between (i) the antiporter Pet9, which imports ATP into and exports ADP out of the mitochondria, and (ii) the hydrophilic (F 1 ) domain of complex V, which instead runs in reverse and hydrolyses ATP.As expected, when both components are functional, mutations in the electron transport chain (ETC) often produce IMM potential comparable to wild-type. 35Upon either Pet9 or F 1 domain malfunction, budding yeast becomes petitenegative.For example, the deletion of YME1 (encoding the catalytic subunit of a mitochondrial protease complex) resulted in a build-up of Inh1 (the F 1 ATPase inhibitory factor), ultimately disrupting the electrochemical gradient in the absence of the mitogenome. 36he orthologues of S. cerevisiae Pet9, Yme1 and Inh1 have been identified in S. pombe, respectively referred to as Anc1, Yme1 and Inh1. 1 S. pombe Anc1 complemented for the loss of Pet9 in S. cerevisiae. 37Interestingly, the expression of S. cerevisiae YME1 in S. pombe converted the petite-negative yeast to petite-positive. 38Spontaneous conversions of S. pombe to petite-positive have also been obtained, termed ptp1-1 (for petite-positive), ptp2-1, rzl4-1 (for rho zero lethality), rzl5-1 and rzl5-1. 13,14Our unpublished data suggests that some of the rzl mutations resided in the F 1 subunits Atp2 and Atp3.The remaining strains contain as yet uncharacterised nuclear mutations.Taken together, the petite-negative property of S. pombe appears to arise from the F 1 domain, more specifically from its inability to hydrolyse ATP when the mitochondrial-encoded F 0 domain is absent.Consistently, Kluyveromyces lactis (similar to fission yeast in terms of mtDNA protein-coding capacity and dependence) could be converted to petite-positive by boosting F 1 ATP hydrolysis, induced upon inh1 deletion, F 1 subunit mutations, or a combination of both. 39Based on these observations, the two-component hypothesis for petitenegativity was proposed: S. pombe (and K. lactis) must possess the mitogenome to build a functional ETC and an F 1 -F 0 assembled complex V, either of which is required to maintain the IMM potential and support viability. 342 | Some of the S. pombe mtDNA repair factors are shared with the nucleus Eukaryotic mitogenomes are prone to mutations, deletion and rearrangements.In fission yeast, several mtDNA repair proteins have been recognised either in vitro (Cmb1 40 ) or in vivo (Cce1 28 and Uve1 41 ). S. pombe Cmb1 is homologous to S. cerevisiae Abf2 and human TFAM (previously mentioned in 'Mitochondrial DNA structure and replication'), and to date, remains as the only HMGbox protein in S. pombe mitochondria.1 Other candidate factors include the mitochondrial isoform of Pfh1 and Exo5.Specifically, the S. pombe pfh1 and exo5 mRNA each produce a nuclear-and a mitochondrial-targeted protein via alternative translation initiation.42,43 The former isoforms are firmly established to participate in nuclear DNA (nDNA) replication and/or repair.Whilst, the mitochondrial counterparts have been inferred by mutant studies to maintain the mitogenome, though the specific stage(s) of action remain(s) ambiguous.In budding yeast, one of the two homologues of S. pombe Pfh1 (termed Pif1) is also present in vivo as a nuclear-and a mitochondrial-localised factor, the latter of which functions in mtDNA replication and repair.29 On the contrary, a single PIF1 in humans contributes to both nDNA and mtDNA replication, while S. cerevisiae Exo5 and human EXO5 are solely and respectively involved in mtDNA replication and nDNA repair.29,43 4 | MITOCHONDRIAL TRANSCRIPTION 4.1 | S. pombe mtDNA transcription employs universally conserved promoters and proteins Transcription of the S. pombe mitogenome starts at three promoters of increasing initiation efficiency, termed intronic (P in , ATATATGTG), minor (P mi , TTATATGTG) and major (P ma , ATATATGTA) (Figures 1 and 2.). 19,44hese are localised to a single mtDNA strand, and situated upstream of the rnl gene, upstream of the cox3 gene and at the start of the cytb-I1 intron, respectively.The positions of the three promoters were established by in vitro capping of the primary transcripts with Vaccinia virus guanylyl transferase, in the presence of radioactively labelled GTP, and subsequently confirmed via 5 0 rapid amplification of cDNA ends (5' RACE).Across S. pombe, S. cerevisiae and humans, the mitochondrial promoter sequences are highly conserved. 18,19,45However, their number and strand distribution vary (Figure 1).The human mitogenome contains two promoters, each located to a different strand. 45In contrast, 14 mitochondrial promoters have been detected in budding yeast. 18hese show considerably skewed strand dispersal, and three promoters (immediately upstream of ORI2, ORI3 and ORI5) solely synthesise primers for mitogenome replication, that is, they do not generate transcripts.
S. pombe mtDNA transcription requires the RNA polymerase Rpo41, and at least the transcription factor Mtf1. 44 Similar to the Δpog1/pog1 strain (discussed in 'Mitochondrial DNA structure and replication'), the Δrpo41/rpo41 and Δmtf1/mtf1 heterozygous diploids could be generated.However, upon tetrad dissection, neither the Δrpo41 nor Δmtf1 haploid survived after a few division cycles, that is, viability necessitates a functional copy of each gene.During the short proliferation period, the cells exhibited distorted shape, abnormal septation, minimum IMM potential and dramatically reduced mt-mRNAs.Electrophoretic mobility shift assay (EMSA) confirmed the specific binding of Rpo41 and Mtf1 to P in , P mi and P ma .Promoter binding and transcription in vitro was most efficient when both proteins were present, emphasising the importance of Mtf1 in mitochondrial transcription initiation, and suggesting a direct interaction between S. pombe Rpo41 and Mtf1.To date, no experimental results are available on the structures and in vivo functions of these proteins.Concerning S. cerevisiae and humans, transcriptions of the mitogenomes respectively recruit Rpo41 and POLRMT (the homologues of S. pombe Rpo41), alongside Mtf1 and TFB2M (the homologues of S. pombe Mtf1), among others. 19,45In each organism, the RNA polymerase has been visualised by cryo-electron microscopy (cryo-EM), and demonstrated to bind the corresponding transcription factor.
Based on AlphaFold2 prediction and cryo-EM structures (when available), the conformation of S. pombe Rpo41 and its homologues (S. cerevisiae Rpo41 and human POLRMT) are rather conserved. 19,45,46Each protein contains an N-terminal extension (NTE) of unknown origin, and a C-terminal catalytic core.A few pentatricopeptide repeat (PPR) motifs are also present, connecting the two protein domains.The NTE of S. cerevisiae Rpo41 has been inferred in mtDNA maintenance, among other functions. 19,45Interestingly, a potential interaction between S. pombe Rpo41 and the mtDNA repair protein Pfh1 (discussed in 'Maintenance of Mitochondrial DNA Integrity') has been indicated. 47Specifically, upon immunoprecipitation (IP) of Pfh1, Rpo41 was recovered as a co-enrichment by mass spectrometry.However, no confirmatory experiment (e.g.co-IP coupled with Western blot) was conducted, and the binding region on the RNA polymerase (if present) remains to be explored.

| The mt-tRNA punctuation model applies to S. pombe and humans
For fission yeast and humans, mtDNA transcription generates two to three large polycistronic transcripts, in which mitochondrial RNase P RNA (S.pombe only), mt-rRNAs and mt-mRNAs are virtually always interspersed with mt-tRNAs 19,44 (Figures 1 and 2.).Consequently, mt-tRNA excision could liberate nearly every constituent from the primary transcripts.This is termed the mt-tRNA punctuation model, and in contrast to the clustered mt-tRNA system observed in budding yeast.Regarding S. pombe, the model has been confirmed independently via primer extension and mitochondrial transcriptomics. 19,48The former experiment revealed the precise correspondence of each mt-rRNA/mt-mRNA 5 0 end to the upstream mt-tRNA 3 0 end.Subsequently, mtRNA sequencing confirmed these observations, and further showed the coincidence between both ends of rnpB mRNA and the cleavage sites Schizosaccharomyces pombe mtDNA transcription and mtRNA processing.Transcriptions could be initiated from any of the three promoters, albeit at varying efficiency (P in < P mi < P ma ).The resulting transcripts are polycistronic, comprising of several mtRNA species (coloured as per Figure 1).Subsequent processing is only shown for the most abundant primary transcript (P ma ), and involves the endoribonucleolytic cleavage of mt-tRNAs according to the tRNA punctuation model, the 3 0 exoribonucleolytic cleavage of mt-mRNAs and the splicing of any introns.For each stage in mtDNA transcription and mtRNA processing, known factors are indicated (more might be uncovered in the future).The mitochondrial 3 0 -5 0 exoribonuclease Par1 (Rpm1, SPCC1322.01)should not be confused with the cytoplasmic serine/threonine phosphatase regulatory subunit Par1 (Pbp1, SPCC188.02).
of its flanking mt-tRNAs.As exceptions to the mt-tRNA punctuation model, the cox1-cox3 and cox2-rnl intergenic regions are devoid of mt-tRNAs.However, these regions respectively contain P mi and P ma , from which transcriptions compensate for the absence of mt-tRNA processing.
In general, the 5 0 endoribonucleolytic cleavage of mt-tRNAs is conducted by mitochondrial RNase P, a group of functionally conserved enzyme complexes, albeit compositionally diverse. 49Concerning S. pombe, homology searches reveal the presence of a mitochondrial-encoded RNase P RNA (rnpB). 19Neither the function of this RNA, nor any protein component of the mitochondrial RNase P has been characterised.The homologues of rnpB are present across the Schizosaccharomyces taxa, except for S. japonicus. 21Meanwhile, S. cerevisiae mitochondrial RNase P is formed by the mitochondrial-encoded ribozyme rpm1 (homologous to S. pombe rnpB 50 ) and the nuclear-encoded PPR protein Rpm2 (no clear S. pombe orthologue 1 ). 51Whilst, human mitochondrial RNase P does not contain an RNA component, instead comprising three protein subunits: TRMT10C (MRPP1), HSD17B10 (MRPP2) and the catalytic PPR protein PRORP (MRPP3), none of which possesses homologues in the budding or fission yeast.
In contrast to mitochondrial RNase P, members of the mitochondrial long form of ribonuclease Z (RNase Z L ) are both structurally and functionally conserved. 49hese are referred to as ELAC2 in humans, Trz1 in S. cerevisiae and Trz2 in S. pombe.While human ELAC2 and S. cerevisiae Trz1 carry out 3 0 processing of both nuclear and mitochondrial tRNAs, S. pombe Trz2 strictly functions in the mitochondria. 52A mutation in trz2 resulted in severely impaired 3 0 mt-tRNA processing, coupled with a build-up of mt-mRNAs. 485 0 maturation of mt-tRNAs was also affected, albeit to a lesser extent.This had previously been reported in budding yeast, and might be due to reduced 3 0 processing of the mt-tRNA directly preceding RPM1 (or rnpB for S. pombe).Taken together, these observations have (i) confirmed the function of S. pombe Trz2 in 3 0 mt-tRNA processing, (ii) supported the tRNA punctuation model and (iii) implicated the involvement of rnpB in the mitochondrial RNase P machinery.

| S. pombe mt-mRNAs further undergo 3 0 exonucleolytic cleavage
As mt-tRNAs are cleaved from the primary transcripts, the liberated mt-mRNAs each contain a 3' C-core motif, which appears to be the signal for downstream cleavage by the mitochondrial 3 0 -5 0 exoribonuclease Par1 (Rpm1), and the mitochondrial DExH/D box RNA helicase Pah1 (Rpm2). 19,48,53Amongst eukaryotes, DExH/D box RNA helicases play crucial roles in both nuclear and mitochondrial RNA metabolism. 54Concerning S. pombe, the absence of Par1 resulted in the accumulation of mt-mRNAs with unprocessed 3 0 ends, extending up to the RNase P cleavage site. 53On the contrary, pah1 deletion brought about random end truncations.Taken together, these results have demonstrated (i) the essential function of Par1 as 3 0 mt-mRNA exoribonuclease, and (ii) the support role of Pah1 in mt-mRNA end protection, C-core motif recognition and/or the loading of mt-mRNAs onto Par1.Moreover, neither par1 nor pah1 overexpression could complement for the absence of Pet127 (an mtRNA catabolic factor conserved in lower eukaryotes), and vice versa.Therefore, S. pombe Par1 and Pah1 are probably not involved in mtRNA degradation, contrary to their homologues in S. cerevisiae (Dss1 and Suv3) and humans (SUPV3L1). 54Apart from S. pombe Pet127, a possible mtRNA catabolic factor is Pnu1, previously demonstrated to degrade ssDNA and RNA in vitro. 55

| S. pombe mt-mRNA introns are generally spliced by mitochondrial intronencoded proteins
As mentioned previously, the S. pombe cytb, cox1 and cox2 genes could each contain one or more introns (Table S1). 20,56These are classified as either group I or II, based primarily on the predicted secondary structures.The prior group resides exclusively in the cox1 gene.Except for the recently discovered cox1-I1b', of which no experimental results have been published, mitochondrial group I introns are incapable of autocatalytic splicing in vitro.Instead, cox-I1b and cox-I2b contain reading frames, and translations from the start codon of the upstream exon could yield intron maturases (Figure 2.).The essentiality of these proteins for mitochondrial splicing has been demonstrated by mutant studies.][59] In addition, the intron-encoded proteins participate in homologous (homing) and ectopic insertion (retrotransposition) of the intron sequences, thereby contributing to evolution via horizontal intron transfer. 20,56For the remaining group I introns, the mechanism of splicing remains ambiguous: the reading frame of cox1-I1b' is out-of-frame with the upstream exon, while cox1-I2a and cox1-I3 appear to be non-coding.
On the contrary to those from group I, mitochondrial introns of group II are distributed among cytb, cox1 and cox2.However, these also appear to encode proteins with maturase, endonuclease and reverse transcriptase activities, respectively responsible for intron splicing, homing and retrotransposition.Autocatalytic splicing of cytb-I1 has been refuted in vitro, while that of other group II introns remains to be explored. 22Recently, via mass spectrometry, a polypeptide uniquely corresponding to cytb-I1 has been unambiguously detected, and shown to enrich among mitoribosomal proteins and mitochondrial translational activators (Supplementary Data in 57 and unpublished).On the one hand, such observation confirmed the in vivo translation of the cytb-I1 reading frame.Furthermore, because cytb-I1 mRNA locates at the beginning of the P in primary transcript (Figure 2.), its translation might employ an internal initiation site, rather than an upstream start codon as per cox-I1b and cox-I2b.On the other hand, a potential interaction between Cytb-I1 and the mitochondrial translation machinery suggests a functional coupling between mt-mRNA splicing and translation.

| S. pombe mtRNA modification is poorly understood
After Trz2-mediated endoribonucleolytic cleavage, to produce mitochondrial aminoacyl-tRNAs, the free 3 0 ends of mt-tRNAs are joined to a CC series by Cca1, appended with an A by Cca2 and eventually attached to a specific amino acid via the appropriate aminoacyl-tRNA ligase. 60ased on sequence homology, 38 aminoacyl-tRNA ligases have been recognised in fission yeast, among which 17 are mitochondrial-specific, and five probably localised to both the mitochondria and cytoplasm (Table S2). 1 On the one hand, similar to humans and unique among yeasts, S. pombe contains two valyl-tRNA ligases, each functioning in a specific cellular compartment. 61On the other hand, fission and budding yeast might resemble one another regarding mitochondrial glutaminyl-tRNA synthesis.Neither species contains a specific mitochondrial glutaminyl-tRNA ligase. 1,62Instead, S. cerevisiae mitochondria first generate glutamyl-tRNA using Mse1, then trans-amidate the molecule to glutaminyl-tRNA via the Her2/Pet112/Gtf1 complex. 62The homologues of these factors in S. pombe, respectively termed Mse1 and Gta1/2/3, are functionally uncharacterised. 1 Sequence homology has also revealed the presence of several mt-tRNA/mt-rRNA methyltransferases and pseudo-uridine synthases, potentially involved in specific mtRNA base modifications. 1 These processes have yet to be investigated in S. pombe, albeit well established in other eukaryotes.Mature mt-mRNAs in fission yeast are subjected to neither 5 0 7-methylguanylate capping nor 3 0 polyadenylation, consistent with those of budding yeast. 19,48On the contrary, 3 0 poly(A) tails have been reported in human mature mt-mRNAs.
5.5 | Several S. pombe penta-tricopeptide repeat proteins are involved in mtRNAspecific stabilisation PPR proteins constitute a family of RNA-binding factors ubiquitous amongst eukaryotes. 63,64Regarding fission yeast, 12 members have been identified by domain prediction algorithms. 57,58,65Each member contains between two (for Rpo41) and at least 16 PPR motifs (Ppr5).Increasing the number of PPR motifs enables the recognition of longer RNA sequences, ultimately improving the binding specificity.So far, S. pombe PPR proteins reside exclusively in the mitochondria, where they contribute to transcription (Rpo41; previously discussed in 'Mitochondrial transcription'), mt-rRNA and mt-mRNA stabilisation (Ppr3, Ppr1, Ppr7 and Ppr6; addressed here) and translation (see 'Mitochondrial general translation factors' for Ppr10, Ppr2 and Ppr5; see 'Mitochondrial specific translation and early complex assembly' for Ppr4, Mtf2 and Cbp8).One member (Ppr8) remains functionally ambiguous. 58. pombe Ppr3 has been demonstrated to preserve 15S rRNA, consistent with its homologue in S. cerevisiae (Ccm1 or Dmr1).63 The absence of ppr3 in S. pombe mitochondria was associated with severe respiratory deficiency, a strong decrease in full-length (coupled with an accumulation of degraded) 15S rRNA and negligible mitochondrial translation.58 There was also visible reduction in the remaining mtRNAs, especially cytb mRNA.These results suggested that (i) Ppr3 plays an important role in mt-SSU biogenesis and (ii) the protein might possess more than one mtRNA targets.Similarly, S. cerevisiae Ccm1 participates in both 15S rRNA stabilisation, and cytb-bI4 and cox1-aI4 intron splicing.67 Several of the other PPR members have been demonstrated to stabilise mt-mRNAs.These are Ppr1 (maintaining cox2 and cox3 mRNAs), Ppr7 and Ppr6 (stabilising atp6 and atp9 mRNAs, respectively).The absence of ppr1 resulted in severe respiratory growth defect, major reductions in cox2 and cox3 mRNA and no detectable translation of these species.58 Whilst, S. pombe deleted for either ppr7 or ppr6 showed sensitivity to antimycin A and minimal respiratory growth.Because antimycin A inhibits complex III, the former phenotype indicated a complex V defect, as per the two-component hypothesis (previously discussed in 'Maintenance of mitochondrial DNA integrity').34 Furthermore, atp6 mRNA was barely detectable in the Δppr7 strain, and atp9 mRNA could not be visualised by Northern blot upon Δppr6.In both mutants, the remaining mt-mRNAs were generally decreased (including urfa), and the synthesis of every mitochondrial-encoded protein was downgraded.These might be secondary defects, resulting from complex V dysfunction. Todate, the homologues of S. pombe Ppr1 and Ppr7 in S. cerevisiae have not been identified.Meanwhile, the homologue of S. pombe Ppr6 in S. cerevisiae (Aep2) also functions in atp9 mRNA stabilisation.63

| THE MITORIBOSOME
Given the endosymbiotic origin of membrane-bound organelles, it comes as no surprise that the mitochondrial translation machinery generally resembles the prokaryotic (rather than the eukaryotic cytoplasmic) system.For example, various antibiotics could inhibit both the organellar and the bacterial ribosomes, while showing no effects against the eukaryotic cytosolic counterpart. 68The majority of general translational factors are also conserved between mitochondria and prokaryotes (see 'Mitochondrial general translation factors').
However, through evolution, the mitochondrial translation machinery has accumulated unique features.Foremost, the mitoribosome possesses a higher protein:rRNA mass ratio (Table 2).Secondly, because the bacterial Shine-Dalgarno sequence is absent in mt-mRNAs, mitochondria must adopt other strategies to initiate translation (e.g.translational activators, see 'Mitochondrial specific translation and early complex assembly').Thirdly, certain mitochondrial codons deviate from the universal genetic code (Table 3).Surprisingly, such difference is minimal in S. pombe: the TGA codon encodes for tryptophan in the yeast mitochondria, instead of the universal 'stop'.Consequently, upon a relocation of the mitochondrial urfa gene to the nucleus, the TGA codon must be replaced by TGG to produce a functional protein. 69However, TGA only occurs in the urfa and intronic transcripts, that is, rarely compared to the other tryptophan codon (TGG). 70The sole mitochondrial trnW(cca) has been proposed to recognise both TGA and TGG, albeit at varying efficiency.

| The majority of universal mitoribosomal proteins bear sequence homologues in S. pombe
No structural information is currently available for the S. pombe mitoribosome.However, in collaboration with PomBase, we have managed to assemble a comprehensive list of putative proteins, based on (i) sequence homology with the human and/or S. cerevisiae mitoribosomal constituents, and (ii) in vivo interactions with the S. pombe translational activator Mrh5 (see 'Mitochondrial specific translation and early complex assembly') (Table S3). 1,57No homologue has been identified for two S. cerevisiae and 19 human mitoribosomal proteins.Structural and functional studies might retrieve more divergent orthologues, alongside any Schizosaccharomyces-specific components.So far, only four proteins of the S. pombe mitoribosomal small subunit (mt-SSU), and one of the mitoribosomal large subunit (mt-LSU) have been experimentally explored.Respectively, these are termed Urfa (uS3m), Bot1 (mS45), Mrp51 and Mug178 (bS1m isoforms) and Aco2-Mrpl49 (Aco2-bL21m) tandem fusion protein.
Urfa is the only mitoribosomal protein encoded by the mtDNA (Figure 1).The function of Urfa in mitochondrial Other RNA -? tRNA Val/Phe -Note: Cryo-EM structures have been published for the cytosolic ribosome of E. coli, 129 and the mitoribosomes of H. sapiens 67,130 and S. cerevisiae. 76In contrast, the S. pombe mitoribosomal composition was solely inferred by sequence homology (see Table S3).
translation remains ambiguous, though its contribution to mitogenome maintenance has been inferred. 69,71,72A single Urfa residue substitution enhanced the occurrence of respiratory-deficient progeny, which contained mtDNA point mutations and/or deletions (the 'mutator' phenotype).Whilst, a non-sense urfa mutation produced offspring with varying degrees of respiratory deficiency, ranging from normal to no respiratory growth. 69,71Such segregation was recorded across generations (i.e.stably inherited), and could be cured by expressing from a nuclear locus the wild-type, mitochondrial-targeted Urfa.Similarly, the S. cerevisiae homologue Var1 (uS3m) is encoded by the mitogenome, and constitutes the mt-SSU (Figure 1).Interestingly, the relocation of var1 to the nucleus promoted mtDNA instability under stress, probably as a strategy to enhance cellular survival. 73The protein was further shown to co-sediment with the mitochondrial nucleoids, suggesting a physical interaction.Among the nuclear-encoded constituents of the S. pombe mitoribosome, Bot1 was the first ever identified.The protein co-sedimented with the mt-SSU, and contains clear sequence homology to S. cerevisiae Mrps35 (mS45). 74The abolition of bot1 could be obtained by tetrad dissection of a Δbot1/bot1 hetero-diploid, and resulted in bottle-shaped haploids (hence the name Bot1), which briefly proliferated before reaching lethality.Similarly, basal bot1 expression (from a thiamine repressive promoter) was associated with altered cellular morphology, abnormal actin distribution, mitochondrial fragmentation and severely reduced mitochondrial translation.On the contrary, S. pombe overproducing Bot1 showed increased cell size, and enhanced oxygen consumption under fermentable condition, though similar mitochondrial translation to the wild-type strain.Morphology alterations commonly occurs upon respiratory defects, 44,75 and did not suggest an extra-mitoribosomal function of Bot1.

| Structural heterogeneity enables S. pombe mitoribosome to regulate translation
The search for the homologues of S. cerevisiae Mrp51 (bS1m) and human MRPS28 (bS1m) yielded two nuclear-encoded proteins in fission yeast, termed Mrp51 and Mug178. 1 These co-sedimented and co-immunoprecipitated with the mt-SSU protein Bot1, albeit never co-immunoprecipitated with one another. 57In wild-type S. pombe, Mrp51 was highly expressed, especially under fermentable condition.In vivo absence of this protein caused lethality.On the contrary, Mug178 showed relatively low and stable expression across the growth conditions.Upon mug178 deletion, cells were viable, albeit deficient in respiratory growth, Cytb accumulation, cox3 translation and complex IV activity.Taken together, these results demonstrated the presence of two mitoribosomal populations: one contained the bS1m isoform Mrp51, and fulfilled a wider role in mitochondrial translation; while the other instead contained Mug178, and rather specialised in cytb and cox3 mRNA translation.Together with transcriptspecific translational activators, the two mitoribosomal populations coordinate the stoichiometric synthesis of OXPHOS complex subunits (see 'Mitochondrial specific translation and early complex assembly').Interestingly, S. cerevisiae Mrp51 and human MRPS28 both constitute the mt-mRNA exit channel, which in budding yeast, appears as a recruitment platform for translational activators. 67,76The prior protein was further shown to supress mutations in the 5' UTR of cox2 and cox3 mRNA. 77ote: Bold, codons specific to mitochondria.a S. pombe mitochondria contain trnI(cau) and trnI( gau), the prior of which decodes the AUA codon as isoleucine. 21A mitochondrial trnI(cau) is absent in S. cryophilus, S. octosporus, S. osmophilus, 20 S. cerevisiae 16 and H. sapiens. 12b In S. pombe mitochondria, UAA is the primary stop codon, while UAG only appears in some intronic reading frames. 20mong the 10 proteins recognised so far, half are structurally and/or functionally connected to the mitoribosome (Table 4). 1 For instance, Aco2-Mrpl49 (SPBP4H10.15)contains an N-terminal aconitase, and a C-terminus homologous to S. cerevisiae Mrpl49 (bL21m) and human MRPL21 (bL21m).The spbp4h10.15transcript is subjected to alternative selection of poly(A) site. 78Upon addition of a poly(A) tail $400 nucleotides (nt) upstream of the 3 0 end, a stop codon is introduced, producing only the Aco2 domain upon translation.On the contrary, when a poly(A) tail is positioned after the 3 0 end, the mRNA is translated into the pre-Aco2-Mrpl49 polypeptide.No internal cleavage of the tandem fusion protein was observed in the mitochondria.As S. pombe mitochondria also contain another aconitase (Aco1), 1 Aco2 is essential to viability solely because of its association with the Mrpl49 domain. 78Consistently, Mrpl49 alone could sustain mitochondrial translation, albeit at reduced efficiency compared to Aco2-Mrpl49.Thus, tandem fusion might serve to optimise the stability and/or import of the mt-LSU constituent.Interestingly, an interactomics study on S. cerevisiae mitochondria has revealed a potential interaction between Aco2 and the mt-LSU. 79eanwhile, the S. pombe mitochondrial Rsm22-Cox11 is present in vivo as two isoforms (SPAC1420.04cand SPAC19B12.13).The prior has been demonstrated to undergo cleavage after mitochondrial transport, releasing the two domains. 80Thus, the tandem fusion might serve to coordinate the expression and/or import of Rsm22 and Cox11, rather than to persistently tether the two proteins.

|
No study has been conducted on the second isoform.The homologues of S. pombe Rsm22 in S. cerevisiae (Rsm22) and humans (METTL17) each contains an iron-sulphur cluster, and is essential for mt-SSU assembly. 81Whilst, S. cerevisiae Cox11 co-fractionated with the mitoribosome, and functions in the (apparently co-translational) shuttle of copper to Cox1. 82Human COX11 similarly delivers copper to COX1, and further participates in the concerted biosynthesis and/or incorporation of complex IV metal redox centres. 83Taken together, mitochondrial tandem fusion proteins could be a mechanism to couple the various stages of mitochondrial gene expression.In humans and Note: Numbers in brackets indicate start and end residue.Proteins related to the mitoribosome are shaded.Sc and Hp , respectively inferred from S. cerevisiae and H. sapiens homologue. 1 The ribosome silencing factor (Rsf) domain is homologous to human mitochondrial assembly of ribosomal large subunit protein 1 (MALSU1).1 The tandem fusion protein is also present in S. cerevisiae, probably as a strategy to prevent the Rsf domain from interfering with the cytosolic translation machinery.134 DINH and BONNEFOY budding yeast, mitochondrial gene expression is spatiotemporally orchestrated within specialised compartments, respectively referred to as RNA granules and mitochondrial organization of gene expression (MIOREX) (Table 1).84,85 These are dynamic, visible by microscopy and composed of numerous factors involved in mitochondrial transcription, RNA processing and translation.Perhaps, equivalent foci are also present in S. pombe.

| S. pombe Mti2 and Mti3 play partially overlapping roles in translation initiation
In prokaryotes, translation initiation requires the methionyl-tRNA formyltransferase FMT, and the initiation factors 1-3 (IF1-3). 86While FMT formylates methionyl-tRNA, IF1 blocks the product from entering the ribosomal A site.IF2 then positions formylmethionyl-tRNA in the ribosomal P site, and IF3 prevents the premature association of the ribosomal subunits.Except for IF1, the homologues of these proteins have each been recognised in S. pombe, S. cerevisiae and humans. 1 However, human mitochondrial IF2 (MTIF2) contains a 37-residue insertion, capable of occupying the ribosomal A site and further stabilising mt-mRNAs. 87An insert sequence is also present in S. cerevisiae 88 and S. pombe mitochondrial IF2 (Mti2) (unpublished), albeit shorter and functionally uncharacterised.
Regarding fission yeast mitochondria, neither FMT (Fmt1), IF2 (Mti2) nor IF3 (Mti3) is required for viability. 89Normal proliferation was observed in the absence of either Fmt1 or Mti3.In contrast, upon mti2 deletion, both fermentative and respiratory growth were negatively affected, the mtDNA level progressively decreased and mitochondrial translation was barely detectable. 89These phenotypes were exacerbated upon an additional mti3 deletion (Figure 3).Furthermore, both Mti2 and Mti3 cosedimented slightly with the assembled mitoribosome, and primarily with the mt-SSU.Mti2 remained enriched alongside the mt-SSU in the absence of Mti3.Whilst, mti2 deletion abolished any potential interaction between Mti3 and the mitoribosome.Taken together, these results suggested that (i) S. pombe mitochondrial translation could be initiated by both formylated and unformylated methionyl-tRNA, (ii) Mti2 and Mti3 might share certain functions and (iii) Mti2 plays a much more prominent role, and might be required for Mti3-mitoribosomal association.The absence of S. pombe Mti2 could be complemented by overexpressing its S. cerevisiae homologue (Ifm1), indicating a functional conservation.

| S. pombe translation elongation represents an intermediary between S. cerevisiae and humans
In bacterial cytoplasm and human mitochondria, translation elongation involves the delivery of an aminoacyl-tRNA to the ribosomal A site, and the translocation of mRNA and aminoacyl-tRNAs within the ribosome. 86,90espectively, these are mediated by elongation factor Tu (EF-Tu, TUFM) and G (EF-G, GFM1), both of which belong to the GTPase protein family.However, only the prior requires a GTP/GDP exchange enzyme, termed elongation factor Ts (EF-Ts, TSFM).In addition, another GTPase protein (LepA, GUF1) is crucial for the fidelity (accuracy) of translation elongation. 91Each factor contains a structural homologue in S. pombe mitochondria (Figure 3). 1 So far, only Tuf1 (mitochondrial EF-Tu) and Tsf1 (mitochondrial EF-Ts) have been functionally characterised. 92The deletion of tuf1 could be conducted solely on the petite-positive background ptp1-1 (previously mentioned in 'Maintenance of mitochondrial DNA integrity'), that is, a functional copy of the gene is absolutely required for viability.The Δtuf1 ptpt1-1 strain exhibited drastic mtDNA depletion, though most clones stably maintained 1-2% of residual mtDNA, perhaps as storage copies.In contrast, S. pombe could survive in the absence of Tsf1, albeit severely deficient in respiratory growth and mitochondrial translation.The basal translational activity was sufficient to maintain a wild-type mtDNA level.
Interestingly, while both mitochondrial EF-Tu and EF-Ts are present in human and S. pombe mitochondria, only the former was found in S. cerevisiae (Tuf1).In other words, S. cerevisiae Tuf1 functions independently of a GTP/GDP exchange factor.The overexpression of S. cerevisiae Tuf1 complemented for S. pombe tuf1 deletion (Figure 3).Furthermore, S. pombe Tsf1 could be rendered redundant by either overproducing Tuf1, or introducing mutations around its GTP-binding site.These results suggested a strong, albeit incomplete dependency of S. pombe Tuf1 on its GTP/GDP exchange factor.On the other hand, human TSFM could functionally replace its homologue in fission yeast.In contrast, human TUFM complemented neither S. pombe nor S. cerevisiae Δtuf1 mutants, even when its own GTP/GDP exchange factor was co-expressed.Thus, while human mitochondrial EF-Ts could activate GTP/GDP exchange on S. pombe mitochondrial EF-Tu, human mitochondrial EF-Tu appears incapable of delivering aminoacyl-tRNAs to the S. pombe mitoribosome.

| An interplay of factors acts in S. pombe translation termination, salvage and mitoribosome recycling
During translation termination, bacteria recruit the class I release factors 1 (RF1) and 2 (RF2) for stop codon recognition and peptidyl-tRNA hydrolysis. 86Both proteins carry a conserved glycine-glycine-glutamine (GGQ) motif, responsible for cleaving the ester bond between the nascent polypeptide and the tRNA in-ribosome.Subsequently, RF1 and RF2 are discharged from the ribosome, either via the class II release factor 3 (RF3, a GTPase) or spontaneously (for RF2).In some cases, the translation machinery is stalled on the mRNA, and must be salvaged by peptidyl-tRNA hydrolase (PTH), among other factors. 93Contrary to RF1 and RF2, PTH does not possess the GGQ motif, hence could only hydrolyse peptidyl-tRNA already released from the ribosome.Regarding S. pombe mitochondria, a class I release factor (Mrf1) and several peptidyl-tRNA hydrolases (Pth1-4) have been recovered in silico. 1 Pth1 and Pth2 are respectively homologous to the bacterial and archaeal PTH, and neither contain a GGQ motif 94 nor appear essential for viability. 2No further experimental data is available.Interestingly, the S. cerevisiae homologue of S. pombe Pth2, apart from its peptidyl-hydrolase activity, further participates in the removal of non-imported proteins from the translocase of the outer mitochondrial membrane (TOM). 95n the contrary, S. pombe Mrf1, Pth3 and Pth4 each contains a GGQ motif.The independent abolition of F I G U R E 3 General translation factors in Schizosaccharomyces pombe mitochondria.The majority of these factors contain structural homologues in Saccharomyces cerevisiae and/or humans (names as per UniProt).Notably, S. pombe and S. cerevisiae Mrf1 are homologous to two mitoribosomal release factors in human mitochondria (MTRF1 and MTRF1L).Whilst, the human homologue of yeast Pth1 rather acts on the cytosolic ribosome (asterisk, *). 141Based on physical and genetic interaction studies, numerous S. pombe factors (bold) were functionally confirmed and assigned to a specific translational stage.So far, human diseases have been associated with MTFMT, TUFM, TSFM, GFM1, GUF1, GFM2, PTRH2 and MTRFR.The names of some human factors might vary between UniProt and publications: MTRF1L (mtRF1a), MTRFR (C12orf65) and MRPL58 (ICT1).Homologues of S. pombe Ppr10, Mpa1 and Tsf1 have either not been identified (question mark,?) or proven absent (hyphen, À).
these proteins respectively produced cells with severe, slight and no respiratory deficiency. 94,96Increasing the pth4 copy number showed no effect on the Δpth3 strain, though considerably improved the respiratory growth and mitochondrial translation of Δmrf1 (Figure 3). 94hilst, overproducing Pth3 could not complement for mrf1 deletion.To further investigate the genetic interactions, double mutants were constructed.Additive defect was only observed for Δpth4 Δpth3 and Δpth4 Δmrf1.Taken together, these results indicated a partial overlap in function between S. pombe Pth4 and Mrf1.Recently, S. cerevisiae Rso55 (homologous to S. pombe Pth3) and Pth4 have been demonstrated to hydrolyse peptidyl-tRNA in the mitoribosome, preferentially and respectively salvaging the 'no-go' (e.g.under antibiotics inhibition) and 'non-stop' mitoribosome (e.g.loaded with an mt-mRNA without a stop codon). 97Regarding human cells, two mitochondrial release factors have been identified, termed MTRF1L (MTRF1a) and MTRF1. 96While the prior significantly complemented for mrf1 deletion in the fission and budding yeast, the latter could achieve neither, and was subsequently shown to instead decode AGG and AGA as 'stop' codons. 98Other genetic interactions have also been explored, revealing an intricate, partially conserved network of factors involved in the final stages of mitochondrial translation (Figure 3).
After dissociation from the peptidyl-tRNA, the bacterial LSU and SSU are recovered by the ribosome recycling factor RRF, alongside EF-G and IF3 (discussed above). 86S. pombe mitochondria contain a mitoribosome recycling factor (Rrf1) 99 and a GTPase protein (Mef2, paralogous to the mitochondrial EF-G Mef1), 1 none of which is essential for viability. 2Upon S. pombe rrf1 deletion, impaired proliferation on respiratory medium was observed, and could be reverted by overproducing the human homologue MRRF (Figure 3). 99On the contrary, the overexpression of neither fission yeast rrf1 nor human MRRF complemented for the absence of budding yeast rrf1.Thus, S. pombe Rrf1 is more functionally related to human MRRF than the S. cerevisiae homologue.

| Three PPR proteins are important for S. pombe mitochondrial translation
Apart from their involvements in mtDNA transcription and mtRNA stabilisation (previously and respectively discussed in 'Mitochondrial transcription' and 'Mitochondrial RNA processing and stabilisation'), S. pombe PPR proteins also act as general translation factors.On the one hand, Ppr10 participates in mitochondrial translation initiation.The deletion of ppr10 abolished growth on both respiratory medium, and fermentable medium supplemented with antimycin A. 65 Mitochondrial translation showed severe reduction, and mitochondrialencoded OXPHOS subunits were barely detectable.Moreover, mature cytb and cox1 mRNA were less abundant, a result of deprived intron-encoded maturases. 100Tandem affinity purification (TAP) of Ppr10 recovered every mt-mRNAs, several mt-LSU and mt-SSU proteins and a novel nuclear-encoded, mitochondrial-localised protein Mpa1. 65,101Consistently, both Ppr10 and Mpa1 cosedimented with the assembled mitoribosome.Deletion of the two central PPR motifs in Ppr10 compromised its interaction with Mpa1.In addition, Ppr10 co-immunoprecipitated with the mitochondrial translation initiation factor Mti2 (discussed above).Upon either ppr10 or mpa1 deletion, Mti2 (and Mti3) no longer co-sedimented with the mt-SSU. 101Taken together, these data suggested that Ppr10 and Mpa1 cooperatively enhance mitochondrial translation initiation, by physically connecting Mti2 and Mti3 to the mt-SSU.
On the other hand, the contributions of S. pombe Ppr2 and Ppr5 to mitochondrial translation remain enigmatic.Upon ppr2 deletion, cells barely showed any respiratory growth or mitochondrial translation. 58However, the associated Northern blot and prolonged 35 S labelling profile were similar to wild-type.Thus, the absence of Ppr2 might have lowered the efficiency, without affecting the fidelity, of the mitochondrial translation machinery.S. pombe Ppr2 could be the structural homologue of human MRPS27 (mS27, an mt-SSU protein) or PTCD1 (an RNR2 stabilisation factor). 102,103Based on the low expression of Ppr2 in vivo, 1 we would favour the second hypothesis.On the contrary, the absence of ppr5 did not produce any visible defects on cellular proliferation and mtRNA accumulation. 58Surprisingly, mitochondrial translation was enhanced for Δppr5, and suppressed in ppr5 overexpression.Therefore, the corresponding protein plays a general, inhibitory role on mitochondrial translation via unestablished mechanisms.

| S. pombe translational activators cooperate with the mitoribosome to balance Cytb and Cox1 production
During bacterial translation initiation, the 16S rRNA binds to the Shine-Dalgarno sequence, located within the mRNA 5' UTR. 86To date, no Shine-Dalgarno sequence has been detected in the mt-mRNAs of S. pombe, S. cerevisiae and humans.Concerning budding yeast, mitochondrial translation initiation instead employs nuclear-encoded, transcript-specific translation activators. 104,105These proteins facilitate interactions between the mt-mRNA 5' UTRs and the mitoribosome, and in some cases, further participate in translational autoregulation.Similar factors have also been uncovered in fission yeast, through the search for either (i) S. pombe PPR proteins (Ppr4), (ii) the homologue (Cbp7) of an S. cerevisiae mitochondrial translational activator (Cbs2) or (iii) the physical and genetic interactors (Cbp8, Mtf2, Mrh5 and Sls1) of the S. pombe mitochondrial translation machinery.
The cox1 translational activators encompass Ppr4, Mtf2, Mrh5 and Sls1.While Ppr4 and Mtf2 both belong to the PPR protein family, Mrh5 is a DExH/D box RNA helicase.No obvious domain has been found for Sls1.9]106 Across the strains under study, severe respiratory growth defect was observed, coupled with abolished cox1 mRNA translation, un-or barely detectable Cox1 and Cox2, and a secondary defect on Cytb accumulation.While the intronless strains might also exhibit a slight reduction in mature cox1 mRNA, the intron-containing mutants consistently accumulated un-spliced cox1 species.Furthermore, via co-IP and mass spectrometry, the four proteins were shown to bind each other and the mt-SSU (at least through Mrh5) without interacting with Cox1. 57In another study, Sls1 co-precipitated with the cox1 mRNA. 106Thus, S. pombe Ppr4, Mtf2, Mrh5 and Sls1 form a stable complex of translational activators, essential for the synthesis of both Cox1 and its associated intron-encoded maturases 57,106 (Figure 4).
Meanwhile, Cbp7 and Cbp8 play crucial roles in the normal accumulation and translation of S. pombe cytb mRNA.Respectively, the proteins are orthologous to the S. cerevisiae translational activators Cbs2 and Cbp1 (a PPR protein). 57,104,105The absence of either Cbp7 or Cbp8 resulted in severe respiratory deficiency, reduced cytb mRNA level, barely detectable Cytb amount, decreased complex III activity and minimal presence of supercomplex III/IV. 57Unexpectedly, in both cases, defect on Cytb synthesis was negligible, and the protein appeared resistant to degradation.Perhaps, mitochondrial translation without cbp7 or cbp8 produced misfolded and/or aggregated protein, capable of escaping (i) detection from both in vivo mitochondrial proteases and in vitro Cytb antibody, and (ii) supercomplex integration.In addition, Cbp7 and Cbp8 were shown to form a stable complex via co-IP, coupled with either mass spectrometry or Western blot.The prior factor also roughly co-sedimented with the mt-SSU protein Bot1.However, upon the IP of Cbp7, neither mitoribosomal proteins nor Cytb were significantly co-enriched.Thus, Cbp7 and Cbp8 appear less probable as complex III assembly factors, and more credible as cooperative cytb translational activators, interacting transiently or indirectly with the mitoribosome (Figure 4).
Together with the Mug178/Mrp51 mitoribosomal isoforms (previously discussed in 'The mitoribosome'), cytb and cox1 translational activators regulate the stochiometric production of OXPHOS complex subunits (Figure 4). 57This was deduced from two sets of observations.Firstly, upon mug178 deletion, additional absence of either cytb translational activator (Cbp7 or Cbp8) exacerbated the respiratory growth defect, and rendered cytb mRNA translation undetectable.In contrast, overexpression of mug178 (but not mrp51) improved the respiratory growth and Cytb accumulation of Δcbp7 strain.Thus, Mug178 and the Cbp7/8 complex collaborate in cytb mRNA translation.Secondly, the respiratory growth deficiency associated with mug178, cbp7 or cbp8 deletion was suppressed by the same chromosomal mutations, occurred in either Mrp51 or one of the cox1 translational activators (Ppr4, Mtf2 or Mrh5).The general rescue mechanism was enhanced Cytb synthesis, coupled with lowered productions of one or several complex IV subunits.

| S. pombe transcript-specific translation might not adhere to the autoregulation model
In S. cerevisiae mitochondria, Cbp3 and Cbp6 function as both cytb translational activators and early complex III assembly factors, and generate a negative feedback loop on cytb mRNA translation. 104,105Whilst, the S. pombe (and human) homologues of these proteins solely contribute to the correct assembly of complex III 107,108 (Figure 3).Upon the absence of either Cbp3 or Cbp6, S. pombe respiratory growth was abolished, though cytb mRNA remained at normal cellular levels.The Δcbp6 strain was further analysed, revealing no deficiency in cytb translation, albeit undetectable Cytb subunit and its associating complex III.Thus, Cbp6 (and perhaps also Cbp3) is crucial for Cytb stabilisation, hence complex III assembly.
Similarly, while S. cerevisiae Mss51 mediates the autoregulation of cox1 mRNA translation, 104,105 the S. pombe homologue functions solely in early complex IV assembly 107 (Figure 4).The deletion of S. pombe mss51 resulted in severe respiratory growth defect, coupled with enhanced Cox1 and Cox2 degradation, as well as dramatically reduced complex IV presence and activity.The accumulation and translation of cox1 and cox2 mRNAs showed no or very little deficiency.Thus, S. pombe Mss51 functions primarily in the stabilisation and assembly of complex IV subunits.The homologue of S. pombe Mss51 in humans (MSS51 or ZMYND17) is instead a mitochondrial metabolic regulator. 1093 | Members of the YidC/ALB3/OXA1/ COX18 family play vital roles in early OXPHOS complex assembly Another assembly factor of S. pombe complex IV is Cox18.110 The absence of this protein abolished growth on respiratory medium, and destabilised Cox2, despite showing a similar mitochondrial translation profile to the wild-type.Cox18 belongs to the YidC/ALB3/OXA1/ COX18 membrane insertase family, whose members are evolutionarily conserved from prokaryotes to eukaryotes.111 S. cerevisiae Cox18 and human COX18 also participate in complex IV assembly, more specifically in the translocation of the Cox2/COX2 C-terminus in the intermembrane space.Overexpression of the corresponding genes partially complemented for S. pombe cox18 deletion.110 The other YidC/ALB3/OXA1/COX18 mitochondrial member is OXA1.In S. cerevisiae and humans, Oxa1/ OXA1L is responsible for the IMM translocation of Cox2/ COX2 N-and C-terminus, and of other mitochondrialencoded and some nuclear-encoded proteins. 111To perform these functions, the protein interacts with the mitoribosome, and several OXPHOS complex assembly factors (e.g. S. cerevisiae Cbp3/6 and Mss51), among other proteins.S. pombe Oxa1 is present in vivo as two isoforms, both of which could partially complement for a disruption in the sole S. cerevisiae OXA1.112 The isoforms are encoded on different chromosomes, and as diverged from one another as they are from the S. cerevisiae and human homologues.Upon a disruption in the S. pombe oxa1 on chromosome 1 (SPAC9G1.04,isoform I), normal growth was observed on fermentable and respiratory medium, coupled with slight reductions in complex III and IV activities.On the contrary, disruption of the chromosome II oxa1 (SPBP4H10.03,isoform II) resulted in severe respiratory deficiencies: neither growth on respiratory medium nor resistance to antimycin A, absence of complex III and IV cytochrome spectral absorption and F I G U R E 4 Schizosaccharomyces pombe mitochondrial transcript-specific translation and early OXPHOS complex assembly.S. pombe mitochondria contain two mitoribosomal populations.These are distinct by virtue of the bS1m isoform (Mug178 or Mrp51), and cooperate with translational activators (Cbp7/8 and Ppr4/Mrh5/Sls1/Mtf2) to initiate the translation of specific mt-mRNAs.During and after mitochondrial translation, the nascent polypeptides are subjected to stabilisation and IMM insertion by general (Oxa1) and protein-specific factors (Cbp3/6, Mss51 and Cox18).Eventually, mitochondrial-and nuclear-encoded subunits are assembled into OXPHOS complexes and supercomplexes.Unknown translational activators and complex-specific assembly factors are shown as question marks (?).Some representations have been inferred from Saccharomyces cerevisiae and/or humans, and remain to be confirmed in S. pombe: the mitoribosome is tethered to the inner mitochondrial membrane (IMM) via the mt-LSU and Oxa1; translational activators and early complex assembly factors are either peripheral or integral IMM proteins; and Cox18 specifically inserts the C-terminus of Cox2 to the IMM.substantial reductions in complex III and IV activities. Theoverexpression of isoform I could partially rescue the growth phenotypes, and the double isoform disruptions were lethal, regardless of the presence or absence of the ptp1-1 petite-positive mutation.Moreover, overexpression of neither oxa1 isoforms could complement for cox18 deletion, consistent with observations on the S. cerevisiae homologues.111 Therefore, despite close structural homology, the YidC/ALB3/OXA1/COX18 mitochondrial members within each species exhibit certain functional divergence.Isoform I of S. pombe Oxa1 could be a backup for isoform II.Alternatively, the proteins might associate with different mitoribosomal populations (previously discussed in 'The mitoribosome').The presence of two oxa1 genes is not a hallmark of petite-negative yeasts, because K. lactis contains a single oxa1 coding sequence.9 | CONTRIBUTIONS OF S. POMBE TO MITOCHONDRIAL RESEARCH 9.1 | S. pombe is a tool for mitochondrial biomedical research S. pombe has been successfully employed in biomedical research, whose applications are relevant to mitochondrial disorders.Firstly, studies conducted on fission yeast contributed to the recognition of (i) a novel cancer susceptibility gene ( pif1), 113 (ii) the toxicity off-target (POLG) of sunitinib (an anticancer molecule) 114 and (iii) the action mechanism of osthole (another chemotherapeutic). 115condly, using the S. pombe single-gene deletion library, multiple proteins have been found to promote resistance against doxorubicin, a chemotherapeutic agent.116 Among the significant factors were Ppr1, Cbp6 and Cox6, whose roles are cox2 and cox3 mRNA stabilisation, complex III assembly and complex IV subunit, respectively (previously discussed in 'Mitochondrial RNA processing and stabilisation' and 'Mitochondrial specific translation and early complex assembly').The study also revealed an extensive network of molecules, residing in numerous cellular compartments, and acting in conjunction to ensure cellular survival in the presence of doxorubicin.Thirdly, S. pombe has also assisted disease modelling and drug repurposing.For example, human OPA1 functions in mitochondrial fusion, and is structurally and functionally homologous to S. pombe Msp1.14 Mutations in the prior could cause dominant optic atrophy, a neurodegenerative disorder with no effective treatment.Upon screening of a drug library on an S. pombe msp1 mutant, hexestrol and clomifene were shown to suppress the associated phenotypes (respiratory growth deficiency, mitochondrial fragmentation and/or mtDNA depletion).117 Because hexestrol and clomifene have already been used on humans, the drugs could be repurposed for OPA1-, and perhaps other mitochondrial fusion-related disorders.

| S. pombe is a highly pertinent model and tool to study mitochondrial translation
Fission yeast has established itself as a valuable model for fundamental research, especially regarding mitochondrial translation.Compared to S. cerevisiae, S. pombe better resembles human cells in terms of general translation factors.For example, mitochondrial EF-Ts are present in fission yeast (Tsf1) and humans (TSFM), though absent in budding yeast (previously discussed in 'Mitochondrial general translation factors') (Figure 3). 92Concerning mitoribosomal recycling factors, human MRRF could complement for S. pombe rrf1 deletion, albeit had no effect on S. cerevisiae ΔRRF1. 99Whilst, the human release factor MTRF1L, when expressed with its own mitochondrial targeting sequence, better accumulated in fission yeast (rather than budding yeast) mitochondria. 96Upon the absence of either human MTRF1L or S. pombe mrf1, cells exhibited respiratory defects less severe than S. cerevisiae ΔMRF1.This might be attributed to the lengths of mt-mRNA 3' UTRs, which range from hundreds of base pairs in budding yeast, to a couple of dozens in fission yeast and mostly absent in humans (Table 1).Consistently, the double deletion of S. pombe pah1 and mrf1 was lethal, 94 the former of which functions in the 3 0 exoribonucleolytic cleavage of mt-mRNAs (previously discussed in 'Mitochondrial RNA processing and stabilisation').In other words, by increasing the length of mt-mRNA 3' UTRs, fission yeast becomes more dependent on Mrf1.
Although S. pombe is petite-negative, the majority of respiratory mutants are viable.Moreover, strong mitochondrial defects often select for mitogenome conservation in this species, rather than mtDNA instability as per S. cerevisiae.For example, fission yeast devoid of mti2, tsf1 or mrf1 stably maintained a pool of residual mtDNA, despite minimal mitochondrial translation (previously discussed in 'Mitochondrial general translation factors'). 89,92,96In some cases, respiratory mutations are lethal.These could be confirmed by tetrad dissection of a hetero-diploid containing a wild-type allele (e.g.Δbot1/ bot1, 74 previously discussed in 'The mitoribosome'), and/or investigated in a petite-positive background (e.g.tuf1 ptp1-1 92 ).In the latter example, a strong mtDNA depletion homogenously occurred across the population (previously discussed in 'Mitochondrial general translation factors').However, the residual mitogenome was wild-type, and brought about cellular proliferation once mitochondrial translation was restored.By retaining some wild-type mtDNA, S. pombe respiratory mutants enable genetic interaction studies (reversion, complementation, suppression, etc.), which are vital for functional characterisation.
Research on S. pombe mitochondrial translation encounters three major drawbacks.Foremost, 35 S labelling of newly synthesised proteins is rather inefficient in fission yeast, rendering short pulse difficult to implement.In our experience, this could be improved by replacing cycloheximide with anisomycin for cytosolic translation inhibition. 57Secondly, because S. pombe mitochondrial transformation has yet to be achieved, mitochondrial translation defects could not be studied using reporter genes.An example is Arg8 m , which has been extensively applied to S. cerevisiae. 118Once mitochondrial transformation is established for fission yeast, the expression of heterologous genes would greatly benefit from the near universal genetic code in S. pombe mitochondria.Thirdly, similar to S. cerevisiae, S. pombe contains no complex I, instead possessing nuclear-encoded, single-polypeptide NADH dehydrogenases (Table 1). 1,30Consequently, any study concerning human complex I must employ an alternative model (e.g.Yarrowia lipolytica, Neurospora crassa or Podospora anserina).

| S. pombe is a tool to investigate novel concepts in mitoribosomal biology
As previously discussed (in 'The mitoribosome'), S. pombe mitochondria contain at least two populations of mitoribosomes, which carry either the Mrp51 or Mug178 isoform, and preferentially translate cytb and cox3 mRNA, or the remaining mt-mRNAs, respectively. 57reviously, only cytoplasmic ribosomes were shown to induce transcript-specific translation via compositional paralogues. 119For instance, to synthesise nuclearencoded, mitochondrial-localised proteins, S. cerevisiae cytoplasmic ribosomes must contain the b isoforms of uL1, uL2 and eS26.Considering the nomenclature of known cytosolic ribosomal paralogues, and the functions of S. pombe Mrp51 and Mug178, we propose a second name for these isoforms: bS1ma and bS1mb, respectively.
Mitoribosomes had been demonstrated to regulate translation, though not via structural heterogeneity.Specifically, in S. cerevisiae, the absence of the mt-SSU protein Cox24 (mS38) modified the decoding centre of the mitoribosome, and was associated with primary defects in cox1-3 mRNA translation initiation. 120Such phenotypes occurred regardless of modifications in translational activators, and overexpression of cox24 showed wild-type (rather than enhanced) Cox1-3 synthesis.Moreover, the position of Cox24 within the mitoribosome would have prevented any interaction of the mitoribosomal protein with translational activators, and only one population of mitoribosome has been detected in S. cerevisiae mitochondria. 76Therefore, the preference towards cox1-3 mRNA translation is an intrinsic property of S. cerevisiae mitoribosome, mediated by Cox24, and dependent on neither translational activators nor structural heterogeneity.It would be particularly interesting to investigate the role of the Mrx6-Cox24 (Mrx6-mS38) tandem fusion protein (Table 4) in the two S. pombe mitoribosomal populations.

| CONCLUSION AND PERSPECTIVES
Fission yeast is an invaluable model for studies on mitochondrial biogenesis and diseases, due to the availability of experimental and database support, and its inherent resemblance to human cells.Notably, the two organisms share a similar structure of, and dependency on, the mitogenomes.Expression of these genomes generally employs conserved mechanisms (e.g.mt-tRNA punctuation) and protein machinery (e.g.general translation factors).However, compared to the amount of knowledge from S. cerevisiae and humans, our understanding of S. pombe mitochondrial gene expression is still at an early stage.The participating factors remain obscure for numerous processes, and only two multimeric protein complexes have had their structures resolved: OXPHOS complex III (published as part of supercomplex III/IV) 121 and complex IV (retrieved as either a supercomplex III/IV constituent 121 or a stand-alone complex 122 ).
Further application of fission yeast to mitochondrial research necessitates structural information on the mitoribosome.To date, the recognition of S. pombe mitoribosomal constituents have solely or predominantly relied on sequence homology.Structural resolution of the mitoribosome would (i) contribute to the functional confirmation of these proteins, (ii) enable the identification of Schizosaccharomyces-specific components, (ii) further elucidate the biological significance of the bS1m and Oxa1 isoforms and (iv) provide information on the physical partners of and conformational changes within the mitoribosome.Previously, cryo-EM analyses have uncovered several novel ligands (e.g.iron-sulphur clusters) in the human mitoribosome, 123 and a protein recruitment platform on the S. cerevisiae counterpart. 76The former discovery emphasises the importance of functional coupling in mitochondria, more specifically between mitochondrial gene expression and iron metabolism.Interestingly, the absence of several S. pombe mitochondrial PPR proteins has been shown to perturb iron homeostasis. 124,125esearch efforts should also be dedicated to other elements in S. pombe mitochondrial biogenesis.Firstly, studies on the leucine-tyrosine-arginine motif (LYRM) should recover novel complex assembly factors, and/or improve our understanding of the aforementioned functional coupling.In humans, mitochondrial LYRM proteins are critical to the assembly and function of multimeric protein complexes, especially those containing iron-sulphur clusters. 126Several members have been recognised in budding and fission yeast, none of which are functionally characterised. 1,30Secondly, while replication origins, nucleoids and gene expression foci have been well established in S. cerevisiae and human mitochondria, they remain understudied or undocumented in fission yeast.Thirdly, various S. pombe mitochondrial complexes are structurally and/or functionally ambiguous, including the mitochondrial RNase P, the mitochondrial contact site and cristae organising system (MICOS) and the endoplasmic reticulum-mitochondria encounter structure (ERMES).In addition, S. pombe mitophagy is only starting to be investigated, using for instance, the general translation factor Tuf1. 127 In conclusion, considering its versatility, originality and biological relevance, fission yeast is a powerful model for biomedical and fundamental studies related to mitochondria.We are convinced that through time, as our understanding of S. pombe mitochondrial biogenesis improves, the model system would carry on contributing to these areas of research, and gain even more traction in the scientific community.

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Despite containing similar gene content to other yeasts, Schizosaccharomyces pombe mtDNA is exceptionally compact Comparison of mitochondrial-related features between yeasts and humans.
T A B L E 1 Several tandem fusion proteins from S. pombe appear crucial to the mitoribosome S. pombe mitochondria contain an abnormally high number of nuclear-encoded tandem fusion proteins.Moreover, Schizosaccharomyces pombe mitochondrial tandem fusion proteins.
T A B L E 4