Mitochondria are generated by the expression of genes on both nuclear and mitochondrial genome. Mitochondrial biogenesis is highly plastic in response to cellular energy demand, developmental signals and environmental stimuli. Mechanistic target of rapamycin (mTOR) pathway regulates mitochondrial biogenesis to co-ordinate energy homeostasis with cell growth. The local translation of mitochondrial proteins on the outer membrane facilitates their efficient import and thereby allows prodigious mitochondrial biogenesis during rapid cell growth and proliferation. We postulate that the local translation may also allow cells to promote mitochondrial biogenesis selectively based on the fitness of individual organelle. MDI–Larp complex promotes the biogenesis of healthy mitochondria and thereby is essential for the selective transmission of healthy mitochondria. On the other hand, PTEN-induced putative kinase 1 (PINK1)–Pakin activates protein synthesis on damaged mitochondria to maintain the organelle homeostasis and activity. We also summarize some recent progress on miRNAs' regulation on mitochondrial biogenesis.
Mitochondria are organelles bound by a double membrane that convert energy from carbohydrates and fatty acids to ATP through oxidative phosphorylation. They contain their own genome, mitochondrial DNA (mtDNA), which, in most animals, is a small (<20 kb), closed circular double-stranded DNA molecule . Despite its small size, mtDNA is vital for life, encoding 13 essential polypeptides of the electron transport chain (ETC) complexes, as well as 2 rRNAs and 22 tRNAs of the mitochondrial translation machinery . However, mitochondria are not automatous organelle. The majority of the estimated 1500 mitochondrial proteins, including all factors required for mtDNA maintenance and expression, and the remaining components of the ETC complexes are encoded in the nuclear genome. These proteins are synthesized by cytosolic ribosomes and imported into mitochondria . Thus, the generation and function of mitochondria require not only just the expression of both nuclear and mitochondrial genomes, but also some coordination between the two. The amounts of mitochondria and mtDNA are tightly maintained in a given cell type or tissue to meet its specific energy demand . Mitochondrial biogenesis is critical for development and differentiation, and for selective inheritance, the process that limits the transmission of harmful mtDNA mutations . Deregulated mitochondrial biogenesis can lead to severe disorders, including various neuromuscular diseases and reproductive failures [1,5].
In accordance with the essential roles of mitochondria in health and diseases, sophisticated mechanisms are employed to control mitochondrial biogenesis at transcriptional, translational and post-translational levels. Peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and related transcription co-activators are the master transcriptional regulators of mitochondrial biogenesis. PGC-1α can be activated in response to growth signals or energy deprivation . It interacts with specific transcription factors to activate the transcription of myriad nuclear-encoded mitochondrial genes . The targets of PGC-1α include enzymes of energy metabolism as well as factors essential for the replication and transcription of mtDNA . Mice with whole-body deletion of PGC-1α are viable, but show decreased mitochondrial mass and impaired respiratory capacity in the muscle and liver [9,10]. Consistently, mutant flies lacking PGC-1α homolog, Spargel, have reduced body mass and are female sterile with defective mitochondrial respiration. These genetic studies attest the critical roles of PGC-1α family members in mitochondrial biogenesis .
Most mitochondrial protein precursors synthesized in the cytoplasm are transported across the outer membrane (OM) through a multiprotein complex: the translocase of the OM (TOM) [12–14]. In the intermembrane space (IMS), distinct sorting pathway then targets mitochondrial proteins to their final destinations based on the signal sequences within the transported peptides. β-Barrier proteins on the OM are reinserted into the OM through sorting and assembly machinery [13,15]. Proteins containing cysteine-rich signal will be retained and assembled in the IMS via the mitochondrial IMS assembly pathway . Proteins containing N-terminal mitochondrial targeting sequence will be either imported into the matrix or inserted into the inner membrane (IM) by the translocase of the inner membrane (TIM23) complex [12,13]. Some inner membrane carrier proteins are integrated into the IM with the help of the translocase of inner membrane (TIM22) complex . The import of nuclear-encoded components into mitochondria provides another opportunity to fine-tune mitochondrial biogenesis. Several core components of the TOM complex can be phosphorylated by PKA, which inhibits the proteins' import and thereby down-regulates mitochondrial biogenesis . PKA phosphorylates mitochondrial transcription factor A (TFAM) in response to intramitochondrial cAMP signaling. The phosphorylation of TFAM triggers its degradation and thereby down-regulates mitochondrial genome maintenance and expression .
While transcription and translocation both play crucial roles in mitochondrial biogenesis, the object of this review is to describe the role of translation regulation in mitochondrial biogenesis. We particularly emphasize the importance of the local protein synthesis on mitochondrial surface and postulate its potential roles in mitochondrial homeostasis and quality control.
Regulations of cytoplasmic translation of mitochondrial proteins
Energy and mass are two critical elements, driving every single activity of living cells. Given the essential roles of ribosomes in protein synthesis and of mitochondria in energy metabolism, it is conceivable that these two organelles are co-regulated in some level to maintain cellular homeostasis. mechanistic target of rapamycin (mTOR) complex I (mTORC1), a key regulator of cell growth and ribosomal biogenesis, also controls mitochondrial biogenesis and activity. Activation of mTORC1 relieves the inhibitory effect of 4E-BP1 on eIF4E and stimulates the translation of nuclear-encoded mRNAs of mitochondrial proteins, including TFAM and subunits of ETC complexes . It is unclear how mTORC1 regulates these mitochondria-related mRNAs specifically. Some unidentified RNA-binding proteins are probably involved. Clueless (Clu) proteins are highly conserved RNA-binding proteins in eukaryotes. Loss of Clu leads to mitochondrial clustering and abnormal intracellular distribution in several species . clu mutant flies display growth defects and neuromuscular disorders, further corroborating the essential role of Clu in mitochondrial regulation . Clu is located in the cytoplasm and complexed with mRNA-encoding mitochondrial proteins, whose abundance decreases upon the depletion of Clu . Interestingly, some targets of Clu and mTORC1 overlap. It is possible that Clu might be the missing link between mitochondria and mTOR pathway, through which co-ordinates the mitochondrial biogenesis and energy homeostasis with the ribosome activities.
While mTORC and potentially Clu connect mitochondrial biogenesis to cell growth, other cytoplasmic regulators tune mitochondrial biogenesis in response to various physiological stimuli or environmental cues. Chief among those are microRNAs (miRNAs), evolutionarily conserved noncoding small RNA molecules that regulate gene expression posttranscriptionally. Several miRNAs are predicted to target mRNAs of nuclear-encoded mitochondrial proteins, integrating miRNAs into the landscape of translational regulation of mitochondrial biogenesis. For instance, miR-494 is predicted to target TFAM. Consistent with this prediction, knockdown of miR-494 up-regulates TFAM protein level and increases mitochondrial content. Interestingly, the expression of miR-494 is markedly decreased during myocyte differentiation and skeletal muscle adaptation to exercise, both of which are accompanied with an increase in mitochondrial biogenesis. This observation strongly suggests that miR-494 regulates mitochondrial biogenesis via controlling TFAM translation in muscle . Another example is miR-338, a neuronal-specific miRNA that modulates oxidative phosphorylation in the distal axons. miR-338 localizes to axons and down-regulates protein level of cytochrome c oxidase IV, and thereby adjusts energy metabolism, responding to the local need at the distal axons . Besides modulating mitochondrial biogenesis under physiological regulations, miRNAs also regulate mitochondrial functions under environmental stress. miR-210 is induced under hypoxia. It directly represses the expression of several key enzymes in citric acid cycle and ETC complexes [26–28]. The concerted down-regulation of these factors, mediated by miR-210, allows cells to adapt to hypoxia by tuning down the oxidative phosphorylation system and avoiding excessive production of damaging oxidative species. Besides modulating the translation of nuclear-encoded mitochondrial proteins in cytoplasm, miRNAs are also found in the mitochondrial matrix and regulate mitochondrial translation therein. In addition, many more miRNAs have been identified to regulate other mitochondria-related processes, including energy metabolism, mitochondrial dynamics and apoptosis . Nonetheless, the upstream signals regulating miRNAs in response to physiological, developmental or environmental cues remain to be explored.
Localized translation on the mitochondrial surface in yeast
The subcellular localization of mRNAs has been recognized as an effective mechanism to achieve functionally distinct intracellular compartments and structures in diverse organisms and cell types. Large amounts of nucleus-derived mRNAs are associated with the mitochondrial OM in yeast, plant and animal cells [30–32]; most of them encode mitochondrial proteins, and genome-wide analyses uncovered mRNAs for over 400 mitochondrial proteins on the OM in yeast , about a fourth of the ∼1500 known mitochondrial proteins. The mechanisms targeting mRNAs to the mitochondrial OM are partially understood. Puf3, a member of the Pumilio homology domain family proteins, is associated with the mitochondrial OM and preferentially binds to 3′-UTRs of mRNAs that encode mitochondrial proteins [34,35]. Deletion of Puf3 protein or the ablation of Puf3-binding sites on target mRNAs impairs their localization to mitochondria . In addition, Tom complex  and a chaperone protein Ssa1  are also found to be involved in targeting mRNAs to mitochondrial surface.
This localization strongly suggests that these mRNAs are also translated at this site. This idea is consistent with the long-time observation that cytosolic 80S ribosomes are associated with the mitochondrial OM in yeast [39,40]. Recently, an OM protein, OM14, was identified to physically interact with ribosome-associated nascent chain-associated complex, through which to recruit ribosomes to the vicinity of mitochondria . Most importantly, proximity-specific ribosome profiling, indeed, identified that nearly 90% of the proteins synthesized by cytosolic ribosomes, associated with the outer mitochondrial membrane, are mitochondrial proteins; these proteins represent 30% of the annotated yeast mitochondrial proteome . Mitochondrial proteins synthesized in the cytoplasm are imported as unfolded polypeptides. Many chaperones are employed to prevent protein misfolding or aggregation. It is, therefore, a reasonable assumption that the local synthesis of mitochondrial proteins promotes their efficient import and assembly. Recent work in mammals further substantiates it as a conserved mechanism promoting mitochondrial biogenesis in cells or tissues undergoing rapid growth or proliferation.
Localized translation on the mitochondrial surface in animals
Local protein synthesis has also been demonstrated in animals. A mitochondrial OM protein, MDI (short for Mitochondrial DNA Insufficient) encoded by CG3249 locus, was found to be required for mtDNA replication in Drosophila ovary. MDI belongs to a family of conserved A kinase anchoring proteins (AKAPs). AKAPs function as scaffolds to tether PKA, cytoskeleton proteins and other signaling proteins at distinct subcellular locations . However, MDI does not physiologically or genetically interact with PKA. Instead, it complexes with, and recruits the fly homolog of La-related RNA-binding protein, Larp to the mitochondrial surface. The mammalian homolog of Larp, Larp1, stimulates the translation of mRNAs, containing a 5′-terminal oligopyrimidine motif . The MDI–Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial OM . MDI–Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins and ETC subunits. The mitochondrial ribosomes are responsible for synthesizing the ETC subunits encoded on the mtDNA. Thus, MDI–Larp regulates mitochondrial biogenesis by both promoting mtDNA replication and co-ordinating the expression of the nuclear- and mitochondria-encoded components of the ETC complexes. The lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency and reduced mitochondrial contents in mature eggs.
Translation at the mitochondrial OM may facilitate rapid mitochondrial biogenesis in cells with high-energy demand. During Drosophila oogenesis, the mtDNA copy number in the ovary increases from about one hundred to tens of millions in a span of 3 days. This prodigious amplification of mitochondria, which is required to fuel the early stages of embryogenesis, demands vast amounts of proteins. However, the levels of nuclear-encoded mitochondrial mRNAs are either unchanged, or only slightly increase in female germline compared with somatic tissues. Thus, the major driver of massive mitochondrial biogenesis appears to be a translational boost. Given that many mitochondrial proteins, in particular the subunits of the ETC, are highly hydrophobic; local synthesis of these proteins at the mitochondrial surface could efficiently couple translation and import and thereby allow prodigious mitochondrial biogenesis during oogenesis. The mammalian homologs of MDI, AKAP1/AKAP149 in human and AKAP121 in mouse localize to the mitochondrial OM . Interestingly, AKAP121 knockout mice are female semi-sterile , which is similar to mdi mutant flies. Additionally, mammalian mitochondrial AKAPs bind to RNA and hence have been proposed to regulate protein translation on mitochondrial surface [48,49]. Furthermore, the yeast homolog of Larp, Slf1P, associates with and activates the expression of many mRNAs of nuclear-encoded mitochondrial proteins . Thus, the translational boost by MDI–Larp on the mitochondrial OM likely represents a conserved mechanism promoting mitochondrial biogenesis.
Local protein synthesis on the mitochondrial surface may also allow the control of mitochondrial homeostasis at the individual organelle level. We found that the selective inheritance process limiting the transmission of deleterious mtDNA mutations was impaired in MDI mutant flies (Zhang and Xu, unpublished observations). It is possible that MDI–Larp senses mitochondrial fitness and selectively accumulates on healthy mitochondria, thus preferentially promoting the biogenesis of healthy mitochondria. Other findings support this idea, while suggesting an even more discriminating effect of local protein synthesis on mitochondrial selection. The major players of mitophagy, the pathway that eliminates defective mitochondria, have been shown to promote translation at the mitochondrial OM in both cultured mammalian cells and flies . One of them, PTEN-induced putative kinase 1 (PINK1), interacts with Tom20 to localize mRNAs of nuclear-encoded ETC subunits on the mitochondrial surface. The other one, Parkin, displaces translation repressors bound to mRNAs of nuclear-encoded ETC subunits, thereby activating their translation. Parkin selectively localizes to de-energized mitochondria and subsequently triggers their removal through mitophagy . Many OM proteins, including Tom20, are targets of Parkin and rapidly degraded under metabolic stress . Thus, it is counterintuitive that Parkin would promote translation on the surface of defective mitochondria. Recent findings suggest that mitophagy removes only the portions of mitochondrial structure containing damaged proteins [54–56]. Perhaps, PINK–Parkin pathway is only partially activated on mildly damaged mitochondria. Instead of triggering the total disposal of mitochondria, PINK1–Parkin promotes the protein synthesis that might repair the damage and compensate for the loss of proteins caused by mitophagy or other quality control processes.
Translational control in the mitochondrial matrix
The assembly of the respiratory chain requires the co-ordinated expression of nuclear and mitochondrial-encoded subunits. In parallel to the multifaceted translational regulations in the cytoplasm and on the mitochondrial surface, several mechanisms also exist in the mitochondrial matrix to regulate the translation of mtDNA-encoded proteins. Saccharomyces cerevisiae mitochondria genome encodes seven proteins in ETC complexes: cytochrome c oxidase subunits I, II and III, ATP synthase subunits 6, 8 and 9, and apocytochrome b. At least one nuclear-encoded translational activator has been reported for each of them. Most activators bind to the 5′-UTR of the mitochondrial mRNAs, through which they stabilize the mRNAs, facilitate their loading onto mitochondrial ribosomes and activate their translation .
In metazoans, mt-mRNAs usually do not have a significant 5′-UTR, if they have any. It is thereby not surprising that very few mitochondrial translational activators have been identified in metazoans so far. Instead, miRNAs are found inside mitochondria isolated from mammalian tissues and cultured cells and emerged as novel regulators of mitochondrial translation. miR-1 is a nuclear-encoded miRNA, but localizes in the mitochondrial matrix with AGO2. miR-1/AGO2 complex binds to mRNAs of COX1, ND1, CYTB, COX3 and ATP8, and surprisingly enhances, rather than represses, their translation during muscle differentiation . It is unclear how a miRNA could boost protein translation in mitochondria. miR-181c, another miRNA found in mitochondria, appears even more perplexing. The overexpression of miR-181c significantly decreased COX1 protein level, but increased COX2 mRNA and protein content in cardiac myocyte . Given the opposite impacts of the miRNAs on mitochondrial biogenesis, future studies are warranted for a better understanding of miRNAs' functions inside mitochondria and to elucidate underlying mechanisms. Mitochondrial translation is also controlled at the level of ribosomal biogenesis. RNA granules, the ribonucleoparticles that are often found in nucleus and cytoplasm, are involved in various processes of RNA metabolism, including ribosomal RNA transcription, RNA splicing and mRNA degradation. RNA granules are also present in the mitochondrial matrix of mammalian cultured cells and have emerged as centers for RNA processing and ribosomal assembly [60–63]. These granules link transcriptional apparatus with mitochondrial ribosomes and provide a platform for spatiotemporal coupling of several sequential processes of mtRNA expression: RNA processing, ribosome assembly and translation initiation . miRNAs are essential components of processing bodies, RNA granules involved in translational repression and mRNA degradation in the cytoplasm  . Whether miRNAs in the matrix are physically and functionally associated with mitochondrial RNA granules is an interesting, yet unexplored idea.
In conclusion, recent progress has revealed sophisticated translational mechanisms regulating mitochondrial biogenesis and demonstrated their essential roles in controlling energy metabolism, mitochondrial inheritance and quality control. Mitochondrial biogenesis relies on the concerted activities of both nuclear and mitochondrial genomes. A recent study showed that mitochondrial and cytosolic translations are synchronously regulated, implicating translational regulation as a potential orchestrator of the nuclear–mitochondrial co-ordination . Specifically, the synchronization is unidirectionally controlled by an unknown signal from cytoplasm to mitochondria . It would be interesting to test whether any process described above (Figure 1), particularly these mitochondrial mRNA transcription activators or nuclear-encoded miRNAs, might be involved.
An overview of translational regulations of mitochondrial biogenesis.
eukaryotic translation initiation factor 4E binding protein 1
A kinase anchoring proteins
ATP synthase subunit 8
cytochrome c oxidase subunit I
cytochrome c oxidase subunit III
eukaryotic translation initiation factor 4E
electron transport chain
mTOR complex I
- ND1 NADH dehydrogenase
PTEN-induced putative kinase 1
protein kinase A
mitochondrial transcription factor A
translocase of the outer membrane
The work in authors' laboratory is supported by the intramural program at National Heart, Lung and Blood Institute.
The Authors declare that there are no competing interests associated with the manuscript.
We thank Dr Francoise Chanut for critical reading and editing the manuscript.