Post-transcriptional regulation of mitochondrial function

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ScienceDirect Post-transcriptional regulation of mitochondrial function De´sire´e Schatton and Elena I Rugarli RNA-binding proteins (RBPs) can control each step of the mRNA life cycle, such as splicing, nuclear export, stability, degradation, localization, and translation efficiency. Thus, RBPs can modulate the final amount of protein level and play an important role in fine-tuning many cellular processes. Surprisingly, the role of specific RBPs to allow the dynamic and coordinated expression of functionally related mitochondrial proteins has begun to emerge only recently. These RBPs define specific post-transcriptional RNA regulons that fine-tune mitochondrial gene expression, thus tailoring the effects of broad transcriptional responses to specific demands or affecting mitochondrial biogenesis independently from de novo transcription. Moreover, a handful of RBPs promote translation of mitochondrial proteins in close proximity to the organelle. Here, we review the molecular components that play a role in these elaborate and flexible mechanisms, and discuss the physiological implications of post-transcriptional regulation for mitochondrial function. Address Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany Corresponding author: Rugarli, Elena I ([email protected])

Current Opinion in Physiology 2018, 3:6–15 This review comes from a themed issue on Mitochondria biology Edited by John Elrod and A˚sa Gustafsson

https://doi.org/10.1016/j.cophys.2017.12.008 2468-8673/ã 2017 Elsevier Ltd. All rights reserved.

demands. Moreover, transcriptional cascades control broad gene programs and cannot fully account for the dynamic and plastic nature of mitochondria, which can quickly adapt to environmental conditions without necessarily increasing in number. Finally, in certain physiological situations the translation of mitochondrial proteins is enhanced without a corresponding increase in the transcript levels [2,3]. Here, we review how elaborate post-transcriptional mechanisms, involving binding of RBPs to specific transcripts, fine-tune the expression of nuclear-encoded mitochondrial proteins (NEMPs), thereby affecting physiological processes. Post-transcriptional mechanisms operating inside mitochondria have been recently reviewed elsewhere [4].

Alternative splicing of pre-mRNA regulates the function of mitochondrial proteins The expression of protein variants through alternative splicing contributes to the diversification of the mitochondrial proteome, and may explain in part the tissue specific functions of mitochondria. For example, OGDH, encoding a-ketoglutarate dehydrogenase, has a different splicing pattern in neural precursor cells and in differentiated neurons, resulting in the presence or loss of a stretch of amino acids containing a Ca2+-binding motif, and therefore in differential sensitivity to regulation by Ca2+ [5,6]. A hint for the existence of specific molecular components that regulate mitochondrial function by influencing splicing of pre-mRNAs comes from a recent study that has implicated the RBPs TIA1 and TIA-R in enhancing inclusion of exon 4b in mRNA variants of the OPA1 gene, encoding the GTPase that controls fusion of the inner mitochondrial membrane [7]. The biological significance of this splicing isoform and the conditions that require these RBPs to modulate OPA1 expression remain to be clarified.

Introduction

The fate of mRNAs encoding mitochondrial proteins: to translate or to decay

Mitochondria are crucial organelles not only to produce ATP, but also to regulate fundamental processes, such as Fe-S cluster formation, Ca2+ signaling, and apoptosis. Not surprisingly, mitochondrial biogenesis, usually defined as an increase in the mitochondrial mass to cope with increased metabolic and energy requirements, is essential during development and adult life. Mitochondrial biogenesis is governed by a well-characterized network of transcription factors and regulators (reviewed in [1]). Transcriptional responses, however, operate over a time frame that might be too slow for mitochondria to respond to physiological stressors and transient changes in energy

Protein synthesis depends both on the efficiency of translation and on the availability of the corresponding mRNA, which ensues not only from the rate of transcription but also from the stability of the transcript. Intriguingly, mRNA lifetime and translation are intimately coupled and often concomitantly affected [8–10]. In recent years, RBPs that determine decay versus translation of transcripts encoding NEMPs have been identified. These molecular components coordinate the fate of functionally related post-transcriptional operons or RNA regulons and allow a more sophisticated regulation to mitochondrial gene expression (Figure 1).

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Post-transcription regulation of mitochondrial function Schatton and Rugarli 7

Figure 1

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RBPs define RNA regulons that orchestrate the expression of a specific subset of NEMPs. Transcription factors (TFs) control the production of a broad set of nuclear-encoded mitochondrial mRNAs implicated in different functional pathways (exemplified by different colors). mRNAs are sorted

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8 Mitochondria biology

The best characterized example of an RBP controlling the fate of transcripts encoding NEMPs is the yeast Puf3, a member of the highly conserved Pumilio and FBF (PUF) family of RBPs. PUF proteins harbor a C-terminal RNA-binding domain, the Pumilio homology domain, which is composed of eight tandem imperfect repeats of a 36 amino acid sequence motif [11]. Puf3 specifically binds to the 30 UTR of a large number of mRNAs encoding NEMPs involved in import, translation, and respiration [12–14]. As other Puf family members, Puf3 represses translation by recruiting the cytoplasmic deadenylase complex, Ccr4-Pop2-Not, and decapping factors to its bound transcripts, thus promoting deadenylation, decapping and degradation [13,15–18] (Figure 2a). This explains why Puf3 deletion increases the levels of target mRNAs [19]. The role of Puf3 in regulating the expression of mitochondrial genes is however much more complex. When yeasts are grown in carbon sources that require mitochondrial function, Puf3 remains bound to its transcripts but fails to mediate decay of targeted mRNAs [16,18]. Under these conditions, phosphorylation of Puf3 turns the molecule into a translational activator via an as yet unclarified mechanism [20], exemplifying how a single RBP can act as a molecular switch to mediate degradation or translation of the same set of functionally related mRNAs under different metabolic conditions without the need of de novo transcription (Figure 2a). Puf3 is not the only RBP that affects the fate of transcripts for NEMPs by promoting deadenylation. In a Drosophila model of oculopharyngeal muscular dystrophy (OPMD), the RBP Smg recruits the CCR4-NOT complex selectively to mRNAs encoding oxidative phosphorylation (OXPHOS) subunits, causing their instability. OPMD is caused by a trinucleotide expansion that leads to an abnormal polyalanine stretch in the poly(A) binding protein nuclear 1 (PABP1). This mutation leads to a widespread reduction of the length of poly (A) tails in several transcripts, however only those encoding for OXPHOS components are recognized by Smg and targeted to degradation [21]. This aberrant post-transcriptional regulation may rationalize the earlyonset mitochondrial dysfunction observed in the muscles of fly and mouse OPMD models, as well as of human patients [21]. Despite the fact that two Pumilio family members are present in the human genome, there is no evidence of a functional conservation of Puf3. Recently, however, the evolutionary conserved CLUH protein was found in HeLa cells to act as a RBP that specifically binds hundreds of mRNAs encoding NEMPs [22]. A role as RBP was also attributed to clueless, the fly orthologue, and the TPR domains in the C-terminus of the protein were

linked to RNA-binding [23]. Originally identified for the mitochondrial clustering phenotype caused by its deletion in several organisms (hence the gene name) [22,24–28], CLUH is required to promote stability and translation of mRNAs encoding proteins involved in OXPHOS, the TCA cycle, b-oxidation, ketogenesis, ketolysis, amino acid degradation, and proteolytic quality control [22,29]. In contrast, structural components of mitochondria and the import machinery (which notably are Puf3 targets) did not appear as CLUH targets, suggesting that they may be subject to a different type of regulation, and that CLUH does not simply support programs to increase the number of mitochondria. In absence of CLUH, target mRNAs and the respective encoded proteins are decreased in abundance in mammalian, fly, and plant models [27,29,30]. At least in mammalian cells, this reduction of steady-state levels is due to a decreased lifespan of target mRNAs [29]. It is worthwhile to point out that in absence of CLUH translation of the target mRNAs seems to be impaired to a higher degree than predicted by their increased decay [29]. A direct role of this protein as translational activator is indeed supported by the interaction of both yeast Clu1 and Drosophila clueless with components of the eukaryotic initiation factor 3 [23,31]. Clueless also binds subunits of the small and large ribosomes and co-sediments with polysome fractions [23]. In contrast, in mammalian cells CLUH is found mainly in fractions containing free RNPs and the 40S ribosome [22], suggesting that recruitment to polysomes may be transient or subject to regulation. Analyses of loss-of-function phenotypes linked to CLUH orthologues in multicellular organisms highlight the physiological relevance of post-transcriptional responses in orchestrating mitochondrial metabolic shifts. Whole-body and postnatal liver-specific Cluh knock-out mice have revealed a crucial role of CLUH during the first hours after birth and starvation to reprogram the mitochondrial proteome (Figure 3). CLUH enhances the post-transcriptional expression of mitochondrial enzymes involved in catabolic pathways, such as ketone body production, amino acid degradation, b-oxidation and gluconeogenesis, which mediate survival under nutrient deprivation [29] (Figure 3). A metabolic remodeling was observed also in HeLa cells deleted for CLUH, which showed decreased OXPHOS, increased glycolysis, and a metabolite signature characteristic for a dysfunctional TCA cycle and b-oxidation [32]. In agreement with a role of CLUH under specific metabolic conditions, Drosophila clueless mutants show no abnormalities during development when metabolism is mainly glycolytic, but display an adult muscular phenotype characterized by mitochondrial

(Figure 1 Legend Continued) by RBPs (e.g. Engrailed, Lin28, Puf3, YB-1 and CLUH) into specific RNA subsets determining the degree of translation expression and/or turnover rate. This ultimately modulates the mitochondrial proteome and function. Current Opinion in Physiology 2018, 3:6–15

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Post-transcription regulation of mitochondrial function Schatton and Rugarli 9

Figure 2

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RBPs can switch the fate of their target transcripts depending on the metabolic condition. (a) Under basal conditions Puf3 acts as a translational repressor by recruiting the deadenylase complex Ccr4-Pop2-Not and decapping factors to its bound target mRNAs inducing their deadenylation, decapping and degradation. Under glucose deprivation Puf3 is phosphorylated switching its function from promoting degradation to activating translation [20]. (b) YB-1 is repressing the translation of mRNAs encoding a subset of NEMPs by inhibiting the accession of components of the eIF complex and polysome assembly. Serum stimulation induces Akt-mediated phosphorylation of YB-1, which releases it from target mRNAs, thus allowing formation of polysomes and promoting translation [42]. Depicted in gray are degradation factors; in dark blue ribosomes; in orange the nascent polypeptide chain.

abnormalities, ATP depletion, and decreased levels of mRNAs encoding specific mitochondrial enzymes [27]. Despite evidence for a dual function of CLUH in protecting bound mRNAs from degradation and promoting their translation, the molecular details of how this occurs await clarification: learning which motifs on the mRNA targets are recognized by CLUH and which position they occupy on the transcripts is necessary to untangle its function. It is an open possibility that CLUH and other www.sciencedirect.com

RBPs may act in concert with miRNAs, by binding similar motifs on the mRNAs. Notably, a recent study has identified considerable overlap between miRNA and human Pumilio binding sites [33], suggesting that the interplay between RBPs and miRNAs in human cells may be an important regulatory mechanism. Consistently, there is evidence for the binding of miRNAs in a sequence-dependent manner to transcripts encoding NEMPs to silence their expression and translation under specific conditions [34–39]. Current Opinion in Physiology 2018, 3:6–15

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Figure 3

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CLUH mediates a mitochondrial metabolic switch upon nutrient deprivation. During the post-birth starvation period in neonates and nutrient deprivation in the adult CLUH protects its target mRNAs from degradation and ensures their efficient translation. This leads to an increase in the expression of encoded NEMPs involved in metabolic pathways like OXPHOS, TCA cycle, amino acid catabolism, ketogenesis and gluconeogenesis. This upregulation of specific pathways induces a metabolic switch of mitochondria toward production of ATP and energy equivalents like glucose and ketone bodies [29]. The liver is the main organ performing this metabolic switch. It produces glucose and ketone bodies and secretes them into the blood vessels to sustain the energy demands of peripheral tissues thereby promoting the survival during the starvation period. Depicted in gray are degradation factors; in dark blue ribosomes.

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Post-transcription regulation of mitochondrial function Schatton and Rugarli 11

Regulation of the translation of NEMPs can also occur independently from changes in the steady state of the corresponding mRNAs. The mTOR complex 1 selectively promotes the translation of several NEMPs involved in transcription, translation, and dynamics, via inhibition of the eukaryotic translation initiation factor 4E-binding proteins [40,41], thus providing a feed-forward mechanism to increase OXPHOS to support the high energy demand of mRNA translation during anabolic conditions. Upon serum stimulation, the translational inhibitor YB-1 is phosphorylated and detaches from mRNAs encoding NEMPs, thus allowing their recruitment to polysomes and increased translation [42] (Figure 2b). Other RBPs, such as Engrailed and Lin28, have been proposed to mediate metabolic reprogramming of mitochondria in different physiological conditions by binding to selected mRNAs and enhancing their translation [43,44] (Figure 1). Finally, TIA1 and HuR have been implicated in translational control of specific mitochondrial proteins, mainly by binding the 30 UTR of target genes [45–47]. However, there is no evidence for an action of these RBPs solely in mitochondrial gene expression.

How mRNAs encoding mitochondrial proteins find their destination Another crucial layer of regulation of mitochondrial biogenesis involves the localization of subsets of mRNAs encoding NEMPs to the outer mitochondrial membrane (OMM), allowing localized translation and possibly even co-translational translocation. These phenomena have potentially several advantages [48,49], such as preventing protein misfolding or activity in sites where it might be harmful and reducing the energy cost for protein transport to distant locations, a problem faced especially by polarized cells like neurons. Translation of pre-existing mRNAs already close to mitochondria would not only mediate efficient and fast adaptation to sudden changes in metabolism or under stress, but could in principle spatially restrict remodeling of the mitochondrial proteome to subsets of organelles. Pioneering studies in yeast determined that translationally active cytosolic ribosomes (80S) associate to mitochondria, and suggested a role of receptors on the OMM and the nascent polypeptide emerging from the ribosome [50,51]. More recently, proximity-specific ribosome profiling revealed that most yeast inner mitochondrial membrane proteins are cotranslationally targeted to mitochondria [52]. Ribosomes have been observed on the OMM in yeast cells by electron microscopy [53]. Intriguingly, mRNAs encoding NEMPs were found to localize to the OMM in yeast, plants and animals [54–61]. How does this targeting occur? The knowledge is still very poor and RBPs that may specifically mediate trafficking of mRNAs for NEMPs by linking them to specific vesicular pathways or to cytoskeletal components have www.sciencedirect.com

not been discovered so far [62]. A recent study in yeast has implicated trafficking involving COPI vesicles in the mitochondrial localization of some mRNAs, including Oxa1, however how COPI components would interact with mRNAs and be involved in trafficking to the mitochondria has not been elucidated yet [63] (Figure 4a). Several studies have instead begun to shed light on how mRNAs may be anchored to the OMM (Figure 4a). One of the required receptors for this process in several species is Tom20, a soluble accessory subunit of the translocase of the OMM [64]. Tom20-mediated localization of mRNAs depends on translationally active ribosomes and requires the mitochondrial targeting signal [64]. In addition, in yeast, the OMM protein Om14 serves as receptor for the nascent chain-associated complex (NAC) while it is associated with translating ribosomes [65]. This interaction allows the binding of the emerging proteins with mitochondria and may enhance protein import [65–67] (Figure 4a). Moreover, the cytosolic chaperone Ssa1 appears to function as a connector between Tom70 and nascent hydrophobic proteins and hence supports mRNA localization to the mitochondria [68]. The transport of mRNAs to the mitochondrial OMM implies that translation must be shut off during transport and activated at the mitochondrial surface. Puf3 may play an important role in these processes. A fraction of Puf3 associates to the OMM together with Mdm12, a component of the ERMES complex, and with the Arp2/3 complex, thus linking mitochondrial biogenesis to actin-mediated motility of the organelle [12,69]. Deletion of puf3 causes in fact mislocalization of its target transcripts and alteration of the organelle morphology and motility [14,69], but impairs growth on oxidative carbon sources only in combination with loss of Tom20 [64]. The mitochondrial pool of Puf3 is thus proposed to be a translational activator, in contrast to its function in the cytosol (see above). It has been envisioned that Puf3 either tethers mRNAs to the organelle or aids co-translational import by slowing down translational initiation [70] (Figure 4a). Perhaps the best evidence for a physiological role of localized translation at the mitochondrial surface has been shown in Drosophila, where the MDI-Larp complex is essential to ensure massive mtDNA replication and mitochondrial biogenesis during oogenesis [71]. MDI (mtDNA insufficient) is residing in the OMM where it recruits Larp (La-related protein), a cytosolic RBP already known to be involved in translational activation of transcripts containing 50 terminal oligopyrimidine (TOP) motifs [72]. Loss of MDI impairs Larp recruitment to the OMM and inhibits localized synthesis of NEMPs involved in the electron transport chain, metabolism, mitochondrial quality control, mtDNA replication and translation (Figure 4b). Since both MDI and Larp are conserved in mammals, it will be exciting to test if they Current Opinion in Physiology 2018, 3:6–15

12 Mitochondria biology

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Transport and docking mechanisms of mRNAs to the mitochondrial surface. (a) In yeast, mRNAs encoding NEMPs are actively transported by COPI vesicles to mitochondria [63]. Docking of mRNAs encoding NEMPs on the OMM is mediated by the accessory subunit of the TOM complex, Tom20 and Puf3 [14], whereas Om14 serves as a receptor for the NAC to promote localized translation [65]. (b) In the fly, mRNAs encoding NEMPs might be actively transported by specific RBPs using motor proteins on cytoskeletal tracks. Anchoring of mRNAs encoding NEMPs to the OMM is mediated by PINK1 which binds mRNAs at the mitochondrial surface and activates their translation, in a mechanism that involves the Parkin-dependent displacement of the translational repressors Glorund and Pumilio by ubiquitination [56]. Another way of mRNA docking to the OMM is performed by the MDI-Larp complex which recruits mRNAs encoding NEMPs and the translation machinery to the mitochondrial surface [71]. Furthermore, Drosophila clueless facilitates the localization of both its target mRNAs and ribosomal subunits to the surface of mitochondria by binding to OMM proteins like Tom20 [23]. Ribosomes are depicted in dark blue.

perform similar functions during developmental biogenesis of mitochondria. Finally, an unexpected link has been uncovered between the Parkinson’ disease genes PINK1 and Parkin and translation of a subset of NEMPs in both fly and mammals [56]. PINK1 would directly bind to the 50 cap of mRNAs encoding specific OXPHOS subunits and to components of the translation initiation complex at the OMM, thereby locally activating their translation. The underlying mechanism implicates the Parkin-dependent ubiquitination of translational repressors, like Pumilio and Glorund/hnRNP-F, which would then be displaced from the 30 UTR of the transcripts (Figure 4b). The implication of Pumilio as a translational inhibitor at the mitochondrial surface may therefore underline some degree of evolutionary conservation of function. Also in this context, translation depends on the interaction of PINK1 with Tom20, which again plays a central role for the recruitment of ribonucleoproteins from Current Opinion in Physiology 2018, 3:6–15

the cytosol to the OMM. It is worth mentioning that the Drosophila clueless genetically interacts with both Pink1 and Parkin, in agreement with a role of these proteins in parallel pathways [27,73,74]. Clueless was indeed shown to bind to ribosomes, Tom20 and porin at the OMM [23] (Figure 4b). A small amount of mammalian CLUH also resides in proximity to mitochondria [22]. Whether CLUH plays any role in docking, stabilizing or activating translation of target mRNAs at the OMM is an open question that deserves thorough investigation. Intriguingly, PINK1 is itself a target of CLUH [22].

Regulation of the expression of mitochondrial proteins at the ribosome Post-transcriptional regulation of gene expression can also occur at the level of the synthesis machinery, the ribosome. An exciting new area of research has revealed specialized ribosomes for the translation of groups of proteins with a similar function [75]. A recent study in www.sciencedirect.com

Post-transcription regulation of mitochondrial function Schatton and Rugarli 13

yeast has involved certain ribosomal paralogs in the synthesis of mitochondrial proteins [76]. In particular, rpl1b deletion strains appeared deficient of mitochondrial proteins involved in ATP synthesis, protein import, the TCA cycle, and notably, also Clu1, leading to alteration in mitochondrial function and morphology [76]. Although the exact mechanism remains to be deciphered, this study opens up new possibilities of how translation of mitochondrial proteins could be regulated. Another way to regulate translation at the ribosome is the use of alternative initiation start codons to produce a protein with a different cellular location from the same mRNA. Several mitochondrial proteins are synthesized via a similar mechanism [77]. As an example, the insulindegrading enzyme has cytosolic and mitochondrial isoforms, which derive from alternative AUGs [78,79]. Whether alternative translation depends only on the mRNA sequence or requires the involvement of specific RBPs is still unknown.

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Outstanding questions Several urgent questions remain to be answered. How many other RBPs exist that specifically bind mRNAs for NEMPs? Does a combinatorial code of RBPs and/or miRNAs regulate other aspects of mitochondrial gene expression, and enhance the flexibility of mitochondrial function? Do post-transcriptional mechanisms regulating mitochondrial biogenesis crosstalk with transcriptional cascades and signaling pathways sensing energy status? In which physiological or pathological conditions RBPs mediate reprogramming of mitochondrial function? What is the role of post-transcriptional mechanisms in mitochondrial dysfunction in diseases and during aging? Finally, is it possible to harness these mechanisms for therapeutic purposes? We predict that novel and probably unexpected twists to the control of mitochondrial biogenesis and function will be unraveled in the next years by studying in depth the molecular components that regulate the life cycle of mRNAs encoding NEMPs. These findings will further illuminate the sophisticated life of mitochondria.

Conflict of interest None declared.

Acknowledgements The authors wish to thank all members of the Rugarli lab for helpful comments. Our research is funded by a grant from the Deutsche Forschungsgemeinschaft (SFB1218, project A05) to E.I.R.

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