Moonlighting translation factors: Multifunctionality drives diverse gene regulation

Abstract

Translation factors have been traditionally viewed as proteins that drive ribosome function and ensure accurate mRNA translation. Recent discoveries have highlighted that these factors can also moonlight in gene regulation, but through functions distinct of their canonical roles in protein synthesis. Notably, the additional functions that translation factors encode are diverse, ranging from transcriptional control, extracellular signaling, and RNA-binding, and are highly regulated in response to external cues and intrinsic cellular state. Thus, this multifunctionality of translation factors provides an additional mechanism for exquisite control of gene expression.

Gene regulation through mRNA translation

mRNA translation is the final step of gene expression, and is coordinated through translation factors that function to recruit the ribosome to the mRNA and to regulate elongation of the polypeptide chain. However, as protein synthesis is a hugely energy intensive process, mRNA translation is highly regulated in response to diverse external and intrinsic conditions. This regulation can tune gene expression on both a global and transcript-specific level, thus allowing for both a general decrease in total protein synthesis and the controlled expression of specific proteins. Many translation factors that are critical for protein synthesis also moonlight in additional functions in gene regulation. Indeed, many translation factors exist as gene duplications, and evidence suggests duplications can allow for sub-functionalization or gain of additional functions completely outside of protein synthesis. Here, we will briefly review eukaryotic canonical translation and present examples where translation factors are moonlighting in additional functions outside of their roles in general protein synthesis, both in gene-specific translation regulation (see Glossary) and in other cellular roles.

Translation Initiation: initiation factors in RNA-specific recognition

Translation initiation is a major step of regulation, as it is the rate-limiting step during which the ribosome is recruited to the mRNA (see Text Box 1). Initiation is controlled through careful orchestration of dozens of factors, known as eukaryotic initiation factors (eIFs). Notably, a number of these have been shown to have additional regulatory functions.

Text Box 1.

Translation initiation

Canonical translation initiation is an intricate process that occurs through the necessary coordination of many translation initiation factors, which work together to assemble the ribosomal subunits on the mRNA[59]. Translation initiation starts by the recognition of the 5′ methylated cap structure of the mRNA by the cap-binding multiprotein complex eIF4F, which includes the helicase eIF4A, the scaffolding protein eIF4G, and the cap-binding protein eIF4E. Next, the 43S pre-initiation complex, which includes the 40S ribosomal subunit, eIF1, eIF1A, eIF3, and eIF5, along with the ternary complex (TC), which is made up of eIF2, GTP, and Met-tRNA, is recruited to the mRNA through interactions between eIF4G and eIF3. This association forms the 43S translation complex, which scans the mRNA for the AUG start codon. Recognition of the AUG start codon in a favorable Kozak sequence triggers the TC to release inorganic phosphate from eIF2-GTP, leading to eIF2-GDP dissociation. eIF5B then binds the 48S complex and mediates 60S ribosome subunit, forming a competent 80S ribosome that can begin translation elongation where the initiator aa-tRNA is positioned in the P-site of the ribosomes.

Translation elongation

During translation elongation, the ribosome synthesizes the polypeptide chain, which is encoded by the mRNA engaged by the ribosome.[60] Translation elongation begins with tRNAs being sampled by eEF1A in the A-site of the ribosome for the correct codon-anticodon base pairing. Once the correct aa-tRNA is brought the A-site, eEF1A hydrolyzes GTP, allowing for a conformational change in the aa-tRNA and peptide bond formation. Peptide bond formation is catalyzed by the rRNA in the peptidyl transferase center (PTC) of the ribosome. The uncharged tRNA then moves into the E-site, and the tRNA with the polypeptide chain moves from the A-site to the P-site, which is catalyzed by the GTPase eEF2. The polypeptide chain continues to be synthesized as the ribosome moves along the mRNA until a stop codon is reached.

Eukaryotic initiation factor 4F (eIF4F)

The canonical cap-binding protein eIF4E recognizes the 5′ cap structure through two conserved aromatic residues which coordinate the m⁷G cap[1]. Upon binding the mRNA cap, eIF4E recruits the scaffolding protein eIF4G and the helicase eIF4A to form the eIF4F complex[2]. The eIF4F complex then recruits the 43S preinitiation complex to the mRNA, thus loading the ribosome onto the mRNA (Fig. 1A).

Figure 1. Pathway of canonical translation initiation and elongation.

A. Translation initiation is coordinated by translation initiation factors. Following recognition of the 5′ cap structure by the eIF4F complex, the small ribosomal subunit (40S) in complex with eIF3, eIF1, eIF1A, and eIF2-methionine tRNA, is recruited to the mRNA. The 40S scans to the start codon, release of the initiation factors is catalyzed by eIF5B, and the large ribosomal subunit (60S) can join and form the elongation-competent 80S. B. tRNA synthetases charge tRNAs with amino acids to create a functional aminoacyl tRNA. C. Translation elongation leads to formation of a peptide bond. The incoming tRNA is brought to the A-site of the ribosome through eEF1A after which the polypeptide chain grows in the P-site of the ribosome. The ribosome undergoes translocation mediated through eEF2, and the deacylated tRNA exits through the E-site. Pathways are simplified in the schematics for clarity.

eIF4E also acts outside of this general cap-binding function in regulatory functions during translation initiation. eIF4E can moonlight as an RNA-binding protein, specifically through recognition of a double stem-loop structure located in the 3′ UTR of target mRNAs, called a 4E-sensitive element (4E-SE). In the case of the cyclin D1 mRNA, eIF4E binds to the 4E-SE and the 5′ cap, and this leads to enhanced mRNA transport from the nucleus to the cytoplasm[3]. In contrast, eIF4E also binds to the histone H4 mRNA, and this RNA-binding is essential to orchestrate translation initiation given that the mRNA has a 5′ UTR that is too short to permit conventional ribosome scanning[4]. In this alternative mode of initiation, the H4 mRNA contains a 5′ three-way helix junction that positions the ribosome at the start codon in a cap-independent manner, and eIF4F is provided to the ribosome in cis through binding of eIF4E to the 4E-SE in the open reading frame (Fig. 2A). As eIF4E binds to the H4 RNA through an N-terminal extension upstream of the cap-binding domain, eIF4E can bind to both the H4 RNA sequence and 5′ cap structure simultaneously. It will be of interest to know if there are other examples of this dual usage of eIF4E as an RNA and cap-binding protein and if there is allosteric regulation of these modes of nucleic acid recognition. Interestingly, histone synthesis is rapidly upregulated during the S-phase of the cell cycle, suggesting this alternative usage of eIF4E may be important for temporal regulation of specialized translation.

Figure 2. Translation factors moonlight in functions outside of general protein synthesis.

Many eukaryotic translation factors have evolved diverse functions outside of general protein synthesis, including in RNA-binding, signaling, and transcription regulation. A. Translation initiation factors can moonlight as RNA-binding proteins in gene-specific translation pathways. For example, the Histone H4 mRNA contains a secondary structure that is directly recognized by the eIF4E, which is typically a cap-binding protein. B. During hypoxia, specific stress-response RNAs contain 3′ UTR sequences that are bound by RBM4 and HIF2ɑ. This complex then recruits a non-canonical eIF4F complex which contains the eIF4E paralog eIF4E2. C, D. eIF3 can act outside of its function as a protein scaffold in direct recognition of RNA, either by binding to m⁶A residues or recognizing stem loop motifs. eIF3d additionally is a non-canonical cap-binding protein. E. Translation factors can also be involved in transmitting signals to change gene expression or cellular properties. IFN-gamma treatment of cells leads to the release of rpL13A from the large ribosomal subunit. Cytosolic rpL13A forms the GAIT complex, which binds to conserved 3′ UTR sequences and block translation of those mRNAs. F. During apoptosis, tyrosyl-tRNA synthetase is processed into two fragments, which can induce immune cell migration or act as a leukocyte chemoattractant. G. Translation factors can moonlight in transcription regulation. IgE-Ag-activation causes lysyl-tRNA synthetase to synthesize Ap₄A. This ligand dissociates the HINT1-MITF complex, thus activating MITF-dependent transcription.

eIF4E additionally has paralogs, eIF4E2 and eIF4E3, which are important for supporting translation when eIF4E is inhibited by cellular stress. The eIF4E homologs have differences in cap-binding affinity, with eIF4E3 binding to cap analog ~10 fold less tightly than eIF4E1 and eIF4E2 binding ~100 times less [5, 6]. Correspondingly, eIF4E2 and eIF4E3 rely on distinct mechanisms from eIF4E for recruitment to target RNAs. For example, eIF4E2 mediates translation of hundreds of factors in response to hypoxia, including the epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R) and insulin-like growth factor-binding protein (IGFBP1)[7]. Unlike eIF4E, eIF4E2 cannot bind to eIF4G, and thus its stable binding to 5′ cap structures is mediated through an RNA-binding complex: a complex of the Hypoxia-Inducible Factor 2 ɑ (HIF2ɑ) and the RNA-binding protein RBM4 bind to RNA hypoxia response elements (rHRE) in the 3′ UTR of target mRNAs, and subsequently this recruits eIF4E2 and allows it to stably bind to the 5′ cap (Fig. 2B). As HIF2ɑ expression is regulated by hypoxia and is required for stable eIF4E2 binding to the 5′ cap, this presents an oxygen-regulated switch in cellular usage of eIF4E1 versus eIF4E2. In contrast, unlike both eIF4E and eIF4E2, eIF4E3 has an atypical cap recognition site, with one of the aromatic residues that coordinates the 5′ cap replaced with a cysteine[5]. While the function of eIF4E3 remains poorly understood, it has reduced affinity to eIF4G and thus may instead act as a translation repressor. Beyond the eIF4E paralogs, there are 3 eIF4G (eIF4GI, eIF4GII, eIF4GIII) and 3 eIF4A (eIF4A1, eIF4A2, eIF4A3) homologs. Expression of these proteins are regulated differentially depending on cell state, and in vitro studies have demonstrated that at least eight distinct eIF4F complexes can be formed and mediate translation[8]. It will be of interest to determine how cellular control of eIF4F complexes drives distinct gene programs, and if different post-translational modifications of each eIF4F protein can lead to differential functions.

Eukaryotic initiation factor 3 complex (eIF3a–m)

Following recognition of the 5′ cap structure by eIF4F, the 40S small ribosomal subunit is recruited to the mRNA. This recruitment step relies on interactions between the ribosome-bound 13-subunit eukaryotic initiation factor eIF3 complex (eIF3a–m) and eIF4G (Fig. 1A). Alongside this critical role as a protein scaffold, eIF3 has been widely implicated in many additional functions in regulation of protein synthesis.

eIF3 was originally discovered to have RNA-binding capability through studies of positive-sense RNA virus protein synthesis. eIF3 binds to a highly structured RNA element (domain IIIabc) in the hepatitis C virus internal ribosomal entry site (IRES), and this interaction is required for cap-independent translation initiation[9]. Structural and biochemical studies have revealed that productive eIF3–RNA binding occurs through the eIF3a and eIF3c subunits[10]. Building off these findings, eIF3 is also involved in cellular cap-independent translation. N6-methyladenine (m⁶A) modification of mRNA is the most prevalent post-transcriptional modification on eukaryotic RNAs. eIF3 is recruited to m⁶A modifications in the 5′ UTR of mRNAs, and this allows direct recruitment of the ribosome and cap-independent translation (Fig. 2C) [11].

eIF3 can additionally bind to cellular mRNAs through a multi-subunit interface consisting of the eIF3a, b, d and g subunits. eIF3–RNA binds to ~500 cellular mRNAs, and this leads to translation activation of cell proliferative factors or repression of differentiation factors[12]. These two dichotomous translation phenotypes are due to differences in the modality of eIF3–RNA-binding. In the case of translation activation, eIF3 binds directly to a conserved stem loop in the 5′ UTR, which notably has analogous sequence and structural features to the HCV IRES domain IIIb (Fig. 2D). In contrast, repression cannot be reconstituted in vitro, suggesting this function of eIF3 requires a currently unknown co-factor protein[13].

While eIF3 can target both viral IRESs and cellular mRNAs, translation activation of cellular mRNAs is cap-dependent, and relies on a non-canonical cap-binding domain in the C-terminus of the eIF3d subunit[14]. eIF3d recognizes 5′ cap structures in a distinct mode from the canonical cap-binding protein eIF4E. Instead, eIF3d is structurally homologous to the DXO family of cap endonucleases, and cap-recognition is RNA-specific and regulated by initial recruitment of the eIF3 complex to target mRNAs. Notably, this additional function of eIF3d as a cap-binding protein is critical for cellular control of adaptive translation in response to nutrient deprivation[15]. Under nutrient-rich conditions, eIF3d cap-binding activity is negatively regulated through two phosphorylation sites in the C-terminal tail proximal to the cap-binding domain. Upon chronic glucose deprivation, eIF3d is no longer phosphorylated by CK2, and this drives the increased translation of stress-response proteins required for cell viability.

Outside of these functions of the complex in RNA recognition, open questions remain as to if specific eIF3 subunits may have distinct functions in gene regulation. Indeed, in support of further eIF3 functions to be discovered, individual eIF3 subunits are linked to different tissue-specific cancers, with overexpression of eIF3a, b, c, h, i, and m and reduced expression of eIF3e and f linked to malignancy[16]. Furthermore, certain eIF3 subunits are found to exhibit both cytoplasmic and nuclear localization[17, 18]. It will be important in future studies to study if eIF3 subunits have functions outside of the complex, as part of unique subcomplexes, or through functional single- or multi-subunit interfaces.

Translation Elongation and Termination: multifunctionality both within and beyond protein synthesis

Following recruitment of the ribosome to the mRNA, translation elongation can commence, consisting of mRNA decoding and peptide bond synthesis (see Text Box 1). Elongation requires catalytic activity from the macromolecular RNA-protein ribosome complex, along with the elongation factors eEF1A and eEF2 for charged tRNA delivery and ribosome translocation (Fig. 1B,C). Regulation of elongation is critical to ensure translation accuracy and proteostasis, and is controlled through correct tRNA selection and speed of ribosome translocation.

Ribosome

The critical catalytic machinery for protein synthesis is the ribosome, which is composed of ribosomal proteins and ribosomal RNAs (rRNA). The functional core of the ribosome includes the A, P, and E sites for tRNA binding, the peptidyl transferase center, and the peptide exit tunnel (Fig. 1C). While this functional core is highly conserved throughout eukaryotic, archaeal, and bacterial ribosomes, the ribosomes of higher eukaryotes have evolved increased complexity of expansion segments in the RNA and surface-exposed ribosomal proteins, suggesting these additions may allow for more gene regulation.

While much interest has been placed on the functional significance of heterogeneity of ribosomal protein composition[19], here we will focus on moonlighting functions of ribosomal proteins in an extra-ribosomal context. Extra-ribosomal functions of ribosomal proteins are defined as additional functions executed as individual subunits, in a capacity not bound to the ribosome. One of the best characterized examples of extra-ribosomal protein function comes from studies of the large ribosomal subunit protein rpL13A (Fig. 2E) [20–22]. Upon treatment of human myeloid cells with IFNγ, the zipper-interacting protein kinase (ZIPK) is activated and phosphorylates rpL13a. Phosphorylation of rpL13a leads to its dissociation from the ribosome, allowing it to form the IFN-gamma activated inhibitor of translation (GAIT) complex together with glutamyl prolyl tRNA synthetase (EPRS), NS1-associated protein 1 (NSAP1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The GAIT complex subsequently blocks the translation of mRNAs required for the inflammatory response such as ceruloplasmin and VEGF-A by binding to conserved sequence elements in the 3′ UTR. As ribosomes lacking rpL13a continue to be functional for general protein synthesis, this suggests the ribosome could have a role in “holding” proteins involved in regulating transcript-specific translation.

Outside of translation regulation, there are numerous examples of ribosomal proteins having extra-ribosomal functions outside of protein synthesis. For example, rpS3 can translocate into the nucleus and become part of the NF-kB complex in response to TNF stimulation[23]. Furthermore, rpS7 binds to the E3 ubiquitin ligase MDM2 and blocks its E3 ligase activity, thus stabilizing p53 in response to nucleolar stress[24]. Understanding how cellular perturbation change ribosomal protein occupancy, RNA-binding and translation efficiency will help identify further potential extra-ribosomal functions of ribosomal proteins.

Aminoacyl tRNA synthetases

Aminoacyl tRNA synthetases (aaRS) are a family of 20 enzymes responsible for the charging of substrate tRNAs with the cognate amino acid through ligation of amino acids to the 3′ end of their corresponding tRNA (Fig. 1B). This aminoacylated tRNA is then active to be recruited to the ribosome and used during peptide bond synthesis. Compared to prokaryotic aaRS, human aaRS have evolved domains appended onto the catalytic core. These domains introduce new additional activities to aaRS, ranging from RNA-binding to protein-protein interaction, and allow functionality as both single subunits or as part of larger complexes. As one example of aaRS moonlighting in alternative functions in translation regulation, the threonyl-tRNA synthetase evolved a vertebrate-specific N-terminal extension which interacts with eIF4E2, and recruits this complex to specific mRNAs involved in vascular development. In agreement, genetic dysregulation of threonyl-tRNA synthetase leads to defective angiogenesis in human umbilical vein endothelial cells and the zebrafish model system[25].

Eukaryotic aaRS can also exist in a multi-synthetase complex (MSC), which in eukaryotes consists of nine aaRS, (methionyl-, glutaminyl-, lysyl-, arginyl-, aspartyl-, leucyl-, isoleucyl-, prolyl-, glutamyl-tRNA synthetase) and three aaRS-interacting multifunctional proteins (AIMP1-3). For canonical protein synthesis, the MSC can act as a channeling complex to link tRNA charging to delivery to the ribosome, and hence enhance translation efficiency[26]. For example, AIMP1 directly interacts with arginyl-tRNA synthetase, enhances the recruitment of tRNA, and increases synthetase aminoacylation activity[27]. Similarly, AIMP3 forms a complex between methyionyl-tRNA synthetase, initiator methionine tRNA, and eIF2, thus linking tRNA charging to translation initiation[28]. MSC components can additionally become dissociated in response to cellular stimuli, and thus can also act as a signaling hub for sensing stresses. For instance, UV irradiation causes methyionyl-tRNA synthetase to become phosphorylated by the General Control Nonderepressible 2 (GCN2) kinase, which blocks synthetase charging ability and therefore contributes to a stress-induced decrease in global protein synthesis. Methyionyl-tRNA synthetase phosphorylation additionally leads to the dissociation of AIMP3 from the MSC. AIMP3 enters the nucleus and activates Ataxia telangiectasia mutated (ATM) and ATM and RAD3-related (ATR), thus allowing methyionyl-tRNA synthetase to link the cellular responses to UV stress and DNA damage[29].

Outside of translation, aaRS additionally have non-canonical functions in the nucleus and in extracellular contexts. On an extracellular level, human tyrosyl-tRNA synthetase is secreted from apoptotic cells and processed into two fragments (Fig. 2F). Both fragments exhibit cytokine activities, with the C-terminal fragment containing an endothelial monocyte-activating polypeptide II domain that induces immune cell migration, and the N-terminal fragment acting as a leukocyte chemoattractant[30]. Several aaRS synthesize diadenosine polyphosphates (Ap_nA) in response to thermal and genotoxic stresses[31]. In mast cells, lysyl-tRNA synthetase forms a nuclear complex with Histidine Triad Nucleotide Binding Protein 1 (HINT1) and the microphthalmia-associated transcription factor (MITF), and this sequestration blocks MITF-dependent transcription (Fig. 2G). Upon IgE-Ag-activation, lysyl-tRNA synthetase synthesizes Ap₄A to incredibly high levels, reaching nearly half of the intracellular concentration of ATP. Ap₄A binding to HINT1 leads to the release of MITF and activation of MITF-dependent transcription[32]. Given the wide range of already characterized functions of aaRS outside of tRNA charging, it will be of interest to study how diverse cellular stimuli or developmental contexts affect catalytic tRNA charging activity, cellular localization, or interaction with the MRS or novel cellular co-factors to control functions of aaRS both within and outside of protein synthesis.

eEF1A/eEF2

Translation elongation requires the delivery of the aa-tRNA to the ribosome, which allows elongation of the peptide chain, and translocation of the ribosome to decode the next mRNA codon (Fig. 1C). This process involves the elongation factors, with the aa-tRNA brought to the ribosome by the eukaryotic Elongation Factor 1 (eEF1) complex, and the translocation of the ribosome to the next mRNA codon for decoding driven by eukaryotic Elongation Factor 2 (eEF2). Aa-tRNA delivery occurs as part of a ternary complex consisting of the eEF1 complex component eEF1A bound to GTP and tRNA. Correct formation of the codon-anticodon pair between the mRNA and the incoming tRNA leads to GTP hydrolysis, release of eEF1A-GDP, and subsequent recycling of eEF1A by active GDP-GTP exchange through the guanine nucleotide exchange factor eEF1B.

eEF1A is highly modified, with status changing in response to external stimuli and cellular state. These post-translational modifications regulate the non-translation related functions of eEF1A as well as its function in global translation, and suggest the possibility of an “eEF1A code” linking cellular signaling to translation regulation. eEF1A is phosphorylated by at least eight different kinases and five methyltransferases, and this alters its functions in translation elongation[33–35]. As one example, insulin treatment of cells leads to phosphorylation of eEF1A through S6 kinase and increases translation elongation by 2-fold. In addition, dimethylation of eEF1A at lysine 55 in the GTPase domain by methyltransferase-like 13 (METTL13) increases its GTPase activity and stimulates protein synthesis. This regulatory pathway is taken advantage of by pancreatic and lung cancers, with upregulated levels of METTL13 or increased methylation status of eEF1 both linked to poor prognosis[36]. This modification-induced alteration to function can also be transcript-specific, such as in the case of Lysine-specific Methyltransferase 4 ( KMT4)-directed methylation of eEF1A, which alters translation of proteins involved in the unfolded protein response, the endoplasmic reticulum, ribosome biogenesis and chromatin[33]. eEF1A methylation can additionally regulate the A-site occupancy rates of certain codons[35]. These data altogether raise the possibility that methylation of eEF1A acts as a regulator of gene-specific translation on top of its critical function in global translation. However, the biochemical mechanism by which eEF1A modification affects translation in a gene-specific manner, and what signals controls modification status remain to be elucidated. It will be important to use structural and biochemical approaches to understand if modification changes conformation or affinity of eEF1A for specific tRNAs to allow for codon-specific changes in translation regulation outside of its canonical role in general protein synthesis.

As a further complexity, in mammals, eEF1A exists as two paralogs, eEF1A1 and eEF1A2, which have 98% sequence similarity but exhibit very different tissue and developmental expression profiles. eEF1A1 is ubiquitously expressed in all tissues during embryogenesis and in most adult cells, except for fully differentiated myotubes, neurons, and cardiomyocytes, which instead express eEF1A2 [37]. While both proteins bind to tRNA and direct translation in vitro to similar levels, eEF1A2 binds GDP to higher affinity than eEF1A1. This difference in nucleotide binding may regulate the distinct moonlighting functions of the eEF1A1 and eEF1A2. In particular, eEF1A has additional functions in regulation of the cytoskeleton and cell death decisions. eEF1A has microtubule-severing activity, binds and bundles actin, and also inhibits actin polymerization [38, 39]. However, regulation of actin polymerization by eEF1A is blocked when bound to tRNA[40]. As eEF1A-tRNA binding can only occur in a GTP bound state, the tighter interaction of eEF1A2 with GDP may be important for regulation of this non-translation function. In addition, eEF1A1 and eEF1A2 have opposing roles in cell survival. Deletion of eEF1A2 in mice leads to a wasting phenotype, where motor neurons degenerate and there is loss of muscle. Also, during serum deprivation of myotubes, stable overexpression of eEF1A2 is protective, while eEF1A1 leads to apoptosis [41].

eEF2 is regulated by the eukaryotic elongation factor 2 kinase (eEF2K), which phosphorylates eEF2, reduces its binding to the ribosome, and thus blocks translation elongation. eEF2K activity itself is modulated in a cell stimuli-responsive manner and thus provides a mechanism for the cell to modulate general protein synthesis. For example, eEF2K is turned off by mitogenic signaling pathways, thus activating eEF2 and translation elongation; and correspondingly turned on during hypoxia and nutrient deprivation to block general protein synthesis to conserve energy and as a protective mechanism [42]. Additionally, eEF2 is uniquely modified by a post-translational modification called diphthamide. This unusual modification involves first modification of a conserved histidine (position 715 in mammals) by addition of a 3-amino-3-carboxypropyl group, followed by enzymatic processing to diphthamide through a seven-subunit pathway (DPH1-DPH7)[43]. Diphthamide modification does not seem to be essential for general function, as inhibitory mutants support yeast growth and function like wild-type protein in vitro for mediating ribosome translocation and polypeptide chain synthesis[44, 45]. However, diphthamide modification appears to tune specific eEF2 function and have important biological consequence. For example, mutations to the enzymes responsible for diphthamide modification are linked to human developmental disorders and ovarian cancer[46]. Furthermore, cap-independent initiation of the viral Cricket paralysis virus (CrPV) IRES is dependent on diphthamide-modified state of eEF2[47]. Structural data suggest that the modification stabilizes the interaction between eEF2 and the codon-anticodon interaction, and this could be critical for the unusual translocation steps prior to the first peptidyl transfer that occur during IRES-mediated initiation. Further studies of eEF2 in various developmental and cancer conditions and states will help illuminate how diphthamide regulates multiple functions of eEF2.

eIF5A

The eukaryotic translation initiation factor 5A (eIF5A) family consists of eIF5A1 and eIF5A2, which are ~80% identical but encoded from different chromosomal locations. eIF5A1 is ubiquitously expressed and essential for cell viability, while eIF5A2 is expressed in the testis, brain, and upregulated in specific cancers. The eIF5A proteins are the only known proteins to be hypusinated, which is a two-step process during which the aminobutyl group of spermidine is transferred to a lysine residue and hydroxylated by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH).

eIF5A1 was initially identified as an initiation factor that could stimulate methionyl-puromycin formation, but now is known to be also involved in translation elongation and termination[48, 49]. During elongation, eIF5A1 is important for resolving ribosome pausing at geometrically problematic codons. For instance, consecutive proline motifs would introduce an unusually rigid Pro-Pro bond in the ribosome exit tunnel and lead to difficulties with peptide bond synthesis with the incoming A-site substrate[50]. Structural data have revealed that eIF5A1 binds in the ribosome between the E and P-sites, and the modified hypusine residue interacts with the CCA-end of the P-site tRNA. This interaction allows the P-site tRNA to be stabilized in a conformation that is optimal for peptide-bond formation, and thus lead to productive elongation instead of ribosome stalling[51]. eIF5A1 also enhances termination by stimulating peptide release mediated by the eukaryotic translation termination factor 1 (eRF1)[50].

Notably, knockdown of eIF5A1 in human cells only leads to a ~30% decrease in total protein synthesis, indicating that it is not essential for global translation elongation and instead may be important for specific cellular conditions or regulation of distinct functional pathways. Indeed, as hypusination is required for eIF5A1 functionality and spermidine is a substrate for the rate-limiting enzyme DHPS during hypusination, polyamine biosynthesis pathways exhibit crosstalk with eIF5A1-regulated protein synthesis. For instance, inhibition of eIF5A1 hypusination by treatment with GC7, a DHPS inhibitor, leads to defective mitochondrial respiration in MEFs. This is due to disrupted translational control of specific mitochondrial proteins that contain mitochondrial targeting sequence, which tend to have repetitive, charged amino acids that can stall the ribosome[52]. Correspondingly, GC7 treatment of renal proximal cells blocks mitochondrial function and oxidative phosphorylation, and causes cells to switch to anaerobic glycolysis[53]. Outside of metabolism, eIF5A1 is also linked to diverse cellular phenotypes. For example, eIF5A1 depletion blocks autophagy by controlling the translation of Autophagy Related 3 (ATG3), which contains a difficult-to-translate DDG tripeptide motif[54]. Additionally, eIF5A1 interaction with ribosomes increases upon nutrient starvation, suggesting eIF5A1 may additionally be recruited to translation machinery to mediate a stress response, although it remains unknown why this dynamic alteration occurs[55].

Unlike eIF5A1, the molecular function of eIF5A2 remains relatively uncharacterized. eIF5A2 knockout mice are viable, while eIF5A2 overexpression transgenic mice exhibit accelerated aging, manifesting in a shorter lifespan, faster weight loss, poor wound healing, and skeletal degeneration upon adulthood[56]. eIF5A2 is upregulated in lung, bladder, cervical, and gastric cancers[57]. Intriguingly, this oncogenic link may be due to an unexpected role of eIF5A2 in transcription. Indeed, in esophageal squamous cell carcinoma, eIF5A2 is overexpressed in response to hypoxia and localized in the nucleus, where it binds to the HIF1ɑ promoter and increases HIF1ɑ expression[58]. Given their high sequence identity, it will be of great interest to understand how eIF5A1 and eIF5A2 have diverged to have functions in translation versus transcription regulation, and whether the overlap as hypusination substrates contributes to any feedback regulation in the two functions.

Concluding Remarks

Translation factors and their function in regulating protein synthesis have been studied extensively, revealing how dozens of factors coordinate the loading and translocation of the ribosome on mRNA. Here, we described the diverse functions of translation factors and ribosomal proteins in cellular processes outside of mRNA translation, and discussed how moonlighting is regulated through post-translational modifications, alternative complex formation, and cellular localization (see Outstanding Questions). Notably, many translation factors are associated with human disease, including cancer and developmental disorders. As these phenotypes do not reflect general inhibition of total protein synthesis, one possibility is that the diseases manifest through disruption of the non-canonical moonlighting functions of the translation factors. However, a major difficulty is segregating specific functions of translation factors from their general roles in total protein synthesis. Likely, it will be important to develop genetic or biochemical tools to disrupt specifically moonlighting functions of translation factors. Altogether, understanding how translation factors moonlight in alternative gene regulation functions, and how general versus moonlighting functions are regulated by the cell is critical for our understanding of gene regulation.

Outstanding Questions Box.

• How are translation initiation factors that have many paralogs within the cell regulated to perform their function?

• What are the mechanisms of subunit specificity of translation complexes for moonlighting functions?

• How do external cues induce or change the moonlighting functions of translation factors?

• What other translation factors have moonlighting functions and are they involved in disease phenotypes?

Highlights.

• Translation factors can act outside of their canonical roles and have moonlighting functions that occur normally and during cellular stress.

• Many translation factors have post-translational modifications that are induced by cellular stress and change the function of these proteins.

• Dysregulation of translation factor moonlighting functions can lead to cancer and disease.

• Understanding the moonlighting functions of translation factors is critical for understanding human disease.

Glossary

Gene-specific translation regulation

Mechanisms of translation that allow for regulation of the expression of specific proteins, rather than general changes to the global proteome. These mechanisms typically involve recognition of distinct mRNA sequences of structures by RNA-binding proteins or translation factors.

Moonlighting

Functions of factors outside of their canonical roles. Here, we describe how translation factors can act outside of general protein synthesis to be involved in other cellular functions, including signaling, transcription, or cell fate decisions.

Translation initiation

Translation initiation is the step during which the ribosomal subunits are loaded onto an mRNA and positioned to the start codon. This rate-limiting step of protein synthesis is regulated through dozens of translation initiation factors.

Translation elongation

Translation elongation is the step at which mRNA is decoded into proteins. It is important to control translation accuracy, as incorrectly made proteins can lead to protein misfolding or defective protein function.