You are here: Home > Dever Lab

Mechanism and Regulation of Eukaryotic Protein Synthesis

• Thomas E. Dever, PhD, Head, Section on Protein Biosynthesis
• Ivaylo P. Ivanov, PhD, Staff Scientist
• Byung-Sik Shin, PhD, Staff Scientist
• Arya Vindu, PhD, Visiting Fellow
• Chune Cao, Biological Laboratory Technician
• Joo-Ran Kim, BS, Special Volunteer
• Thomas Saba, BS, Predoctoral Intramural Research Training Award Fellow

We study the mechanism and regulation of protein synthesis, focusing on GTPases, protein kinases, translation factors, and mRNA features that control this fundamental cellular process. We use molecular-genetic and biochemical studies in yeast and human cells to dissect the structure-function properties of translation factors, elucidate mechanisms that control protein synthesis, and characterize how mutations in the protein-synthesis apparatus cause human disease. Of special interest are the translation initiation factors eIF2 (eukaryotic initiation factor 2), a GTPase that binds methionyl-tRNA to the ribosome, and eIF5B, a second GTPase that catalyzes ribosomal subunit joining in the final step of translation initiation. We also investigate stress-responsive protein kinases that phosphorylate the eIF2 subunit eIF2alpha, as well as viral regulators of these kinases, and how cellular phosphatases are targeted to dephosphorylate eIF2alpha. We are characterizing eIF2gamma mutations that are associated with the MEHMO syndrome, a rare X-linked intellectual disability syndrome, and we are investigating the function of the translation factor eIF5A, with a focus on its ability to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates such as proline. We are also examining the role of the hypusine modification on eIF5A and the role the factor plays in polyamine-regulated gene-specific translational control mechanisms, and we are characterizing metabolite control of translation via non-canonical upstream open reading frames (uORFs) in select mRNAs.

Molecular analysis of translation start-site selection stringency

A key interest of the lab is to study the regulation of translation start-site selection. While translation typically initiates at an AUG codon, the efficiency of initiation at a particular AUG codon is influenced by context nucleotides flanking the AUG codon and by levels of the factors eIF1 and eIF5. Interestingly, eIF1 and eIF5 auto- and trans-regulate the translation of their own mRNAs to control the levels of these factors in cells. We are exploiting such autoregulation to generate reporters to assess start-codon selection stringency, and we are also searching for mRNAs whose translation would be sensitive to changes in stringency. In a previous search of mammalian genes, we identified five homeobox (Hox) gene paralogs initiated by AUG codons in conserved suboptimal context, as well as 13 Hox genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context [Reference 1]. Our collaborator Rachel Green mapped the 5′ end of the Hox mRNAs, revealing that the mRNAs are much shorter than previously reported and lack proposed alternative translation elements. We found that the conserved uORFs inhibit Hox reporter expression and that altering the stringency of start-codon selection by overexpressing translation initiation factors eIF1 or eIF5 modulates the expression of Hox reporters. We also showed that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin lowers the fidelity of start-codon selection. Given that the Hox genes encode developmental regulators of animal body plans, our studies reveal that alterations in start-codon selection stringency could control body plan formation in animals.

In parallel with these studies, we are collaborating with Jody Puglisi and colleagues to molecularly characterize the key processes in translation start-site selection including ribosomal scanning, AUG selection, and ribosomal subunit joining. Using single-molecule fluorescence spectroscopy, the Puglisi lab was able, for the first time, to directly track binding of the small 40S ribosomal subunit to an mRNA, scanning of the ribosome down the mRNA, and then joining of the 60S subunit. Their studies revealed that 40S binding to the mRNA is slow, whereas, once bound, the 40S ribosome scans rapidly. Interestingly, RNA hairpin sequences near start codons forced scanning ribosomes at start codons to move backward in the 5′ direction, and in vivo, we showed that these secondary structures enhanced initiation at upstream near-cognate CUG or UUG start codons positioned 15-nucleotides before the stem-loop structure. Thus, RNA structures can influence the stringency of translation start-site selection [Reference 2].

The translation factor eIF5B is a GTPase required for the last step of translation initiation: the joining of the large 60S ribosomal subunit to the small subunit poised on the start codon of an mRNA. The eIF5B binds to the 40S subunit and collaborates in the correct positioning of the initiator Met-tRNA_i^Met on the ribosome in the later stages of translation initiation, gating entrance into elongation. Our ongoing studies with the Puglisi lab reveal that, in addition to promoting 60S subunit joining, eIF5B also controls a checkpoint that helps monitor the fidelity of translation start-site selection, a critical determinant in establishing the reading frame for translation on an mRNA.

Study of translational control by metabolite-sensing nascent peptides

Our search of genes with poor start codons identified several mRNAs containing noncanonical uORFs initiated by near-cognate start codons that differ from AUG by a single-nucleotide change or by AUG codons in poor context. One candidate was identified in plants in the mRNA encoding GDP-L-galactose phosphorylase (GGP), a control enzyme in the vitamin C biosynthetic pathway. Using reporter assays in mammalian cells and, in vitro, using rabbit reticulocyte lysates, we revealed that a uORF–like element in the GGP mRNA mediates translational control by vitamin C. We propose that interaction of vitamin C with the GGP uORF nascent peptide in the ribosome exit tunnel causes the ribosome to pause and that queuing of subsequent scanning ribosomes results in increased initiation on the uORF and prevents ribosome access to the GGP ORF. We are currently studying two additional examples of conserved uORFs that control translation in response to specific metabolites, and we hypothesize that similar mechanisms of nascent peptide recognition of the metabolite mediate the translational control.

Characterization of the MEHMO syndrome, an X-linked intellectual disability associated with mutations in translation initiation factor eIF2gamma

The human disease MEHMO syndrome is caused by mutations in the translation initiation factor eIF2gamma. We are characterizing yeast, mammalian cell, and mouse models of the MEHMO syndrome to better understand how the mutations impair eIF2 function and cause disease. In previous studies, we showed that the MEHMO syndrome (named based on the patient phenotypes: mental [intellectual] disability, epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity) is caused by mutations in the EIF2S3 gene, which encodes the gamma subunit of eIF2. Using genetic and biochemical techniques in yeast, we showed that the mutations linked to the MEHMO syndrome impair eIF2 function, disrupt eIF2 complex integrity, and alter the stringency of translation start-codon selection. Over the past year, we have been characterizing additional novel EIF2S3 mutations identified in patients with the MEHMO syndrome. Our studies aim to link genetic and biochemical properties of the broad clinical expressivity of the MEHMO syndrome.

In previous studies, we characterized induced pluripotent stem (iPS) cells derived from a patient with the MEHMO syndrome. Our studies revealed defects in general protein synthesis, constitutive induction of the integrated stress response (ISR), a cellular stress-response pathway that alters protein synthesis to mount an adaptive response, and hyper-induction of the ISR under stress conditions. The EIF2S3 mutation also impaired neuronal differentiation by the iPS cells. We showed that the drug ISRIB, an activator of the eIF2 guanine nucleotide exchange factor, rescued the cell growth, translation, and neuronal differentiation defects associated with the EIF2S3 mutation, offering the possibility of therapeutic intervention for the MEHMO syndrome [Reference 3]. Recently, we generated a mouse model of the MEHMO syndrome, and we are currently characterizing the phenotypes and pathologies of the mouse to gain further insights into this rare disease and to identify potential new targets for therapeutic intervention.

Molecular analysis of the hypusine-containing protein eIF5A and polyamine control of protein synthesis

Translation factor eIF5A is the sole cellular protein containing the unusual amino acid hypusine [N^e-(4-amino-2-hydroxybutyl)lysine]. We previously found that eIF5A promotes translation elongation and translation termination and that these activities are dependent on the hypusine modification. Moreover, using in vivo reporter assays and in vitro translation assays, we showed that eIF5A in yeast, like its bacterial homolog EF-P, is especially critical for the synthesis of proteins containing runs of consecutive proline residues. Using our in vitro reconstituted assay system, we also showed that the structural rigidity of the amino acid proline contributes to its heightened requirement for eIF5A and that eIF5A could functionally substitute for polyamines to stimulate general protein synthesis. Given that we previously found that eIF5A binds in the ribosome E site with the hypusine residue projecting toward the acceptor stem of the P-site tRNA, we propose that eIF5A and its hypusine residue function to reposition the acceptor arm of the P-site tRNA to enhance reactivity towards either an aminoacyl-tRNA, for peptide bond formation, or a release factor, for translation termination.

In ongoing studies, we are focusing on three areas:

1. identification of the function of hypusine;
2. elucidation of the role of eIF5A in controlling cellular polyamine levels;
3. characterization of the fungal polyamine transporter Hol1.

To address the first aim, we are investigating the hypusine modification on eIF5A. The modification is formed in two steps: first, an n-butylamine moiety from spermidine is transferred to a specific Lys side chain on eIF5A, whereupon hydroxylation of the added moiety completes the formation of hypusine. In contrast to the essential deoxyhypusine synthase, which catalyzes the first step in hypusine formation, the LIA1 gene, encoding the hydroxylase, is non-essential in yeast. We identified and are now characterizing mutations in eIF5A that cause synthetic growth defects in cells lacking the hydroxylase. The mutations map to the ribosome-binding face of eIF5A and near to magnesium ions that coordinate eIF5A binding to the ribosome. Our results are consistent with the notion that the hydroxyl modification helps bind and position eIF5A and its hypusine residue to effectively promote the reactivity of the peptidyl-tRNA on the ribosome.

Towards the second aim, we linked eIF5A to the regulation of polyamine metabolism in mammalian cells. The enzyme ornithine decarboxylase (ODC) catalyzes the first step in polyamine synthesis. ODC is regulated by a protein called antizyme, which, in turn, is regulated by another protein called antizyme inhibitor (AZIN1). The synthesis of AZIN1 is inhibited by polyamines. We identified a regulatory uORF in the leader of the AZIN1 mRNA and found that high polyamine levels enhance translation initiation from the near-cognate start site of the uORF. Remarkably, this polyamine induction of uORF translation depends on the sequence of the encoded polypeptide, including a highly conserved Pro-Pro-Trp (PPW) motif, which causes polyamine-dependent pausing of elongating ribosomes. The polyamine-induced translation of the uORF blocks ribosome access to the AZIN1 start codon and thereby inhibits synthesis of AZIN1.

In addition to elucidating the importance of the cis-acting amino acid motif in the uORF, we identified eIF5A as a sensor and effector for polyamine control of uORF translation. Using reconstituted in vitro translation assays, we found that synthesis of a PPW peptide, like translation of polyproline sequences, requires eIF5A. Moreover, the ability of eIF5A to stimulate PPW synthesis was inhibited by polyamines and could be rescued by increasing eIF5A levels. Taken together, our studies showed that eIF5A functions generally in protein synthesis and that modulation of eIF5A function by polyamines can be exploited to regulate specific mRNA translation. In ongoing studies, we have found that polyamine control of eIF5A function underlies the translational control of mRNAs encoding other regulators and enzymes in the polyamine biosynthetic pathway.

Regarding the third aim, we recently identified Hol1 as the high-affinity polyamine transporter in yeast [Reference 4]. Using ribosome profiling, we identified HOL1 in the group of mRNAs whose translation was repressed in high polyamines. The Hol1 protein is a member of the drug-proton antiporter (DHA1) family of transporters, and we showed that HOL1 was required for yeast growth under limiting polyamine conditions and for high-affinity polyamine uptake by yeast. Together with Anirban Banerjee’s lab, we showed that purified Hol1 transports polyamines. We identified a conserved uORF encoding the peptide MLLLPS in the leader of the HOL1 mRNA, and we found that polyamine inhibition of eIF5A impairs translation termination at the Pro-Ser-stop (PS) motif of the uORF to repress Hol1 synthesis under conditions of elevated polyamines. Thus, polyamine transport, like polyamine biosynthesis, is under translational autoregulation by polyamines in yeast, highlighting the extensive control cells impose on polyamine levels. In ongoing studies, we are characterizing HOL1 homologs in the pathogenic yeast Candida albicans, and our preliminary data indicate that polyamines are critical for C. albicans pathogenesis, raising the possibility that combined inhibition of Hol1 and polyamine synthesis might be an effective means to block growth of this pathogenic yeast.

Analysis of the role of eEF2 and its diphthamide modification in translation elongation

Like its bacterial ortholog EF-G, the eukaryotic elongation factor eEF2 promotes translocation of tRNAs and mRNA from the A site to the P site on the ribosome following peptide bond formation. In most eukaryotes and archaea, a conserved histidine residue at the tip of eEF2 is post-translationally modified to diphthamide through the action of seven non-essential proteins. The function of diphthamide and the rationale for its evolutionary conservation are not well understood. The name diphthamide is derived from diphtheria, a disease of the nose and throat caused by the bacterium Corynebacterium diphtheriae. Infections with C. diphtheriae can lead to respiratory distress and even death; however, an effective vaccine is available. The bacterium expresses a toxin that ADP–ribosylates the diphthamide residue, leading to inactivation of eEF2. Several additional bacterial pathogens, including Pseudomonas aeruginosa and Vibrio cholerae, express distinct toxins that also modify the diphthamide residue and inactivate eEF2.

Based on a cryo-electron microscopy structure of eEF2 bound to the yeast 80S ribosome, obtained during our previous collaboration with Venki Ramakrishnan’s lab (Cambridge, UK), we hypothesized that diphthamide has at least two functions: first, to disrupt the decoding interactions of rRNA with the codon-anticodon duplex in the ribosomal A site; and second, to help chaperone the codon-anticodon interaction as the A-site tRNA is translocated to the P site. In recently published studies [Reference 5], we found that diphthamide enhances translational fidelity.

Characterizing Saccharomyces cerevisiae mutants that lack diphthamide or that show synthetic growth defects in the absence of diphthamide, we found that loss of diphthamide increases -1 ribosomal frame-shifting at programmed frame-shifting sites in the HIV and SARS-CoV-2 viruses. In addition, using reporter assays we observed increased rates of frame-shifting at non-programmed sites during normal translation elongation. Ribosome profiling of yeast and mammalian cells lacking diphthamide revealed increased ribosomal drop-off during elongation with fewer ribosomes translating to the end of mRNAs. Interestingly, removal of out-of-frame stop codons restored ribosomal processivity on the ultralong yeast MDN1 (gene encoding an essential ATPase required for ribosome biogenesis) mRNA. Our results reveal that loss of diphthamide impairs the fidelity of translocation during translation elongation, resulting in increased rates of ribosomal frame-shifting throughout elongation and leading to premature termination at out-of-frame stop codons. We propose that diphthamide, despite its non-essential nature in yeast or mammalian cells in culture, has been conserved throughout evolution to maintain the fidelity of translation elongation and block spurious frame-shifting events that would impair the production of native proteins and generate novel frame-shifted proteins that might be deleterious to the cell. Moreover, we propose that the beneficial effects of diphthamide on translational fidelity have ensured its retention during evolution despite its being a target for inactivation by bacterial toxins. Using reconstituted biochemical assays, we also showed that ADP–ribosylation of diphthamide by diphtheria toxin impairs protein synthesis by blocking the productive binding of eEF2 to elongating ribosomes.

Additional Funding

• Intramural Targeted Anti-COVID-19 (ITAC) Award (2021–2023): “Control of Ribosomal Frameshifting on the SARS-CoV-2 mRNA”

Publications

1. Ivanov IP, Saba JA, Fan CM, Wang J, Firth AE, Cao C, Green R, Dever TE. Evolutionarily conserved inhibitory uORFs sensitize Hox mRNA translation to start codon selection stringency. Proc Natl Acad Sci USA 2022 119:e2117226119.
2. Wang J, Shin BS, Alvarado C, Kim JR, Bohlen J, Dever TE, Puglisi JD. Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation. Cell 2022 185:4474–4487.
3. Young-Baird SK, Lourenço MB, Elder MK, Klann E, Liebau S, Dever TE. Suppression of MEHMO syndrome mutation in eIF2 by small molecule ISRIB. Mol Cell 2020 77:875–886.
4. Vindu A, Shin BS, Choi K, Christenson ET, Ivanov IP, Cao C, Banerjee A, Dever TE. Translational autoregulation of the S. cerevisiae high-affinity polyamine transporter Hol1. Mol Cell 2021 81:3904–3918.
5. Shin BS, Ivanov IP, Kim JR, Cao C, Kinzy TG, Dever TE. eEF2 diphthamide modification restrains spurious frameshifting to maintain translational fidelity. Nucleic Acids Res 2023 51:6899–6913.

Collaborators

• John Atkins, PhD, University College Cork, Cork, Ireland
• Anirban Banerjee, PhD, Unit on Structural and Chemical Biology of Membrane Proteins, NICHD, Bethesda, MD
• Harold Burgess, PhD, Section on Behavioral Neurogenetics, NICHD, Bethesda, MD
• An N. Dang Do, MD, PhD, Unit on Cellular Stress in Development and Diseases, NICHD, Bethesda, MD
• Adam Geballe, MD, The Fred Hutchinson Cancer Research Center, Seattle, WA
• Terri Goss Kinzy, PhD, Rutgers University, Piscataway, NJ
• Rachel Green, PhD, The Johns Hopkins University School of Medicine, Baltimore, MD
• Vera Kalscheuer, PhD, Max Planck Institut für Moleculare Genetik, Berlin, Germany
• Michail Lionakis, MD, ScD, Fungal Pathogenesis Section, NIAID, Bethesda, MD
• Karl Pfeifer, PhD, Section on Epigenetics, NICHD, Bethesda, MD
• Joseph Puglisi, PhD, Stanford University, Palo Alto, CA
• Matthew Sachs, PhD, Texas A&M University, College Station, TX
• Naomi Taylor, MD, PhD, Pediatric Oncology Branch, Center for Cancer Research, NCI, Bethesda, MD
• Daniel Wilson, PhD, Institut für Biochemie und Molekularbiologie, Universität Hamburg, Hamburg, Germany
• Sara Young-Baird, PhD, Uniformed Services University of the Health Sciences, Bethesda, MD

Contact

For more information, email thomas.dever@nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/dever.

Top of Page