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Mechanism and Regulation of Eukaryotic Protein Synthesis

• Thomas E. Dever, PhD, Head, Section on Protein Biosynthesis
• Byung-Sik Shin, PhD, Staff Scientist
• Ivaylo P. Ivanov, PhD, Research Fellow
• Arya Vindu, PhD, Visiting Fellow
• Sara Young-Baird, PhD, Postdoctoral Research Associate Program (PRAT) Fellow
• Chune Cao, Biological Laboratory Technician
• Joo-Ran Kim, BS, Special Volunteer
• Leda Lotspeich-Cole, BS, Graduate Student

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, 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 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 novel 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 upstream Conserved Coding (uCC) regions in select mRNAs.

Analysis of eIF2gamma mutations that link intellectual disability with impaired translation initiation

Protein synthesis plays a critical role in learning and memory in model systems, and our studies have linked a human X-linked intellectual disability (XLID) syndrome to altered function of eIF2. 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 models of human MEHMO–syndrome mutations, we previously characterized several mutations that impair eIF2 function, disrupt eIF2 complex integrity, and alter the stringency of translation start-codon selection. Our studies on the EIF2S3–I259M mutation, identified in patients with a severe form of the disease, revealed that the mutation impaired binding of the initiator methionyl-tRNA to eIF2 [Reference 1]. Over the past year, we generated yeast models of two additional EIF2S3 mutations linked to the MEHMO syndrome [Reference 2]. These new mutations, which lie in the G domain of eIF2gamma, impaired yeast cell growth, altered translation and reduced stringency of translation start-site selection. Our collaborators in Germany linked the EIF2S3 mutations with variable levels of motor delay, microcephaly, ID, epilepsy, central obesity and diabetes, thus broadening the genetic spectrum and clinical expressivity of MEHMO syndrome.

In additional studies from the last year, we characterized induced pluripotent stem (iPS) cells derived from a patient with the MEHMO syndrome [Reference 3]. In addition to a general reduction in protein synthesis, the cells constitutively induced the integrated stress response, with elevated expression of a translational regulatory response typically associated with eIF2alpha phosphorylation, and heightened expression of the transcriptional activators ATF4 and CHOP and the protein phosphatase-regulatory subunit GADD34. Under stress conditions, hyperactivation of the integrated stress response in the mutant iPS cells triggered apoptosis.

In addition, upon differentiation into neurons, the mutant cells exhibited reduced dendritic arborization. Our studies linking altered protein synthesis with intellectual disability are consistent with the critical role of protein synthesis in learning and memory in model systems. Based on our studies, we propose that the mutations in eIF2gamma impair the efficiency and fidelity of protein synthesis, and that such altered control of protein synthesis underlies the MEHMO syndrome. Addition of 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].

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

The translation factor eIF5A is the sole cellular protein containing the unusual amino acid hypusine [N^e-(4-amino-2-hydroxybutyl)lysine]. In previous studies, we showed that eIF5A promotes translation elongation and that such activity depends on its hypusine modification. Moreover, using in vivo reporter assays, we showed that eIF5A in yeast, like its bacterial homolog EF-P, is especially critical for the synthesis of proteins containing runs of three or more consecutive proline residues. Moreover, using reconstituted in vitro translation assays, we showed that eIF5A was necessary for the synthesis of peptides containing polyproline sequences. In collaboration with Rachel Green, we reported that eIF5A functions globally to promote both translation elongation and termination. Moreover, using our in vitro reconstituted assay system, we 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 [Reference 4]. The function of eIF5A to promote peptide bond formation is consistent with our structural studies with the X-ray crystallographer Marat Yusupov. The eIF5A was bound 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 to enhance reactivity towards either an aminoacyl-tRNA, for peptide bond formation, or a release factor, for translation termination.

In ongoing experiments, we are further 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 on 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 mutations in eIF5A that cause synthetic growth defects in cells lacking the hydroxylase. Interestingly, the mutations map to the ribosome-binding face of eIF5A. 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.

Recently, we linked eIF5A to the regulation of polyamine metabolism in mammalian cells [Reference 5]. 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, and an element in the leader of the AZIN1 mRNA is critical for the regulation. The element resembles an upstream open reading frame (uORF); however, we refer to it as an upstream Conserved Coding (uCC) region, because the element lacks an AUG start codon and initiates at a near-cognate codon instead. Whereas translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate start codons, we found that high polyamine levels enhance translation initiation from the near-cognate start site of the uCC. Remarkably, the polyamine induction of uCC translation depends on the sequence of encoded polypeptide, including a highly conserved Pro-Pro-Trp (PPW) motif. Ribosome profiling revealed polyamine-dependent pausing of elongating ribosomes on the PPW motif in the uCC, and mutation of the PPW motif impaired initiation at the near-cognate AUU start codon of the uCC and abolished polyamine control, leading to constitutive high-level expression of AZIN1. We proposed that scanning ribosomes typically bypass the near-cognate start codon of the uCC without initiating and then translate AZIN1. However, occasionally a ribosome will initiate translation at the uCC start codon. Under conditions of high polyamine levels, the elongating ribosomes pause on the PPW motif. The paused ribosome serves as a roadblock to subsequent scanning ribosomes that bypass the near-cognate start codon. The resulting queue of scanning ribosomes behind the paused elongating ribosome positions a ribosome near the start site of the uCC, providing greater opportunity for initiation at the weak start site. Consistent with this queuing model, we found that impairing ribosome loading, and thus queue formation, reduced uCC translation and derepressed AZIN1 synthesis.

In further studies on the AZIN1 regulatory mechanism, we identified eIF5A as a sensor and effector for polyamine control of uCC 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. We propose that polyamines interfere with eIF5A binding on the ribosome and that inhibition of eIF5A serves as the trigger to cause the ribosome pause that governs uCC translation. 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 [Reference 5]. We are now exploring the possibility that polyamine regulation of eIF5A underlies translational control of mRNAs encoding other enzymes and regulators of polyamine biosynthesis.

Translational control by metabolite-sensing nascent peptides

In ongoing studies, we searched for additional mRNAs containing potential uCCs. One such 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 is a uCC. We propose that interaction of vitamin C with the GGP uCC 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 uCC and prevents ribosome access to the GGP ORF. We hypothesize that the mechanism by which a paused elongating ribosome promotes initiation at an upstream weak start site via ribosome queuing may underlie the control of translation of other mRNAs, especially those whose translation is derepressed by conditions that impair ribosome loading.

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

We are also studying the translation elongation factor eEF2. Like its bacterial ortholog EF-G, eEF2 promotes translocation of tRNAs and mRNA from the A site to the P site on the ribosome, following peptide bond formation. In all 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. Of note, several additional bacterial pathogens, including Pseudomonas aeruginosa and Vibrio cholerae, express distinct toxins that likewise modify the diphthamide residue and inactivate eEF2.

To gain insights into the role of eEF2 and diphthamide, we screened for mutations that sensitize eEF2 to loss of the diphthamide modification and are currently characterizing the mutants. In previous studies, we showed that loss of diphthamide does not affect in vitro peptide synthesis directed by the canonical initiation pathway; however, synthesis of the same peptide directed by the novel cricket paralysis virus (CrPV) internal ribosome entry site (IRES), which bypasses canonical translation initiation and instead requires novel eEF2–directed pseudo-translocation reactions prior to peptide bond formation, is sensitive to the loss of diphthamide. Based on a cryo-electron microscopy structure of eEF2 and the CrPV–IRES bound to the yeast 80S ribosome obtained by our collaborators in Venki Ramakrishnan’s lab, we hypothesize 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 ongoing studies, we are further exploring the role of diphthamide in promoting the accuracy and efficiency of translation elongation. Our preliminary data indicate that loss of diphthamide impairs the fidelity of translation leading to increased levels of ribosome frameshifting. Accordingly, we propose that diphthamide, despite its non-essential nature in yeast, has been conserved throughout evolution to maintain the fidelity of translation elongation and block spurious frameshifting events that would limit the production of the proteins.

Publications

1. Young-Baird SK, Shin BS, Dever TE. MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2. Nucleic Acid Res 2019;47:855-867.
2. Kotzaeridou U, Young-Baird SK, Suckow V, Thornburg AG, Wagner M, Harting I, Christ S, Strom T, Dever TE, Kalscheuer VM. Novel pathogenic EIF2S3 missense variants causing clinically variable MEHMO syndrome with impaired eIF2gamma translational function, and literature review. Clin Genet 2020;98:507-514.
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. Shin BS, Katoh T, Gutierrez E, Kim JR, Suga H, Dever TE. Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis. Nucleic Acids Res 2017;45:8392-8402.
5. Ivanov IP, Shin BS, Loughran G, Tzani I, Young-Baird SK, Atkins JF, Dever TE. Polyamine control of translation elongation regulates start site selection on antizyme inhibitor mRNA via ribosome queuing. Mol Cell 2018;70:254-265.

Collaborators

• John Atkins, PhD, University College Cork, Cork, Ireland
• Israel Fernandez, PhD, Columbia University, New York, NY
• Adam Geballe, MD, The Fred Hutchinson Cancer Research Center, Seattle, WA
• Rachel Green, PhD, The Johns Hopkins University School of Medicine, Baltimore, MD
• Vera Kalscheuer, PhD, Max Planck Institute for Molecular Genetics, Berlin, Germany
• Terri Goss Kinzy, PhD, Western Michigan University, Kalamazoo, MI
• Eric Klann, PhD, New York University, New York, NY
• Stefan Leibau, MD, Eberhard Karls Universität Tübingen, Tübingen, Germany
• Joseph Puglisi, PhD, Stanford University, Palo Alto, CA
• Venkatraman Ramakrishnan, PhD, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
• Matthew Sachs, PhD, Texas A&M University, College Station, TX
• Hiroaki Suga, PhD, University of Tokyo, Tokyo, Japan
• Marat Yusupov, PhD, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg, Strasbourg, France

Contact

For more information, email thomas.dever@nih.gov or visit http://deverlab.nichd.nih.gov.

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