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, 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 this 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, with collaborators in Israel, Germany, Slovakia, and at the US Walter Reed National Military Medical Center, we showed that the MEHMO syndrome (named based on the constellation of 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. Our prior studies, using genetic and biochemical techniques in yeast models of human MEHMO syndrome mutations revealed that the mutations disrupt eIF2 complex integrity and translation start-codon selection. Over the past year, we generated yeast models of two additional EIF2S3 mutations linked to MEHMO syndrome. One of the mutations impaired methionyl-tRNA binding to eIF2 [Reference 1], and both mutations impaired eIF2 function, altered translational control of specific mRNAs, and reduced the stringency of translation start-site selection. Our collaborators in London linked a novel EIF2S3 mutation with hypopituitarism and glucose dysregulation, potentially expanding the clinical symptoms of MEHMO syndrome [Reference 2]. More recently, we studied induced pluripotent stem (iPS) cells derived from a patient with MEHMO syndrome. We observed a general reduction in protein synthesis, constitutive induction of the integrated stress response, 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 in the cells. Moreover, upon differentiation into neurons, the mutant cells exhibited reduced dendritic arborization. Based on our studies, we propose that the mutations in eIF2gamma impair the efficiency and fidelity of protein synthesis, and that this altered control of protein synthesis underlies the MEHMO syndrome. 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 more severe EIF2S3 mutations cause the full MEHMO phenotype, while less deleterious mutations cause a milder form of the syndrome with only a subset of symptoms. Ongoing studies are examining additional MEHMO syndrome mutations in eIF2gamma, using both yeast and mammalian cell systems.
Molecular analysis of the hypusine-containing protein eIF5A and polyamine control of protein synthesis
In a series of molecular-genetic and biochemical studies we found that the translation factor eIF5A, the sole protein containing the unusual amino acid hypusine [Ne-(4-amino-2-hydroxybutyl)lysine], promotes translation elongation and that this activity depends on the hypusine modification. 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. Consistent with these in vivo findings, we showed that eIF5A was necessary for the synthesis of polyproline peptides in reconstituted yeast in vitro translation assays, and, using directed hydroxyl-radical probing experiments, we mapped eIF5A binding to near the E site of the ribosome. In collaboration with Rachel Green, we reported that eIF5A functions globally to promote both translation elongation and termination. Moreover, utilizing 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 3]. Working with the X-ray crystallographer Marat Yusupov, we obtained a 3.25 Å–resolution crystal structure of eIF5A bound to the yeast 80S ribosome. With the hypusine residue projecting toward the acceptor stem of the P-site tRNA, eIF5A occupies the E site of the ribosome. Our studies support a model in which 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.
Over the past year, we further investigated 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 formation of hypusine. Whereas deoxyhypusine synthase, which catalyzes the first step in hypusine formation, is essential in yeast, the LIA1 gene encoding the hydroxylase is non-essential. We identified mutations in eIF5A that cause synthetic growth defects in cells lacking the hydroxylase. 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 4]. 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 such regulation is dependent on an element in the leader of the AZIN1 mRNA. The element resembles an upstream open reading frame (uORF); however, we refer to it as an upstream Conserved Coding (uCC) region, because it 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, such regulation is dependent 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 polyamines, these 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 resultant 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 4]. 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 recent studies, we searched for additional mRNAs containing potential uCCs. Reporter assays in mammalian cells and in vitro revealed that a uORF–like element in the mRNA encoding plant GDP-L-galactose phosphorylase (GGP), a control enzyme in the vitamin C biosynthetic pathway, 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 believe that the mechanism of a paused elongating ribosome promoting 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 posttranslationally modified to diphthamide through the action of seven nonessential proteins. The function of diphthamide and the rationale for its evolutionary conservation are not well understood, and to date the only clear function of diphthamide is to serve as a substrate for inactivation by diphtheria toxin. To gain insights into the role of eEF2 and diphthamide, we screened for mutations that sensitize eEF2 to loss of the diphthamide modification and we are currently characterizing the mutants. We also examined peptide synthesis in a reconstituted yeast in vitro translation system using unmodified eEF2 or eEF2 containing the diphthamide modification. Using the canonical initiation pathway to direct the synthesis of the peptide Met-Phe-Lys revealed no distinction between unmodified eEF2 and eEF2 with the diphthamide modification. In contrast, 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 pseudotranslocation reactions prior to peptide bond formation, was sensitive to the loss of diphthamide. We propose that the precise phasing of pseudotranslocation, in which a tRNA–mimicking RNA element from the virus is translocated through the ribosome, is dependent on the diphthamide modification on eEF2.
Further insights into the function of diphthamide were obtained by our collaborators in Venki Ramakrishnan’s lab. Using electron cryomicroscopy, they revealed that eEF2 interacts with the CrPV–IRES on the ribosome and stabilizes the IRES in a conformation reminiscent of a hybrid tRNA state. Interestingly, diphthamide appeared to interact directly with the tRNA–mimicking element of the CrPV IRES, perhaps to facilitate its precise translocation in the ribosome and to break decoding interactions between conserved rRNA bases and the IRES. Our studies provide the evidence that diphthamide plays a role in protein synthesis, and we propose that diphthamide has at least two functions: first, to disrupt the decoding interactions of rRNA with the codon-anticodon duplex in the A site; and second, to help chaperone the codon-anticodon interaction as the A-site tRNA is translocated to the P site [Reference 5]. In ongoing studies, we are further exploring the role of diphthamide in promoting the accuracy and efficiency of translation elongation.
Publications
- 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.
- Gregory LC, Ferreira CB, Young-Baird SK, Williams HJ, Harakalova M, van Haaften G, Rahman SA, Gaston-Massuet C, Kelberman D, GOSgene, Qasim W, Camper SA, Dever TE, Shah P, Robinson ICAF, Dattani MT. Impaired EIF2S3 function associated with a novel phenotype of X-linked hypopituitarism with glucose dysregulation. EBioMedicine 2019;42:470-480.
- 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.
- 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.
- Murray J, Savva CG, Shin BS, Dever TE, Ramakrishnan V, Fernández IS. Structural characterization of ribosome recruitment and translocation by type IV IRES. eLife 2016;5:e13567.
Collaborators
- John Atkins, PhD, University College Cork, Cork, Ireland
- Lina Basel-Vanagaite, MD, Tel Aviv University, Tel Aviv, Israel
- Guntram Borck, MD, PhD, Universität Ulm, Ulm, Germany
- Mehul Dattani, MD, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
- Daniela Gasperikova, PhD, Slovak Academy of Sciences, Bratislava, Slovakia
- Rachel Green, PhD, The Johns Hopkins University School of Medicine, Baltimore, MD
- Vera Kalscheuer, PhD, Max Planck Institute for Molecular Genetics, Berlin, Germany
- Venkatraman Ramakrishnan, PhD, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
- Hiroaki Suga, PhD, University of Tokyo, Tokyo, Japan
- Clesson Turner, MD, Walter Reed National Military Medical Center, Bethesda, MD
- Marat Yusupov, PhD, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France
Contact
For more information, email thomas.dever@nih.gov or visit http://deverlab.nichd.nih.gov.
