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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, viral regulators of these kinases, and how cellular phosphatases are targeted to dephosphorylate eIF2alpha. We are characterizing eIF2gamma mutations that are associated with 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 of this factor in polyamine-regulated gene-specific translational control mechanisms.

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

In collaboration with several other researchers including Lina Basel-Vanagaite, Guntram Borck, Vera Kalscheuer, Daniela Gasperikova, and Clesson Turner, we found that the human MEHMO syndrome, an X-linked intellectual disability (XLID) syndrome, is caused by mutations in the EIF2S3 gene, which encodes the translation factor eIF2gamma. MEHMO syndrome is named based on the constellation of patient phenotypes: intellectual (mental) disability, epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity. Genetic and biochemical studies of a yeast model of the first-characterized EIF2S3 mutation linked to MEHMO syndrome revealed that the mutation disrupts eIF2 complex integrity and translation start-codon selection. Our studies on yeast models of additional MEHMO syndrome mutations in eIF2gamma likewise revealed impaired eIF2 function, altered translational control of specific mRNAs, and reduced stringency of translation start-site selection [Reference 1]. Consistent with these properties, the Integrated Stress Response, a translational regulatory response typically associated with eIF2alpha phosphorylation, is induced in patient cells. The findings directly link intellectual disability with impaired translation initiation and provide a mechanistic basis for MEHMO syndrome resulting from partial loss of eIF2 function [Reference 1]. 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 of additional MEHMO syndrome mutations in eIF2gamma reveal protein synthesis defects associated with altered binding of the initiator methionyl-tRNA to eIF2.

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 [N^e-(4-amino-2-hydroxybutyl)lysine], promotes translation elongation, an activity that depends on the hypusine modification. Using in vivo reporter assays, we showed that eIF5A in yeast, like its bacterial homolog EF-P, is critical for the synthesis of proteins containing runs of three or more consecutive proline residues. Consistent with our 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. Thus, we proposed that eIF5A, like its bacterial ortholog EF-P, stimulates the peptidyl-transferase activity of the ribosome and facilitates the reactivity of poor substrates such as proline.

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 2]. 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 [Reference 3].

The eIF5A occupies the E site of the ribosome, with the hypusine residue projecting toward the acceptor stem of the P-site tRNA. Our studies suggest a function for eIF5A and its hypusine residue in repositioning the peptidyl–tRNA [Reference 3]. In related studies, we reported the structure of a diproline–tRNA analog bound to the ribosome, revealing that proline affects nascent peptide positioning in the ribosome exit tunnel. Taken together, our studies support a model in which eIF5A and its hypusine residue function to reposition the acceptor arm of P-site tRNA to promote peptide bond formation and that the body of eIF5A functions like polyamines to enhance general protein synthesis.

Over the last year, 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 this regulation is dependent on a conserved element in the 5′ leader of the AZIN1 mRNA, which we refer to as a uCC (for upstream Conserved Coding region.) 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. Surprisingly, the regulation is dependent on the sequence of encoded polypeptide. Ribosome profiling revealed polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in the uCC. 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. In contrast, substituting an alternate elongation pause sequence restored uCC translation. We proposed that most scanning ribosomes bypass the near-cognate start codon of the uCC without initiating and then translate AZIN1. However, a ribosome will occasionally 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 the scanning ribosomes that bypass the near-cognate start codon. The resultant queuing 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 the notion that ribosome queuing is important for uCC translation, impairing ribosome loading reduced uCC translation and derepressed AZIN1 synthesis. We believe that the mechanism whereby 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.

In further studies on the AZIN1–regulatory mechanism, we identified eIF5A as a sensor and effector for polyamine control of uCC translation. Using our reconstituted in vitro translation assay system, 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. Thus, we propose that polyamine 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].

Publications

1. Skopkova M, Hennig F, Shin BS, Turner CE, Stanikova D, Brennerova K, Stanik J, Fischer U, Henden L, Müller U, Steinberger D, Leshinsky-Silver E, Bottani A, Kurdiova T, Ukropec J, Nyitrayova O, Kolnikova M, Klimes I, Borck G, Bahlo M, Haas SA, Kim JR, Lotspeich-Cole LE, Gasperikova D, Dever TE, Kalscheuer VM. EIF2S3 mutations associated with severe X-linked intellectual disability syndrome MEHMO. Hum Mutat 2017 38:409-425.
2. 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.
3. Melnikov S, Mailliot J, Shin BS, Rigger L, Yusupova G, Micura R, Dever TE, Yusupov M. Crystal structure of hypusine-containing translation factor eIF5A bound to a rotated eukaryotic ribosome. J Mol Biol 2016 428:3570-3576.
4. 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.
5. 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
• 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
• Frank Sicheri, PhD, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada
• Hiroaki Suga, PhD, University of Tokyo, Tokyo, Japan
• Clesson Turner, MD, Walter Reed National Military Medical Center, Bethesda, MD
• Marat Yusupov, PhD, L'Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Strasbourg, France

Contact

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

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