Mechanism and Regulation of Eukaryotic Protein Synthesis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

We study the mechanism and regulation of protein synthesis in eukaryotic cells. Of special interest are the regulation of protein synthesis by GTP-binding (G) proteins and protein phosphorylation. In addition, we are studying unusual post-translational modifications of the factors that assist the ribosome in synthesizing proteins as well as the mRNA features and translational components that mediate the fidelity of translation start site selection. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is composed of three subunits including the G protein eIF2gamma. During translation initiation, the GTP bound to eIF2gamma is hydrolyzed to GDP, and the factor eIF2B recycles eIF2-GDP to eIF2-GTP. Phosphorylation of eIF2alpha on serine 51, by a family of stress-responsive protein kinases, coverts eIF2 into an inhibitor of eIF2B and triggers the integrated stress response (ISR). Our recent studies on eIF2 have provided insights into the human disease MEHMO syndrome. 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 from multiple countries, we showed that MEHMO syndrome, a human XLID syndrome with additional symptoms including epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity as well as hypopituitarism and hypoglycemia is caused by mutations in the EIF2S3 gene encoding the gamma subunit of eIF2. Over the past year we have been characterizing additional novel EIF2S3 mutations identified in patients with MEHMO syndrome to gain further insights into the molecular defects in eIF2 that cause the varied symptoms of the disease. In previous studies using induced pluripotent stem (iPS) cells derived from a patient with MEHMO syndrome, we uncovered defects in general protein synthesis, constitutive induction of the ISR, and hyper-induction of the ISR under stress conditions. The EIF2S3 mutation also impaired neuronal differentiation by the iPS cells. Interestingly, 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 MEHMO syndrome. Over the past year we successfully generated a mouse model of MEHMO syndrome, and we are now characterizing the behavioral, hormonal, and metabolic phenotypes of the mutant mice to gain further insights into the disease. A second major focus is the translational GTPases eIF5B, required for joining the large ribosomal subunit to the small subunit poised on the mRNA start codon, and eEF2, required for translation elongation. In collaboration with researchers at Stanford, we helped show that eIF5B reorients the initiator Met-tRNAi to enable joining of the large ribosomal subunit. Our studies on eEF2 revealed the role of a novel post-translational modification on the factor. A conserved histidine residue in eEF2 is post-translationally modified to diphthamide by a set of evolutionarily conserved enzymes. Interestingly, the disease diphtheria is caused by a bacterial toxin that ADP-ribosylates the diphthamide residue to inactivate eEF2. Characterizing yeast and mammalian cells lacking diphthamide, we found that the modification helps maintain translational fidelity. We observed increased rates of 1 ribosomal frameshifting in cells lacking diphthamide, including on the programmed 1 frameshifting site in the SARS-CoV-2 viral mRNA. Ribosome profiling of yeast lacking diphthamide revealed increased ribosome drop-off, and we showed that this drop-off was due to ribosomes shifting reading frames and terminating at out-of-frame stop codons. We propose that diphthamide has been conserved through evolution to maintain translational fidelity despite conferring sensitivity to bacterial toxins. A third major research focus involves the translation factor eIF5A and polyamines. The eIF5A is the sole cellular protein containing the unusual amino acid hypusine. We previously showed that eIF5A promotes translation elongation and termination, that this activity is dependent on the hypusine modification, and that certain amino acid motifs like polyproline sequences show a heightened requirement for eIF5A. Based, in part, on our studies with x-ray crystallographers in France, we propose that eIF5A and its hypusine residue function to reposition the acceptor arm of the P site tRNA on the ribosome to enhance reactivity towards either an aminoacyl-tRNA, for peptide bond formation, or a release factor, for translation termination. Interestingly, the hypusine modification on eIF5A is derived the polyamine spermidine, and we have discovered several connections between eIF5A and polyamines. In ongoing studies, we have identified eIF5A as a sensor and effector for polyamine control of translation of mRNAs encoding enzymes or regulators of polyamine biosynthesis. The enzyme ornithine decarboxylase (ODC) catalyzes the first step in polyamine synthesis. ODC is inhibited by the protein antizyme (OAZ), which, in turn, is regulated by the protein antizyme inhibitor (AZIN). The synthesis of OAZ is stimulated by polyamines while AZIN synthesis is inhibited. Our studies indicate that polyamines interfere with eIF5A function on the ribosome, causing ribosomes to pause on a uORF in the AZIN mRNA and thereby represses AZIN synthesis. Recently, we also linked polyamine inhibition of eIF5A to stimulation of ribosomal frameshifting on the OAZ mRNA and to uORF-mediated inhibition of S-adenosylmethionine decarboxylase (AMD1) synthesis. We also showed that polyamine inhibition of eIF5A controls translation of the yeast HOL1 mRNA. Together with Anirban Banerjee in NICHD, we showed that Hol1 is the high-affinity polyamine transporter in fungi, and thus our studies identify eIF5A as a general sensor and effector for autoregulation of polyamine uptake and biosynthesis. In recent studies in collaboration with Michail Lionakis in NIAID, we are characterizing HOL1 orthologs in the pathogenic yeast Candida albicans. We have found that combined inhibition of polyamine synthesis and transport prevents hyphal growth and suppresses C albicans virulence in a mouse infection model. Finally, we are also studying translation start site selection. The small ribosomal subunit with bound Met-tRNAi associates with an mRNA at the 5 cap and then scans down the mRNA in search of a start (typically AUG) codon. Our collaborators at Stanford showed that scanning occurs rapidly and that the ribosome can backtrack when encountering impediments; working together, we showed that precisely positioned secondary structures can enhance initiation at upstream weak start sites like the near-cognate start codons CUG or UUG. Selection of the translation start sites is also influenced by context nucleotides flanking the AUG codon and by levels of the factors eIF1 and eIF5. In a search of mammalian genes, we identified several homeobox (Hox) gene paralogs in which the main ORF or a regulatory upstream open reading frame (uORF) is initiated by an AUG codon in conserved suboptimal context. Working with collaborators at Johns Hopkins University, we found that the conserved uORFs inhibit Hox reporter expression in a manner controlled by eIF1 or eIF5. We also showed that reducing ribosome levels or inhibiting general translation lowers the fidelity of start codon selection. Our findings reveal that alteration in start codon selection stringency has the potential to regulate global gene expression programs, including Hox gene-directed body plan formation in animals.