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. 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. 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 in Israel, Germany, Slovakia, the United Kingdom and at Walter Reed National Military Medical Center, 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 begun characterizing a novel EIF2S3 mutation identified in a patient with MEHMO syndrome. We generated a yeast model of the EIF2S3 mutation, and we are examining the impacts of the mutation on yeast growth and translation. This work complements our previous studies characterizing other EIF2S3 mutations linked to MEHMO syndrome and will further our understanding of the molecular defects in eIF2 that cause the varied symptoms of the disease. We have also studied induced pluripotent stem (iPS) cells derived from a patient with MEHMO syndrome. Our studies revealed defects in general protein synthesis, constitutive induction of the integrated stress response (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. A second major focus is the translation factors eIF5B, a GTPase required for the last step of translation initiation joining of the large ribosomal subunit to small subunit poised on the start codon of an mRNA and eEF2, a GTPase that promotes translation elongation. The eIF5B is binds to the 40S subunit and appears to stabilize binding of the initiator Met-tRNAi to the complex. Working with collaborators in California, New York, and Spain, we helped show that eIF5B plays an important role in translation start site selection ensuring high fidelity in this process which establishes the reading frame for translation on an mRNA (1). Studies on the diphthamide modification of eEF2 revealed a critical role in reading frame maintenance including on the programmed frameshift site in SARS-CoV-2. A third major focus is the translation factor eIF5A and polyamines. The eIF5A is the sole cellular protein containing the unusual amino acid hypusine. Using molecular genetic and biochemical studies, we previously showed that eIF5A promotes translation elongation, and that this activity is dependent on the hypusine modification. Moreover, certain amino acid motifs like runs of consecutive proline residues showed a heightened dependency on eIF5A for their translation both in cells and in vitro. In collaboration with researchers at Johns Hopkins University, we reported that eIF5A functions globally to promote both translation elongation and termination. Working with x-ray crystallographers in France, we found that eIF5A occupies the E site of the ribosome with the hypusine residue projecting toward the acceptor stem of the P-site tRNA. Our studies support a model in which 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. 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 are investigating the hypusine modification on eIF5A. Hypusine is formed in two steps: first, transfer of an n-butyl amine moiety from spermidine to a specific Lys side chain on eIF5A, and then second, hydroxylation of the modified residue. Whereas the LIA1 gene encoding the hydroxylase is non-essential in yeast, we identified mutations in eIF5A that caused synthetic phenotypes in the absence of hydroxylation. These mutations alter residues near the magnesium binding sites on eIF5A and thus may impact eIF5A binding to the ribosome. We propose that the hydroxyl modification helps to bind and position eIF5A and hypusine to effectively promote the reactivity of the peptidyl-tRNA. Recently, 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 (OAZ), which, in turn, is regulated by another protein called antizyme inhibitor (AZIN). The synthesis of OAZ is stimulated by polyamines while AZIN synthesis is inhibited. The regulation of AZIN synthesis is dependent on a conserved upstream open reading frame (uORF) in the leader of its mRNA. We showed that polyamines cause ribosomes to pause on a proline-rich motif while translating the uORF. The paused ribosomes trigger queuing of subsequent scanning ribosomes to enhance translation of the uORF and repress AZIN synthesis. We also identified the translation factor eIF5A as a sensor and effector for polyamine control of AZIN synthesis. High polyamine levels inhibited eIF5A stimulation of polyproline synthesis. We propose that polyamines interfere with eIF5A binding on the ribosome triggering the ribosome pause that governs translation of the inhibitory uORF on the AZIN mRNA and thereby represses AZIN synthesis. In ongoing studies, we have also linked polyamine inhibition of eIF5A to translational control of OAZ synthesis, suggesting that eIF5A might be a general sensor for autoregulation of polyamine biosynthesis. In recent studies examining translational control by polyamines, we identified the yeast high-affinity polyamine transporter (2). Using ribosome profiling, we identified mRNAs whose translation was sensitive to changes in polyamine levels. One of the mRNAs encoded a member of the drug-proton antiporter (DHA1) family of transporters called Hol1. We showed that HOL1 was required for yeast growth under limiting polyamine conditions and for high-affinity polyamine uptake by yeast. Together with Anirban Banerjees lab in the NICHD, we showed that purified Hol1 transports polyamines. The leader of the HOL1 mRNA contains a highly conserved upstream open reading frame (uORF) encoding the peptide MLLLPS. We found that polyamine inhibition of the translation factor eIF5A impairs translation termination at the Pro-Ser-stop motif of the uORF to repress Hol1 synthesis under conditions of elevated polyamines. Our findings reveal that polyamine transport, like polyamine biosynthesis, is under translational autoregulation by polyamines in yeast, highlighting the extensive control cells impose on polyamine levels.