Our studies of protein synthesis are focused on three main areas:

Translation GTPases

The GTPases eIF2 and eIF5B promote, respectively, the first and final steps in translation initiation. eIF2 forms a ternary complex with GTP and Met-tRNAiMet and binds Met-tRNAiMet to the small ribosomal subunit in the first step of translation initiation. After the small ribosomal subunit complex binds to an mRNA and via scanning selects the translation start site, eIF5B promotes joining of the large ribosomal subunit. Following GTP hydrolysis and release of eIF5B, the ribosome is poised to elongate and synthesize the protein encoded by the mRNA. The GTPase eEF2 functions in the elongation phase of protein synthesis to promote translocation of the tRNAs and mRNA on the ribosome following each round of peptide bond formation.

We are using molecular genetic, biochemical, and structural approaches to investigate the functions of these factors. In addition, we are studying how variants of eIF2gamma, encoded by the EIF2S3 gene in humans, cause MEHMO syndrome, a rare X-linked disease. Originally identified in patients with X-linked intellectual disability (XLID), patients with MEHMO syndrome can exhibit a range of phenotypes including epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity. We are characterizing yeast, mammalian cell, and mouse models of the MEHMO syndrome to better understand how the mutations impair eIF2 function and cause disease. Moreover, we seek to link the genetic and biochemical impacts of the mutations with the broad clinical phenotypes of MEHMO syndrome and with induction of the cellular Integrated Stress Response (ISR).

eIF2a Phosphorylation

Phosphorylation of eIF2 is a key mechanism of translational control and the central step of the ISR. Upon phosphorylation, eIF2 inhibits its guanine nucleotide exchange factor eIF2B resulting in inhibition of general translation and enhanced translation of specific mRNAs like GCN4 in yeast and ATF4 in mammals that contain novel regulatory upstream open reading frames (uORFs). Whereas yeast contain a single eIF2 kinase, GCN2, humans express four eIF2 kinases that respond to different cellular stress conditions. We previously studied the double-stranded RNA-activated human PKR kinase, an important component in the interferon-mediated antiviral response. Through our studies of PKR and select viral antagonists, we elucidated the mechanism of PKR activation and substrate recognition. In ongoing studies, we are further exploring the mechanisms by which phosphorylation of eIF2 enhances translation of additional uORF-containing mRNAs encoding key stress responsive factors.

eIF5A and Polyamines

Translation factor eIF5A is the sole cellular protein containing the unusual amino acid hypusine [e-(4-amino-2-hydroxybutyl)lysine]. We previously found that eIF5A promotes translation elongation and translation termination and that these activities are dependent on the hypusine modification. Moreover, we showed that eIF5A is especially critical for the synthesis of proteins containing runs of consecutive proline residues. Having previously found that eIF5A binds 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 tRNA to enhance reactivity for peptide bond formation or translation termination.

The hypusine modification on eIF5A is formed in two steps: first, the enzyme deoxyhypusine synthase (DHPS) transfers an n-butylamine moiety from spermidine to a specific Lys side chain on eIF5A to form deoxyhypusine; second, the enzyme deoxyhypusine hydroxylase (DOHH) hydroxylates the added moiety to form hypusine. Recently, biallelic variants in DOHH were found to cause a neurodevelopmental disorder in which patients exhibit global developmental delay, intellectual disability, facial dysmorphism, and microcephaly. To test the pathogenicity of newly identified patient DOHH variants, we developed a yeast-based assay, exploiting the eIF5A mutants that exhibit a growth defect in cells lacking hypusine hydroxylation.

In ongoing studies, we identified eIF5A as a target for homeostatic control of cellular polyamine levels in mammalian cells. The enzyme ornithine decarboxylase (ODC) catalyzes the first step in polyamine synthesis. ODC is regulated by a protein called antizyme (OAZ1), which, in turn, is regulated by another protein, called antizyme inhibitor (AZIN1). The synthesis of AZIN1 is inhibited by polyamines. We also determined that Hol1 is the high-affinity polyamine transporter in yeast and that its synthesis is inhibited by polyamines. We previously showed that polyamine inhibition of eIF5A triggers increased translation of an inhibitory uORF in the AZIN1 mRNA leader and acts through a conserved uORF in the HOL1 mRNA to inhibit the synthesis of AZIN1 and Hol1, respectively. In ongoing studies, we found that polyamines target eIF5A to promote the ribosomal frameshifting needed to synthesize OAZ1 and to enable uORF–mediated inhibition of S-adenosylmethionine decarboxylase (AMD1) synthesis. We propose that eIF5A is a target for homeostatic regulation of cellular polyamines.

Translational Control by Start-site Stringency and uORFs

Selection of the translation start codon is crucial for synthesis of the proper protein encoded by each mRNA. If the ribosome translates the incorrect reading frame there will be reduced levels of the proper protein and synthesis of potentially toxic proteins encoded in the alternate reading frames. Following its binding to the 5′ end of an mRNA, the small ribosomal subunit with bound Met-tRNAi scans down the mRNA in search of a start codon (typically AUG). In previous work we showed that precisely positioned secondary structures can enhance initiation at upstream weak start sites such as the near-cognate start codons CUG or UUG. The context nucleotides flanking the start codon and the translation factors eIF1 and eIF5 also influence the efficiency of initiation at a particular start codon. We previously identified several Hox genes encoding developmental regulators of animal body plans with poor context uORFs or main ORFs, and we showed that their translation is sensitive to changes in stringency. We are studying additional examples of stringency-controlled mRNAs and identifying conditions where stringency is changed.

Our search of genes with poor start codons identified several mRNAs containing noncanonical uORFs initiated by near-cognate start codons that differ from AUG by a single nucleotide change and encode evolutionarily conserved peptides. We are examining the mechanism of translational control conferred by these conserved uORFs.