Publications by authors named "Thomas E Dever"

Translation initiation defines the identity of a synthesized protein through selection of a translation start site on a messenger RNA. This process is essential to well-controlled protein synthesis, modulated by stress responses, and dysregulated in many human diseases. The eukaryotic initiation factors eIF1 and eIF5 interact with the initiator methionyl-tRNA on the 40S ribosomal subunit to coordinate start site selection.

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In addition to the main, protein-coding, open reading frame (mORF), many eukaryotic mRNAs contain upstream ORFs (uORFs) initiated at AUG or near-cognate codons residing 5' of the mORF start site. Whereas translation of uORFs generally represses translation of the mORFs, a subset of uORFs serves as a nexus for regulating translation of the mORF. In this review, we summarize the mechanisms by which uORFs can repress or stimulate mRNA translation, highlight uORF-mediated translational repression involving ribosome queuing, and critically evaluate recently described alternatives to the delayed reinitiation model for uORF-mediated regulation of the / mRNAs.

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Diphthamide (DPH), a conserved amino acid modification on eukaryotic translation elongation factor eEF2, is synthesized via a complex, multi-enzyme pathway. While DPH is non-essential for cell viability and its function has not been resolved, diphtheria and other bacterial toxins ADP-ribosylate DPH to inhibit translation. Characterizing Saccharomyces cerevisiae mutants that lack DPH or show synthetic growth defects in the absence of DPH, we show that loss of DPH increases resistance to the fungal translation inhibitor sordarin and increases -1 ribosomal frameshifting at non-programmed sites during normal translation elongation and at viral programmed frameshifting sites.

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Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine.

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How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A.

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Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome.

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Translation start site selection in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the eukaryotic translation initiation factors eIF1 and eIF5. In a search of mammalian genes, we identified five homeobox () gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. An analysis of published cap analysis of gene expression sequencing (CAGE-seq) data and generated CAGE-seq data for messenger RNAs (mRNAs) from mouse somites revealed that the 5' leaders of mRNAs of interest contain conserved uORFs, are generally much shorter than reported, and lack previously proposed internal ribosome entry site elements.

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Polyamines, small organic polycations, are essential for cell viability, and their physiological levels are homeostatically maintained by post-transcriptional regulation of key biosynthetic enzymes. In addition to de novo synthesis, cells can also take up polyamines; however, identifying cellular polyamine transporters has been challenging. Here we show that the S.

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Recognition of a start codon by the initiator aminoacyl-tRNA determines the reading frame of messenger RNA (mRNA) translation by the ribosome. In eukaryotes, the GTPase eIF5B collaborates in the correct positioning of the initiator Met-tRNA on the ribosome in the later stages of translation initiation, gating entrance into elongation. Leveraging the long residence time of eIF5B on the ribosome recently identified by single-molecule fluorescence measurements, we determine the cryoEM structure of the naturally long-lived ribosome complex with eIF5B and Met-tRNA immediately before transition into elongation.

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Cells utilize transcriptional and posttranscriptional mechanisms to alter gene expression in response to environmental cues. Gene-specific controls, including changing the translation of specific messenger RNAs (mRNAs), provide a rapid means to respond precisely to different conditions. Upstream open reading frames (uORFs) are known to control the translation of mRNAs.

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Rare pathogenic EIF2S3 missense and terminal deletion variants cause the X-linked intellectual disability (ID) syndrome MEHMO, or a milder phenotype including pancreatic dysfunction and hypopituitarism. We present two unrelated male patients who carry novel EIF2S3 pathogenic missense variants (p.(Thr144Ile) and p.

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Dysregulation of cellular protein synthesis is linked to a variety of diseases. Mutations in EIF2S3, encoding the γ subunit of the heterotrimeric eukaryotic translation initiation factor eIF2, cause MEHMO syndrome, an X-linked intellectual disability disorder. Here, using patient-derived induced pluripotent stem cells, we show that a mutation at the C terminus of eIF2γ impairs CDC123 promotion of eIF2 complex formation and decreases the level of eIF2-GTP-Met-tRNA ternary complexes.

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Background: The heterotrimeric GTP-binding protein eIF2 forms a ternary complex with initiator methionyl-tRNA and recruits it to the 40S ribosomal subunit for start codon selection and thereby initiates protein synthesis. Mutations in EIF2S3, encoding the eIF2γ subunit, are associated with severe intellectual disability and microcephaly, usually as part of MEHMO syndrome.

Methods: Exome sequencing of the X chromosome was performed on three related males with normal head circumferences and mild learning difficulties, hypopituitarism (GH and TSH deficiencies), and an unusual form of glucose dysregulation.

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The heterotrimeric eukaryotic translation initiation factor (eIF) 2 plays critical roles in delivering initiator Met-tRNAiMet to the 40S ribosomal subunit and in selecting the translation initiation site. Genetic analyses of patients with MEHMO syndrome, an X-linked intellectual disability syndrome, have identified several unique mutations in the EIF2S3 gene that encodes the γ subunit of eIF2. To gain insights into the molecular consequences of MEHMO syndrome mutations on eIF2 function, we generated a yeast model of the human eIF2γ-I259M mutant, previously identified in a patient with MEHMO syndrome.

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Polyamines are organic polycations that bind to a variety of cellular molecules, including nucleic acids. Within cells, polyamines contribute to both the efficiency and fidelity of protein synthesis. In addition to directly acting on the translation apparatus to stimulate protein synthesis, the polyamine spermidine serves as a precursor for the essential post-translational modification of the eukaryotic translation factor 5A (eIF5A), which is required for synthesis of proteins containing problematic amino acid sequence motifs, including polyproline tracts, and for termination of translation.

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Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis.

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In this review, we highlight the current understanding of translation elongation and recoding in eukaryotes. In addition to providing an overview of the process, recent advances in our understanding of the role of the factor eIF5A in both translation elongation and termination are discussed. We also highlight mechanisms of translation recoding with a focus on ribosomal frameshifting during elongation.

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Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis.

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The eukaryotic translation factor eIF5A, originally identified as an initiation factor, was later shown to promote translation elongation of iterated proline sequences. Using a combination of ribosome profiling and in vitro biochemistry, we report a much broader role for eIF5A in elongation and uncover a critical function for eIF5A in termination. Ribosome profiling of an eIF5A-depleted strain reveals a global elongation defect, with abundant ribosomes stalling at many sequences, not limited to proline stretches.

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Impairment of translation initiation and its regulation within the integrated stress response (ISR) and related unfolded-protein response has been identified as a cause of several multisystemic syndromes. Here, we link MEHMO syndrome, whose genetic etiology was unknown, to this group of disorders. MEHMO is a rare X-linked syndrome characterized by profound intellectual disability, epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity.

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Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling.

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Eukaryotic translation initiation factor eIF5A promotes protein synthesis by resolving polyproline-induced ribosomal stalling. Here, we report a 3.25-Å resolution crystal structure of eIF5A bound to the yeast 80S ribosome.

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In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs.

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Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2).

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Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by eIF2α family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2α family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2α kinase function remains an enigma.

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