Browsing by Subject "Peptide Initiation Factors"
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Item Analysis of the Role of EIF5A in Mammalian Translation(2019-04-10) Manjunath, Hema; Carroll, Thomas J.; Mendell, Joshua T.; Conrad, Nicholas; Amatruda, James F.MYC is a critical growth-promoting gene that is subject to tight post-transcriptional control. However, the genes and mechanisms that mediate this regulation at the mRNA level are poorly understood. In order to identify regulators of MYC that function through the 5' UTR of the transcript, we performed a fluorescent reporter-coupled genome-scale CRISPR/Cas9-mediated loss of function screen. Analysis of screening data identified eukaryotic initiation factor 5A (EIF5A) as novel regulator of MYC translation. eIF5A is a highly conserved translation factor that has been demonstrated to relieve ribosome pauses during translation elongation at 'difficult to translate' peptide sequences in yeast and bacteria. We observed that eIF5A regulates protein isoform distribution of MYC, and that loss of function of this gene results in enhanced upstream non-canonical translation initiation on this transcript. Upon performing ribosome profiling in cells where eIF5A or its upstream activating enzyme were ablated, we discovered that the protein's function as a ribosome pause relief factor is conserved in mammalian cells. Importantly, analysis of ribosome profiling data under conditions of eIF5A depletion revealed not only evidence of enhanced ribosome pausing within coding sequences at elongation stall sites, but also an increase in non-canonical/sub-optimal translation initiation events in 5' UTRs in both yeast and human cells. These data lead us to formulate and test the hypothesis that ribosome pausing resulting from loss of eIF5A increases non-canonical translation initiation at pause-proximal upstream sub-optimal initiation codons. We present data from ribosome profiling experiments in yeast and human cells, as well as luciferase reporter assays that are consistent with this model. Thus, we propose a novel role for the translation elongation factor eIF5A in maintaining appropriate start codon selection during initiation in eukaryotic cells.Item Essentiality and Regulation of Deoxyhypusination in Trypanosoma brucei(2014-07-01) Nguyen, Suong Thu; Orth, Kim; Phillips, Margaret A.; Goodman, Joel M.; De Brabander, Jef K.Human African trypanosomiasis is caused by protozoan parasite Trypanosoma brucei. T. brucei and other trypanosomatids require spermidine for the formation of trypanothione, a unique thiol-redox factor. In other eukaryotes, spermidine is essential for the (deoxy)hypusination of eukaryotic initiation factor 5A (eIF5A). Hypusination, a post-translational modification, occurs via two enzymatic reactions. First, deoxyhypusine synthase (DHS) transfers the aminobutyl moiety of spermidine onto the eIF5A-lysine generating deoxyhypusine which is then hydroxylated by deoxyhypusine hydroxylase to yield the final modification, hypusine. Modified eIF5A has been shown to alleviate ribosome stalling on polyproline tracts. Human and yeast encode two isoforms of eIF5A but only one gene was identified in T. brucei (Tb927.11.740). Herein, I show that TbeIF5A and its modified lysine are essential for parasite growth by gene knockdown and complementation experiments. I have also identified potential proteins whose translation is regulated by eIF5A using proteomic profiling for proline-rich T. brucei proteins. Interestingly, unlike most eukaryotes, trypanosomatids encode two divergent paralogs of DHS (DHSp: Tb927.1.870 and DHSc: Tb927.10.2580), only one of which (DHSc) contains the key catalytic lysine. I showed that both DHS genes are essential for growth of bloodstream-form T. brucei using conditional gene knockouts, further establishing the requirement for deoxyhypusine in these parasites. My biochemical characterization of TbDHS showed that the two T. brucei paralogs form a heterotetrameric complex and that DHSp enhances the activity of recombinant DHSc by 3000-fold. While the essentiality of eIF5A and DHS is consistent with other eukaryotes, the finding that the functional DHS complex is composed of an impaired catalytic subunit (DHSc) and a catalytically dead paralog (termed a prozyme) is novel. This mechanism reiterates the activation and regulation of S-adenosylmethionine decarboxylase by a catalytically dead paralog (AdoMetDC prozyme) in the trypanosomatids, and remarkably, it has independently evolved for two enzymes within the trypanosomatid spermidine biosynthetic pathway. T. brucei seemingly lack several classical eukaryotic transcriptional regulation mechanisms which creates selective pressure to evolve novel strategies to regulate enzyme function. We postulate that many additional examples of 'prozymes' remain to be discovered in the trypanosomatid parasites.