Browsing by Subject "Ribosomes"
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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 Characterization of an Orphan Riboswitch: Identification of a Metal-Sensing Regulatory RNA(2011-09-30) Wakeman, Catherine Ann; Winkler, Wade C.Riboswitches are RNA-based genetic control elements found in untranslated regions of the mRNA transcript that they regulate. These RNA motifs are highly structured and bind metabolites to elicit control of gene expression. Typically, the metabolite sensed by these RNAs is a component of the metabolic pathway in which the regulated gene product resides. The focus of this project has been the identification of the ligand for a riboswitch that was discovered using bioinformatics-based search methods. This riboswitch was designated the ykoK RNA element due to its location in the 5' UTR of the B. subtilis ykoK (mgtE) gene, which appears to be a magnesium transporter. Therefore, the possibility that this RNA senses magnesium levels was explored. The data revealed that the RNA element imparts magnesium-responsive regulation to the ykoK gene. These data also indicated which portions of the RNA are essential for genetic regulation. The results of a battery of biochemical tests demonstrated that magnesium triggers a concerted conformational change in the RNA such that it adopts a compacted tertiary structure. Resolution of the three-dimensional structure of the RNA in the magnesium bound state revealed the basis of this metal-induced tertiary conformation and how this relates to genetic control. Intriguingly, this structure revealed the presence of six magnesium ions, making this the first example of multiple ligands binding to a single riboswitch aptamer. When individual metal-binding sites were eliminated using phosphorothioate substitutions, it became evident that all six of these magnesium-binding sites and up to three additional metal-binding sites are required for function of this RNA. Therefore, these data demonstrate that the ykoK RNA element, now designated the M-box RNA, directly senses intracellular magnesium levels for the purposes of genetic control. These findings should have broad implications given that this RNA element is wide spread among Gram-positive bacteria and appears to regulate many additional gene categories such as ABC transporters, cell division proteins, and proteins of unknown function. The exploration of the connection between magnesium concentration and the expression levels of these proteins might provide insights into previously undefined functional roles.Item Translational Control by the Ribosome-Associated Complex in the Unfolded Protein Response(2020-12-01T06:00:00.000Z) Wu, I-Hui; Shay, Jerry W.; Thomas, Philip J.; Mendell, Joshua T.; Tu, BenjaminRibosome-associated chaperones are ubiquitous and highly conserved. There are two classes of ribosome-associated chaperones in eukaryotes, the nascent polypeptide-associated complex (NAC) and the ribosome-associated complex (RAC). Mammalian RAC consists of Hsp70L1, an Hsp70 chaperone homologue, and Mpp11, a DnaJ cofactor. RAC interacts with the nascent chain near the polypeptide exit tunnel and the decoding center on the 60S and 40S ribosomal subunits, respectively. Its unique position on the ribosome implies the coordinating role of de novo protein folding with translation. Deletion of RAC causes growth defects and sensitizes to osmotic, cold, and aminoglycoside stresses in yeast. Furthermore, studies have shown that Mpp11 is over-expressed in head and neck squamous cell cancer and leukemia. However, the function of RAC in stress responses and its role in oncogenesis remain obscure. The current hypothesis predicts that RAC supports co-translational folding of nascent cytosolic polypeptides. To directly test this hypothesis, I altered levels of RAC components and monitored the cytosolic heat shock response (HSR) and the unfolded protein response (UPR) in the ER, two stress pathways known to be activated by accumulation of misfolded proteins. Contrary to its presumptive role in cytosolic protein folding, the reduction of RAC expression did not activate the cytosolic HSR. Unexpectedly, reduction of RAC sensitizes cells to ER stress by selectively attenuating activation of the IRE1 branch of UPR. When RAC is reduced, Xbp1 mRNA splicing is inhibited upon ER stress. Consistent with this activity, ER stress induces changes in the subcellular distribution of RAC, which coincides with the localization of Xbp1 mRNA. Mechanistically, reduction of RAC affects the pathway at a very early step, as IRE1 self-association is inhibited. Additionally, this study shows that the reduction of RAC enhances cellular mRNA translation, including Xbp1 mRNA translation. Interestingly, reduction of Pelo, a protein involved in recognizing stalled ribosomes, counters the inhibition of Xbp1 mRNA splicing, and IRE1 foci formation due to RAC knockdown. Collectively, these results suggest that RAC plays a central role in the IRE1 branch of the UPR tuning IRE1 clustering and mRNA translation.