Cell Biology & Molecular Genetics
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Item THE IMPACT OF TRANSLATIONAL FIDELITY ON HUMAN HEALTH(2018) Marques dos Santos Vieira, Carolina; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ribosomopathies are a class of diseases resulting from mutations in genes encoding ribosomal proteins and ribosome biogenesis factors. Pleiotropic clinical presentations of different ribosomopathies has been taken as evidence of specialized ribosomes. Alternatively, gene dosage effects have been proposed to account for the observed differences. A yeast genetics approach was used to address this issue. Due to a historical gene duplication event, S. cerevisiae cells harbor two ohnologs for most ribosomal proteins. Deletion of one yeast ribosomal protein ohnolog was used to mimic haploinsufficiency in diploid cells (i.e. pseudo-haploinsufficient yeast). Further, insertion of a second copy of the undeleted ohnolog into the locus of the deleted ohnolog enabled separation of effects due to gene dosage from those due to ribosomal protein ohnolog identity. We found that significant changes in translational fidelity in the ribosomal protein ohnolog deletion strains were corrected by ohnolog duplication. Changes in gene dosage, particularly as they may affect the abundance of an enzyme as central as the ribosome, can impart stress through far reaching effects on cellular metabolism. Thus, as an orthogonal approach, we also examined the stress profiles of cells harboring the cbf5-D95A allele (model of X-linked Dyskeratosis Congenita) and the rps23a-R69K allele (model of MacInnes Syndrome). RNA-seq analysis revealed increased expression of proteins involved in response to oxidative stress in cbf5-D95A cells. Growth curve analysis revealed a longer plateau of the cbf5-D95A cells upon reaching stationary phase, suggestive of a pre-adaptive stress response. Decreased ROS abundance, tunicamycin resistance and increased basal levels of HAC1 mRNA in the mutant cells support this hypothesis. Similar results were observed with regard to the ohnolog deletion strains. Taken as a whole, these data support the gene dosage model as opposed to the specialized ribosome hypothesis with the caveat that this conclusion is limited to yeast cells growing in rich medium.Item Alternate Conformations Regulate Ribosomal Recoding in a Positive-sense RNA Virus(2016) Kuhlmann, Micki Michelle; Simon, Anne E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Positive-sense RNA viruses are important animal, plant, insect and bacteria pathogens and constitute the largest group of RNA viruses. Due to the relatively small size of their genomes, these viruses have evolved a variety of non-canonical translation mechanisms to optimize coding capacity expanding their proteome diversity. One such strategy is codon redefinition or recoding. First described in viruses, recoding is a programmed translation event in which codon alterations are context dependent. Recoding takes place in a subset of messenger RNA (mRNAs) with some products reflecting new, and some reflecting standard, meanings. The ratio between the two is both critical and highly regulated. While a variety of recoding mechanisms have been documented, (ribosome shunting, stop-carry on, termination-reinitiation, and translational bypassing), the two most extensively employed by RNA viruses are Programmed Ribosomal Frameshifting (PRF) and Programmed Ribosomal Readthrough (PRT). While both PRT and PRF subvert normal decoding for expression of C-terminal extension products, the former involves an alteration of reading frame, and the latter requires decoding of a non-sense codon. Both processes occur at a low but defined frequency, and both require Recoding Stimulatory Elements (RSE) for regulation and optimum functionality. These stimulatory signals can be embedded in the RNA in the form of sequence or secondary structure, or trans-acting factors outside the mRNA such as proteins or micro RNAs (miRNA). Despite 40+ years of study, the precise mechanisms by which viral RSE mediate ribosome recoding for the synthesis of their proteins, or how the ratio of these products is maintained, is poorly defined. This study reveals that in addition to a long distance RNA:RNA interaction, three alternate conformations and a phylogenetically conserved pseudoknot regulate PRT in the carmovirus Turnip crinkle virus (TCV).Item ESTABLISHING LINK BETWEEN TRANSLATIONAL RECODING AND HUMAN DISEASE(2015) Advani, Vivek Manoharlal; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Gene expression can be controlled at the level of mRNA stability, and prior studies from our laboratory have explained how Programmed -1 Ribosomal Frameshifting (-1 PRF) fits within this paradigm. Computational analyses suggest that 10-15% of eukaryotic mRNAs contain at least one potential -1 PRF signal. The overwhelming majority of predicted "genomic" -1 PRF events are predicted to direct translating ribosomes to premature termination codons. We have demonstrated that these can function as mRNA destabilizing elements through the Nonsense-Mediated mRNA Decay (NMD) pathway. In published work we have explored the biological significance of the connection between -1 PRF and NMD on telomere maintenance in yeast. More recently we extended this line of inquiry to human cells, demonstrating that a sequence element in the mRNA encoding Ccr5p harbors a -1 PRF signal which functions as an mRNA destabilizing element through NMD. In the current work we are exploring the link between global changes in -1 PRF rates and human health using yeast and human cell-based models of two diseases, X-linked Dyskeratosis Congenita (X-DC) and Spinocerebellar ataxia 26 family (SCA26) as models. Preliminary findings suggest these genetically inherited defects result in translational fidelity defects (i.e. changes in rates of -1 PRF, +1 PRF, and stop codon recognition), with attendant effects on mRNA abundance, gene expression and telomere maintenance. These studies establish a paradigm for understanding the linkage between translational fidelity and human disease.