Yeast Pseudo-Haploinsufficiency as a Model System for Human Ribosomopathies

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Kobylarz, Ryan
Dinman, Jonathan D
Ribosomopathies belong to a class of human diseases caused by mutations in genes that encode ribosomal proteins, ribosomal biogenesis factors, ribosomal RNA (rRNA) or rRNA post-transcriptional modifying factors. Ribosomal Protein S19 (RPS19) is the gene linked to Diamond Blackfan Anemia, the first identified ribosomopathy. Paradoxically, patients suffering from this disorder initially present with insufficient blood cells but later exhibit a proclivity toward developing hyper-proliferative blood cell formation. The other two most common ribosomopathies include Isolated Congenital Asplenia (linked to mutations in the gene encoding for RPS0) and 5q- syndrome (a somatically acquired haploinsufficiency of RPS14). Despite originating in the ribosome, the unique phenotypes that are symptomatic of under-developed cells and the tissue specificity of ribosomopathies are not compatible with ribosomal biogenesis defect etiologies. The unique clinical presentations of each of these diseases are consistent with the presence of “specialized” ribosomes, where each tissue type may require a certain subset of ribosomes. Recent studies into another ribosomopathy, X-linked dyskeratosis congenita (X-DC), revealed that defects in rRNA pseudouridylation patterns result in defects in translational fidelity. In order to study the translational effects of ribosomal protein haploinsufficient ribosomes, we used the Saccharomyces cerevisiae yeast as model for human ribosomopathies. Haploid yeast cells harbor two functional paralogs of RPS0, RPS14 and RPS19, in addition to other ribosomal proteins, due to an ancient whole genome duplication event. The yeast model enables the generation of single knockout of either the A or B paralogous ribosomal protein gene. Yeas also provides the ability to monitor gene specific differences in translational fidelity relative to isogenic wild-type cells. Ribosomal protein gene haploinsufficiency confers gene-specific translational fidelity defects. In assays that monitor recoding event frequencies, the most notable result was an increase in stop codon readthrough for all haploinsufficient strains. -1 or +1 Programmed Ribosomal Frameshift (PRF) recoding events were shown to exhibit isoform and sequence specific events, e.g. one isoform of RPS0 exhibits an increased -1 PRF recoding efficiency while the other demonstrates a decreased -1 PRF efficiency. Steady state mRNA abundance measurements reveals that RPS19 gene pseudo-haploinsufficiency confers a global decrease in mRNA abundance. In steady state mRNA abundance measurements of genes involved in telomere length maintenance, RPS0 and RPS14 were shown to exhibit sequence specific effects, only presenting an increase in mRNA abundance for CDC13 while exhibiting a decrease for others. These idiosyncratic results challenge the prevailing notion of the “monolithic ribosome”. Here, we present a novel model whereby the transcriptome is translated and regulated by a heterogeneous population of ribosomes.