Yeast Models of Diamond Blackfan Anemia and Shwachman Diamond Syndrome Differ in Their Effect on the Synthesis and Function of Ribosomal Subunits.

Nucleolar stress is a frequently invoked mechanism used to describe the pro-apoptotic phenotype of cells affected in human diseases linked to abnormalities of the ribosome. However, the diversity of clinical phenotypes observed in these diseases suggests that there may be different types of nucleolar and/or translational stress that stimulate cell death pathways by alternative mechanisms. We have studied yeast models of Diamond Blackfan anemia (DBA) and Shwachman Diamond syndrome (SDS), two inherited bone marrow failure syndromes linked to defects in ribosome synthesis and/or function, to determine potential underlying molecular mechanisms that distinguish these disease models. To date, all genes identified in DBA encode ribosomal proteins. In contrast, SBDS, the gene affected in SDS encodes a protein that associates with 60S subunits, but is not considered a structural component of the ribosome. We have analyzed the translational capacity of cells harboring mutations in RPL33A and SDO1, yeast orthologs of genes affected in DBA and SDS, respectively. Polysome profiles from cells depleted of Rpl33A have a decrease in the amount of free 60S subunits and the presence of half-mer polysomes, as expected for an essential structural component of the 60S subunit. Polysome profiles from cells depleted of Sdo1 also had half-mer polysomes, but in this case there were significant amounts of free 60S subunits evident. Analysis of the intracellular distribution of 60S subunits by fluorescence microscopy revealed significant differences between the two disease models. In the DBA model, there was no evidence of accumulation of incompletely assembled subunits in the nucleolus indicating that rapid degradation. In contrast, in the SDS model there was significant accumulation of 60S subunits in the nucleoplasm. Thus, the two disease models interfere with the biogenesis of 60S subunits through distinct mechanisms. To determine if these mechanistic differences influence protein synthesis, we analyzed the patterns of proteins synthesized in these two disease models. We found that the expression of the 20S replicon was induced in both models, a sign of general translational stress. However, the two models also showed distinct differences in the synthesis of certain proteins. Thus, the mechanisms by which reductions of Rpl33A or Sdo1 influence levels of functional 60S subunits have differential effects on the patterns of proteins synthesized within cells Together these data indicate that the ribosome-based diseases may result from a composite of effects that include both nucleolar stress mechanisms and changes in translational output. The distinct clinical phenotypes observed in these disorders may result from differences in the relative contributions of either of these two mechanisms.