Translation Initiation Defect and Gene Profiles Linked to Depletion of Shwachman-Diamond Syndrome Gene Product.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, skeletal abnormalities, and bone marrow failure, which can evolve to leukemia. Mutations in SBDS have been shown to cause SDS, and SBDS ’s yeast ortholog, SDO1 , appears to play a role in ribosomal RNA (rRNA) processing. Thus, several bone marrow failure syndromes including dyskeratosis congenita, cartilage hair hypoplasia, and Diamond Blackfan anemia have been linked to defects in ribosome synthesis. In order to clarify the function of SBDS, we have performed studies on the effect of depleting SBDS in yeast, mouse, and human cells. Six plasmids encoding for coral GFP and neomycin resistance were modified to express siRNAs against SBDS and used to establish stable transfectants in human HeLa and mouse NIH3T3 cells. Each siRNA was confirmed to knock down human or mouse SBDS expression, by real-time PCR. The growth of HeLa and NIH3T3 stable transfectants was markedly hindered when compared to that of cells stably transfected with a siRNA against an irrelevant control gene. The growth abnormality in SBDS-knockdown cells appeared to be related to an increased propensity to apoptosis. We then searched for differentially expressed genes between SBDS-knockdown and control cells using a series of 9 Affymetrix GeneChip microarrays. An exhaustive comparison using GenMAPP 2.0 and Gene Ontology software yielded highly significant differences in expression of genes in multiple pathways such as: apoptosis, cell cycle and cell growth, neutrophil chemotaxis, skeletal development, and angiogenesis pathways, among others. Changes (resulting from SBDS silencing) in expression of ribosome-related genes were also evident across experiments with both human and mouse cells. Concurrently, we studied the effect of depleting the yeast ortholog of SBDS , known as SDO1 . Mutants of SDO1 are deficient in early steps in rRNA processing resulting in the accumulation of 35S and 23S pre-rRNA. SDO1 mutants thus have a so-called processome-like phenotype, in which cleavages at sites A 0 and A 1 within the external transcribed sequence-1 and site A 2 within the internal transcribed sequence-1 are severely affected. Processome mutants exhibit a selective deficit in 40S ribosomal subunits, as cleavages at sites A 0 , A 1 , and A 2 are not absolutely required for the maturation of 60S ribosomal subunits. Surprisingly, we found a much more complex pattern than anticipated in polysome profiles from cells harboring null alleles of SDO1 . These profiles contained half-mer polysomes, typical of strains with a 60S subunit deficit and yet, in SDO1 mutants the amount of 60S subunits appeared to be relatively normal. In addition, the pool of free 40S subunits appeared abnormal in SDO1 mutants, with the appearance of two peaks, one of which likely represents an aberrant 40S subunit. This aberrant 40S subunit was apparently capable of early steps in translational initiation but failed to join with 60S subunits to form the 80S initiation complex, resulting in half-mer polysomes. We conclude that the rRNA processing phenotypes of SDO1 mutants are likely secondary to a defect in recruiting a factor or factors to the 40S subunit necessary for subunit joining during initiation. A similar profile for free 40S subunits was seen in NIH3T3 cells in which Sbds had been knocked down, suggesting that at least a part of the molecular phenotype observed in yeast is directly relevant for mammalian cells.