Ribosomal RNA and Gene Expression Abnormalities with Knockdown of the Shwachman-Diamond Syndrome Gene, SBDS.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, skeletal abnormalities, and bone marrow failure, which can evolve to myelodysplastic syndrome and acute myeloid leukemia. Recently, mutations in an uncharacterized gene, SBDS , have been associated with SDS. SBDS is a member of a highly conserved protein family of unknown function, with putative orthologs in species ranging from archaea to plants and vertebrates. The yeast ortholog, YLR022c, is a phospholipid-binding protein that is essential for cell viability and may play a critical role in ribosomal RNA (rRNA) processing. Several bone marrow failure syndromes including dyskeratosis congenita, cartilage hair hypoplasia, and Diamond Blackfan anemia (DBA) have been linked to factors involved in ribosome synthesis. Our studies in yeast show that rRNA processing phenotypes associated with mutations in RPS19 , a gene affected in DBA, overlap with the rRNA processing phenotype reported by others for cells depleted of YLR022c. These data indicate that defects in ribosome synthesis may contribute to the molecular basis of SDS. To study the effect of depleting SBDS in human cells, we created plasmids encoding for coral GFP (cGFP) and neomycin resistance that were further modified to express siRNA against human SBDS . A construct that contains only cGFP/NeoR was used as a control. HeLa cells were transiently transfected with knockdown or control vector for two days and flow-sorted for cGFP expression. In some experiments, neomycin- resistant siRNA expressing cell clones were isolated. Total RNA was extracted from transfected cells, reverse-transcribed, and subjected to real-time PCR for SBDS transcript quantification (using two SBDS primers and a TaqMan 5′ Fam/3′ Tamra probe specific for SBDS transcript). Data from each transfection were normalized to GAPDH expression, and SBDS expression was compared between cells treated with each siRNA vector and cells treated with a control cGFP/NeoR construct lacking the siRNA sequence. After multiple preliminary experiments, we found that one of the siRNAs expressed in cGFP-positive sorted cells decreased SBDS expression to 16% that of cells expressing only cGFP but not the siRNA sequence (84% knock-down). Total RNA was extracted from cGFP-positive knockdown and control cells and analyzed by Affymetrix GeneChip U133A microarrays to search for differentially expressed genes. In general, comparing the knockdown and control cells, we found many more genes with increased rather than decreased expression. However, both 28S and 18S rRNAs were repeatedly found significantly decreased in the knockdown cells (5–10% that of cGFP-positive control cells lacking the siRNA sequence). Although human rRNA processing is not fully understood, inhibition of early processing steps in mammalian cells should affect the production of both 18S and 28S rRNAs. Thus, our results suggest that at least one function of SBDS may be in rRNA processing and furthers the putative link between certain congenital bone marrow failure syndromes and defective ribosomal biogenesis.