Impaired growth, hematopoietic colony formation, and ribosome maturation in human cells depleted of Shwachman-Diamond syndrome protein SBDS

Background ShwachmanDiamond syndrome (SDS), associated with SBDS mutations, is characterized by pancreatic exocrine dysfunction and marrow failure. Sdo1, the yeast ortholog of SBDS, is implicated in maturation of the 60S ribosomal subunit, with delayed export of 60S-like particles from the nucleoplasm when depleted. Sdo1 is needed for release of the anti-subunit association factor Tif6 from 60S subunits, and Tif6 may not be recycled to the nucleus when Sdo1 is absent. Methods To clarify the role of SBDS in human ribosome function, TF-1 erythroleukemia and A549 lung carcinoma cells were transfected with vectors expressing RNAi against SBDS. Results Growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to controls. To analyze the effect of SBDS on 60S subunit maturation in A549 cells, subunit localization was assessed by transfection with a vector expressing a fusion between human RPL29 and GFP: we found a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits. Polysome analysis of TF-1 knockdown cells showed a decrease in free 60S and 80S subunits. We also analyzed the levels of eIF6 (human ortholog of Tif6) following near-complete knockdown of SBDS in TF-1 cells and found an approximately 20% increase in the amount of eIF6 associated with the 60S subunit. Conclusions We conclude that knockdown of SBDS leads to growth inhibition and defects in ribosome maturation, suggesting a role for wild-type SBDS in nuclear export of pre-60S subunits. Furthermore, knockdown of SBDS may interfere with eIF6 recycling. Pediatr Blood Cancer 2013;60:281286. (c) 2012 Wiley Periodicals, Inc.