Identification of RPS14 as the 5q-Syndrome Gene by RNA Interference Screen.

We report here that haploinsufficiency of the ribosomal protein encoding RPS14 gene causes the characteristic hematologic phenotype that defines the 5q- syndrome. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), was first described in 1974 as a distinct clinical entity associated with deletions of the long arm of chromosome 5. Since that time, the common deleted region has been narrowed in search of the causal gene within the region. However, no biallelic inactivation events have been identified for any of the 41 genes within the critical region, suggesting that a haploinsufficiency mechanism may explain the disease phenotype. We addressed this question through a functional genomic approach, utilizing systematic RNA interference (RNAi) to interrogate the function of each gene in the common deleted region. We introduced 3–5 unique, lentivirally expressed short hairpin RNAs (shRNAs) targeting each of the 41 genes in the region into normal CD34+ human bone marrow hematopoietic progenitor cells and assayed the effects of each shRNA on hematopoietic differentiation. We found that knock-down of a single gene, RPS14, recapitulated the phenotype of the 5q- syndrome: namely, a block in erythroid differentiation (leading to erythroid cell apoptosis) with relative preservation of megakaryocyte differentiation as measured by FACS analysis. Importantly, forced expression of an RPS14 cDNA in primary bone marrow cells from patients with the 5q- syndrome rescued the phenotype, yet had no effect on cells from MDS patients without 5q deletions. In addition, we found that RPS14 haploinsufficiency caused a block in the processing of pre-ribosomal RNA and in the formation of the 40S ribosomal subunit. This ribosomal processing defect is highly analogous to the functional defect seen in Diamond Blackfan Anemia (also characterized by an erythroid differentiation defect and predisposition to AML), thereby establishing an unexpected link between the molecular pathophysiology of acquired 5q- syndrome and congenital bone marrow failure syndromes. These results indicate that the 5q- syndrome is caused by a defect in ribosomal protein function, highlighting the importance of translational control in hematologic malignancy. The results further suggest that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes.