Identification of RPS14 as a 5q- syndrome gene by RNA interference screen

Abstract

Somatic chromosomal deletions in cancer are thought to indicate the location of tumor suppressor genes, whereby complete loss of gene function occurs through biallelic deletion, point mutation, or epigenetic silencing, thus fulfilling Knudson's two-hit hypothesis.1 In many recurrent deletions, however, such biallelic inactivation has not been found. One prominent example is the 5q- syndrome, a subtype of myelodysplastic syndrome (MDS) characterized by a defect in erythroid differentiation.2 Here, we describe an RNA interference (RNAi)-based approach to discovery of the 5q- disease gene. We find that partial loss of function of the ribosomal protein RPS14 phenocopies the disease in normal hematopoietic progenitor cells, and moreover that forced expression of RPS14 rescues the disease phenotype in patient-derived bone marrow cells. In addition, we identified a block in the processing of pre-rRNA in RPS14 deficient cells that is highly analogous to the functional defect in Diamond Blackfan Anemia, linking the molecular pathophysiology of the 5q- syndrome to a congenital bone marrow failure syndrome. These results indicate that the 5q- syndrome is caused by a defect in ribosomal protein function, and suggests that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes.

Recurrent genetic defects on chromosome 7q in myeloid neoplasms

“…Several other hematopoiesis-related genes and tumor suppressor genes are located in the CDR (e.g., CTNNA1, PPP2CA, EGR1, SPARC, RPS14 and CDC25C) with previously reported [14,[27][28][29][30][31], however, we were unable to detect hemi-or heterozygous mutations in Oncotarget 6491 www.impactjournals.com/oncotarget these genes. It is possible that they contribute to clinical heterogeneity, shape the clinical phenotype or modulate the growth advantage of the del(5q).…”

Effects of L-leucine in 5q- syndrome and other RPS14-deficient erythroblasts

“…The RPS14 gene, encoding a ribosomal protein and mapping to the commonly deleted region (CDR) of the 5q-syndrome, 1 shows haploinsufficiency in the CD34 þ cells of patients with this disorder. 2,3 The genes in the 5q-syndrome CDR were studied by an RNA-mediated interference-based approach and it was shown that haploinsufficiency of RPS14 in normal CD34 þ cells resulted in a block in erythroid differentiation with relative preservation of megakaryocyte differentiation, suggesting that RPS14 haploinsufficiency is the probable cause of the erythroid defect in this disorder. 3 Several lines of converging evidence suggest that p53 activation secondary to ribosomal haploinsufficiency is the mechanism that underlies the anemia in the 5q-syndrome.…”

“…2,3 The genes in the 5q-syndrome CDR were studied by an RNA-mediated interference-based approach and it was shown that haploinsufficiency of RPS14 in normal CD34 þ cells resulted in a block in erythroid differentiation with relative preservation of megakaryocyte differentiation, suggesting that RPS14 haploinsufficiency is the probable cause of the erythroid defect in this disorder. 3 Several lines of converging evidence suggest that p53 activation secondary to ribosomal haploinsufficiency is the mechanism that underlies the anemia in the 5q-syndrome. 4,5 The bone marrow cells of patients with the 5q-syndrome show a block in the processing of pre-ribosomal RNA 3 and the CD34 þ cells of these patients show deregulation of multiple ribosomal protein genes and genes involved in the control of translation.…”

“…3 Several lines of converging evidence suggest that p53 activation secondary to ribosomal haploinsufficiency is the mechanism that underlies the anemia in the 5q-syndrome. 4,5 The bone marrow cells of patients with the 5q-syndrome show a block in the processing of pre-ribosomal RNA 3 and the CD34 þ cells of these patients show deregulation of multiple ribosomal protein genes and genes involved in the control of translation. 6 These data suggest that the 5q-syndrome represents a disorder of aberrant ribosome biogenesis.…”

“…6 These data suggest that the 5q-syndrome represents a disorder of aberrant ribosome biogenesis. 3,6 Erythroid progenitor cells have a very high rate of proliferation and a requirement for massive globin synthesis, thus necessitating a very high level of ribosome biogenesis and ribosomal activity. This may explain the particular sensitivity of the erythroid lineage to reduced expression levels of ribosomal proteins.…”