The ribosomal protein S19 (RPS19) is located in the small (40S) subunit and is one of 79 ribosomal proteins. The gene encoding RPS19 is mutated in approximately 25% of patients with Diamond-Blackfan anemia, which is a rare congenital erythroblastopenia. Affected individuals present with decreased numbers or the absence of erythroid precursors in the bone marrow, and associated malformations of various organs are common. We produced C57BL/6J mice with a targeted disruption of murine Rps19 to study its role in erythropoiesis and development. Mice homozygous for the disrupted Rps19 were not identified as early as the blastocyst stage, indicating a lethal effect. In contrast, mice heterozygous for the disrupted Rps19 allele have normal growth and organ development, including that of the hematopoietic system. Our findings indicate that zygotes which are Rps19 ؊/؊ do not form blastocysts, whereas one normal Rps19 allele in C57BL/6J mice is sufficient to maintain normal ribosomal and possibly extraribosomal functions.The ribosomal proteins constitute a major component of cellular proteins and are known to be mandatory for cellular growth. A reduced level of one ribosomal protein is rate limiting for the assembly of ribosomes and may constitute a bottleneck for protein synthesis in tissues with a high proliferative activity. Recently, Draptchinskaia et al. found that the gene encoding ribosomal protein S19 (RPS19) was mutated in 25% of patients with Diamond-Blackfan anemia (DBA) (5). DBA is a rare, congenital, and chronic anemia that is characterized by the absence or decreased numbers of erythroid precursors in the bone marrow but an otherwise normal cellularity (1, 4). Approximately 30% of affected patients with DBA show one or several dysmorphic features including growth retardation, hand and/or limb malformations, urogenital anomalies, and congenital heart defects (1, 2, 10). Clinical expression in DBA is highly variable (14,21), and the role of RPS19 in the pathogenesis of the disease is presently unknown.RPS19 is part of the 40S ribosomal unit and shows a high degree of sequence conservation across mammalian species at the protein level (5). In addition to its implication in erythropoiesis, there are indications that RPS19 has extraribosomal functions.A previous study has shown that a dimer of RPS19 may mediate chemotaxis (13). Free RPS19 has also been shown to interact with fibroblast growth factor 2 (FGF-2), and a possible role for RPS19 in embryonic development has been suggested (17). We hypothesized that an Rps19 null mutant would be deleterious for development and that mice heterozygous for Rps19 may present with hematological abnormalities. To clarify the role of RPS19 in erythropoiesis, we created a null mutation for murine Rps19 by homologous recombination in embryonic stem (ES) cells. The targeted Rps19 was introduced on a C57BL/6J background. We present here the results from our studies of this mouse model. MATERIALS AND METHODSConstruction of the Rps19 targeting vector. An Rps19 intron probe was used t...

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. To study effects of RPS19 deficiency in hematopoiesis we transduced CD34 ؉ umbilical cord blood (CB) and bone marrow (

X-linked mental retardation (XLMR) affects one in 600 males and is highly heterogeneous. We describe here a 29-year-old woman with severe nonsyndromic mental retardation and a balanced reciprocal translocation between chromosomes X and 15 [46,XX,t(X;15)(q13.3;cen)]. Methylation studies showed a 100% skewed X-inactivation in patient-derived lymphocytes indicating that the normal chromosome X is retained inactive. Physical mapping of the breakpoints localised the Xq13.3 breakpoint to within 3.9 kb of the first exon of the ZDHHC15 gene encoding a zinc-finger and a DHHC domain containing product. Expression analysis revealed that different transcript variants of the gene are expressed in brain. ZDHHC15-specific RT-PCR analysis on lymphocytes from the patient revealed an absence of ZDHHC15 transcript variants, detected in control samples. We suggest that the absence of the ZDHHC15 transcripts in this patient contributes to her phenotype, and that the gene is a strong candidate for nonsyndromic XLMR.

Diamond-Blackfan anemia (DBA) is a severe congenital anemia characterized by a specific decrease of erythroid precursors. The disease is also associated with growth retardation, congenital malformations, a predisposition for malignant disease and heterozygous mutations in either of the ribosomal protein (RP) genes RPS7, RPS17, RPS19, RPS24, RPL5, RPL11 and RPL35a. We show herein that primary fibroblast from DBA patients with truncating mutations in RPS19 or in RPS24 have a marked reduction in proliferative capacity. Mutant fibroblasts are associated with extended cell cycles and normal levels of p53 when compared to w.t. cells. RPS19 mutant fibroblasts accumulate in the G1 phase, whereas the RPS24 mutant cells show an altered progression in the S phase resulting in reduced levels in the G2/M phase. RPS19 deficient cells exhibit reduced levels of Cyclin-E, CDK2 and retinoblastoma (Rb) protein supporting a cell cycle arrest in the G1 phase. In contrast, RPS24 deficient cells show increased levels of the cell cycle inhibitor p21 and a seemingly opposing increase in Cyclin-E, CDK4 and CDK6. In combination, our results show that RPS19 and RPS24 insufficient fibroblasts have an impaired growth caused by distinct blockages in the cell cycle. We suggest this proliferative constraint to be an important contributing mechanism for the complex extra-hematological features observed in DBA.

Interactive contact between B lymphocytes and T cells is necessary for their expansion during an immune response. It has been shown that B lymphocytes receive signals from T cells, such as IL-4 and cross-linking of CD40, which are crucial for their differentiation. We previously found that these factors induce formation of microvilli on B cells and that this was correlated with increased homotypic adhesion of B lymphocytes. In this study we have investigated if IL-4 induce segregation of proteins to microvilli and lipid rafts. Using immuno-electron microscopy we analyzed cell-surface distribution of molecules involved in B-T cell co-activation. Recruitment to detergent-resistant membrane fractions was analyzed using sucrose gradient centrifugation. We found that microvilli were enriched in ICAM-1 and MHC class II molecules. In contrast, LFA-1 and CD40 were more abundant on the smooth cell surfaces, while B7-2 (CD86) was randomly distributed. We also discovered that depletion of cholesterol, using beta-methyl-cyclodextrin, lowered the number of microvilli, indicating that intact lipid rafts are required for their expression. Moreover, activation of B lymphocytes by lipopolysaccharide (LPS) induced increased expression of GM(1), a marker for lipid rafts. However, although both surface and total levels of GM(1) were similar in B lymphocytes stimulated with either LPS or LPS plus IL-4, GM(1) was mainly expressed on microvilli in LPS plus IL-4-stimulated cells. Taken together, our results indicate that microvilli represent distinct inducible membrane domains that can regulate direct cell-cell interactions via grouping and three-dimensional presentation of cell-surface receptors.