Deficiency of ribosomal protein S19 in CD34+ cells generated by siRNA blocks erythroid development and mimics defects seen in Diamond-Blackfan anemia.

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 (BM) cells with 3 lentiviral vectors expressing small interfering RNA (siRNA) against RPS19 and 1 scrambled control vector. All vectors also express green fluorescent protein (GFP). Transduction with the siRNA vectors reduced RPS19 mRNA levels to various degrees, which resulted in erythroid defects, correlating to the degree of RPS19 down-regulation, and was rescued by expression of an siRNA-resistant RPS19 transcript. Erythroid colony formation capacity conjointly decreased with RPS19 levels in CD34(+) CB and BM cells. In liquid culture supporting erythroid differentiation, RPS19-silenced as well as DBA patient CD34(+) cells exhibited reduced proliferative capacity and impaired erythroid differentiation resulting in fewer erythroid colony-forming units (CFU-Es). When assaying myeloid development, a less pronounced influence on proliferation was seen. This study shows for the first time that RPS19 silencing decreases the proliferative capacity of hematopoietic progenitors and leads to a defect in erythroid development.

Targeted disruption of the ribosomal protein S19 gene is lethal prior to implantation.

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.

Microvilli structures on B lymphocytes: inducible functional domains?

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.