RNA-seq analysis of scat (CbyJ.Cby-Rasa3scat/Llp) and matched control mice in bone marrow and spleen

RASA3 (RAS p21 protein activator 3), also called GAPIII or IP4BP (inositol 1,3,4,5-tetrakisphosphate, IP4, binding protein), is a member of the GAP1 family of RAS-GTPase-activating proteins (GAPs). The RAS superfamily of small GTPases includes the subfamily RAP. Small GTPases act as molecular switches, cycling between active GTP-bound and inactive GDP-bound forms. They are activated by guanine nucleotide exchange factors (GEFs), which stimulate GTP loading, and inactivated by GAPs, which accelerate GTP hydrolysis. A major role of RASA3 in hematopoiesis was first identified upon positional cloning of the co-isogenic autosomal recessive mouse mutation, scat (severe combined anemia and thrombocytopenia. The scat phenotype, in addition to severe anemia and thrombocytopenia, includes significant leukopenia as well. The scat disease progresses episodically, with periods of severe crisis interspersed with one or two periods of remission. The RASA3 mutation (G125V) in scat causes mislocalization of RASA3 to the cytosol, abrogating RASA3 GAP activity and increasing active RAS levels in scat erythroid cells. As part of a broader study to analyze the role of RASA3 in hematopoiesis, we performed RNAseq studies to generate hypotheses regarding the progression of the scat disease from periods of crisis to partial remission. We performed RNAseq using 3-5 biological replicates of whole spleen and bone marrow and flow-sorted HSPC populations from scat homozygotes mice in crisis and their WT littermates. Furthermore, as analysis of transcriptome changes occurring upon partial recovery in scat may suggest targets for disease therapy, we also compared the same tissue/cell populations from scat crisis (cr) and scat partial remission (pr) mice. Mice were classified as pr based on erythroid parameters intermediate to WT and cr with the exception of the reticulocyte percentage and spleen weight, which are expected to remain high during recovery from anemia. Flow-sorted populations obtained were megakaryocyte-erythroid progenitors (MEP) and so-called “stem and myeloid progenitors” (SMP). Because Sca1 (Ly6a) is expressed at very low levels in BALB mice, HSCs cannot be separated reliably from common myeloid progenitors. Therefore, the Lin-Sca-Kit+CD34+, CD16_32lo SMP population was used for these studies.