ZRSR1 cooperates with ZRSR2 in regulating splicing of U12-type introns in murine hematopoietic cells
Recurrent loss-of-function mutations of spliceosome gene, ZRSR2, occur in myelodysplastic syndromes (MDS).  Mutation/loss of ZRSR2 in human myeloid cells primarily causes impaired splicing of the U12-type introns. To investigate further the role of this splice factor in splicing and hematopoietic development, we generated mice lacking ZRSR2. Unexpectedly, Zrsr2-deficient mice developed normal hematopoiesis with no abnormalities in myeloid differentiation evident in either young or =1-year old knockout mice. Repopulation ability of Zrsr2-deficient hematopoietic stem cells was also unaffected in reconstitution assays. Myeloid progenitors lacking ZRSR2 exhibited mis-splicing of U12-type introns, however, this phenotype was moderate compared to the ZRSR2-deficient human cells. Our investigations revealed that a closely related homolog, Zrsr1, expressed in the murine hematopoietic cells, but not human, contributes to splicing of U12-type introns. Depletion of Zrsr1 in Zrsr2 KO myeloid cells exacerbated retention of the U12-type introns, thus highlighting a collective role of ZRSR1 and ZRSR2 in murine U12-spliceosome. We also demonstrate that aberrant retention of U12-type introns of MAPK9 and MAPK14 leads to their reduced protein expression. Overall, our findings highlight that both ZRSR1 and ZRSR2 are functional components of the murine U12-spliceosome, and depletion of both proteins is required to model accurately ZRSR2-mutant MDS in mice. Overall design: RNA-seq of sorted common myeloid precursors (CMP), granulocyte monocyte precursors (GMP) and megakaryocyte erythroid precursors (MEP) from three WT and three ZRSR2 KO male littermates; and murine embryonic fibroblasts (MEF) prepared from three WT and three ZRSR2 KO male embryos. RNA-seq of Lin-Kit+ murine bone marrow cells from either WT or ZRSR2 KO mice transduced with shRNA targeting murine ZRSR1 (sh1 or sh10) or control shRNA; experiment performed in duplicate.