3185 – PROTEOMIC ANALYSIS OF TERMINAL ERYTHROPOIESIS IN THE SF3B1 MUTATION MOUSE MODEL

The splicing factor 3B subunit 1 (Sf3b1) is important for the early steps of RNA splicing catalysis. Mutations in SF3B1 occur in more than 80% of myelodysplastic syndrome with ring sideroblasts (MDS-RS) and in over 25% of all MDS cases. MDS-RS is characterized by progressive macrocytic anemia and ≥ 5% ring sideroblasts if an SF3B1 mutation is present. Among SF3B1 mutations, the K700E hotspot mutation is observed in over 50% of MDS patients. Murine models of mutant Sf3b1 have shown this mutation is sufficient to cause MDS and provided critical insights into how aberrant splicing affects MDS pathogenesis. However, a comprehensive assessment of the impact of mutant Sf3b1 expression on the proteome is still lacking. We used our Sf3b1K700E conditional knockin mouse model to study the effects of Sf3b1K700E on terminal erythropoiesis. Early (proerythroblasts, ProE) and intermediate (orthochromatic erythroblasts, OrthoE) stage erythroblasts were flow-sorted using forward scatter and Ter119 and CD44 surface expression. Protein expression was resolved by tandem mass spectrometry. The proteomic analysis yielded ∼4000 unique proteins expressed at both stages. Comparison of ProE and OrthoE proteomes in the wildtype (WT) condition revealed stage-specific expression of proteins in critical terminal erythropoiesis pathways including heme biosynthesis, cytoskeletal regulation, cell cycle, and translation. Analysis of protein expression in Sf3b1K700E vs. WT showed 5-7% differentially expressed proteins between genotypes (p-value< 0.05) at both stages. Further analysis of the differentially expressed proteins highlighted stage-specific dysregulated pathways in the Sf3b1 mutant compared to WT, e.g., mitochondrial dysfunction (ProE) and nonsense-mediated decay (OrthoE). Taken together, these proteomic data provide valuable insights for the study of erythropoiesis and MDS pathogenesis.