PROJECT SUMMARY In the last decade, significant progress has been made in the understanding of core components involved in splicing catalysis in mature spliceosome. However, major gaps remain in the knowledge of interactions that bring intron ends together to pair splice sites in the early steps of spliceosome assembly. Continued lack of this knowledge is a significant impediment to an improved understanding of mechanisms that regulate constitutive and alternative splicing to shape the cellular transcriptome and how somatic mutations in splicing factors involved in these early steps cause pathogenesis in myeloid malignancies. Our long-term goal is to determine fundamental mechanisms in maintenance of splicing fidelity in the early steps of spliceosome assembly and identify molecular and cellular phenotypes associated with mutations in splicing genes in myeloid diseases. Our central hypothesis is that interactions of SF3A1 with its partner, the U1 small nuclear RNA (snRNA), have crucial functions in splice site pairing and that mutations in SF3A1 disrupt these functions. This hypothesis has been formulated on the basis of our preliminary data demonstrating the role of the cross-intron interaction between SF3A1 and the stem-loop 4 (SL4) of the U1 snRNA in splice site pairing and potential mediation of this interaction by the RNA helicase UAP56. We plan to test our central hypothesis through two specific aims: 1) Determine the molecular mechanism(s) whereby SF3A1-dependent splice site pairing events contribute to spliceosome fidelity and generate normal mRNA profiles, and 2) Determine the impact of SF3A1 mutations on its splicing functions and perform comparative analysis of effects of mutations in SF3A1, U2AF1, and SRSF2 on human hematopoietic stem and progenitor cells. In the first aim, we will delineate the interactions of SF3A1 and UAP56 with U1 snRNA and other components of the splicing machinery by in vitro reconstituted splicing methods and the proximity- dependent biotin identification (BioID) technique. We will determine the impact of SF3A1 and UAP56 on cellular mRNA profiles by siRNA knockdown followed by RNA-seq. In the second aim, we will determine the consequences of SF3A1 mutations on its splicing functions by in vitro reconstituted splicing assays and identify mutation-induced splicing aberrations in human HSPCs by RNA-seq. We will employ ex vivo hematopoietic differentiation assays to identify the abnormal phenotypic effects of SF3A1 mutations on human HSPCs by immunophenotyping. Our strategy includes comparing the influence of myeloid disease mutations in SF3A1 with those in SRSF2 and U2AF1 and is expected to reveal molecular and cellular phenotypic defects that underlie myeloid disease pathogenesis. Completion of the proposed research will advance our understanding of interactions between core spliceosomal components that govern the commitment of an intron to removal and how splicing factor mutations impair splice site pairing lead...