PROJECT SUMMARY A key feature in the splicing of pre-mRNA is the processing step that pair splice sites during the early stages of spliceosome assembly. Yet, major gaps remain in the knowledge of specific molecular interactions that govern RNA splice site pairing, impeding understanding of mechanisms that regulate constitutive and alternative RNA splicing to shape the cellular transcriptome. Importantly, little is known as to how somatic mutations in splicing factors, including SF3A1, SRSF2, and U2AF1, mediating key decisions in the early stages of spliceosome assembly produce myeloid malignancies. The long-term goal of the proposed project is to determine fundamental mechanisms that maintain splicing fidelity during the initial steps of spliceosome assembly and to identify molecular and cellular phenotypes associated with splicing gene mutations that generate myelogenous blood cell diseases. The central hypothesis is that interactions of SF3A1, a pivotal 3-splice site protein that bridges to its 5-splice site partner, U1 small nuclear RNA (snRNA), plays crucial roles in splice site pairing and that mutations in SF3A1 disrupt these functions. The central hypothesis is derived from preliminary data from the PI?s laboratory which reveal cross-intron physical cooperation between SF3A1 and stem-loop 4 (SL4) of U1 snRNA in splice site pairing and novel mediation of this interplay by RNA helicase UAP56. This hypothesis will be tested via 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) Elucidate the impact of SF3A1 mutations on its splicing functions and perform a comparative analysis of the influence of mutations in SF3A1, U2AF1, and SRSF2 on human hematopoietic stem and progenitor cells (HSPCs). Experiments in the first aim, will delineate relevant interactions between SF3A1 and UAP56 with U1 snRNA and other components of the splicing machinery via reconstituted splicing methodology, in vitro, and proximity- dependent biotin identification (BioID) technique. The action of SF3A1 and UAP56 on cellular mRNA profiles will be assessed by siRNA knockdown followed by RNA-seq. Experiments in the second aim are designed to discover the consequences of SF3A1 mutations on its splicing functions by reconstituted splicing assays, in vitro, and to identify mutation-induced splicing aberrations in human HSPCs by RNA-seq. Hematopoietic differentiation assays, ex vivo, coupled with immunophenotyping, will be employed to identify abnormal phenotypic effects of SF3A1 mutations on human HSPCs. The strategy includes comparing the influence of mutations in SF3A1 with those in SRSF2 and U2AF1 and is expected to reveal molecular and cellular phenotypic defects that underlie abnormal hematopoiesis. Impact: Completion of the proposed research will unravel the network of interactions between core spliceosomal components that govern commitment of an intron to removal and reveal how splicing factor mutations impair splice site pairing and lead to splicing alteration, potentially unveiling biochemical interfaces that can be exploited for therapeutic intervention.