PROJECT SUMMARYA key feature in the splicing of pre-mRNA is the processing step that pair splice sites during the early stages ofspliceosome assembly. Yet major gaps remain in the knowledge of specific molecular interactions that governRNA splice site pairing impeding understanding of mechanisms that regulate constitutive and alternative RNAsplicing to shape the cellular transcriptome. Importantly little is known as to how somatic mutations in splicingfactors including SF3A1 SRSF2 and U2AF1 mediating key decisions in the early stages of spliceosomeassembly produce myeloid malignancies. The long-term goal of the proposed project is to determinefundamental mechanisms that maintain splicing fidelity during the initial steps of spliceosome assembly and toidentify molecular and cellular phenotypes associated with splicing gene mutations that generate myelogenousblood cell diseases. The central hypothesis is that interactions of SF3A1 a pivotal 3-splice site protein thatbridges to its 5-splice site partner U1 small nuclear RNA (snRNA) plays crucial roles in splice site pairing andthat mutations in SF3A1 disrupt these functions. The central hypothesis is derived from preliminary data fromthe PIs laboratory which reveal cross-intron physical cooperation between SF3A1 and stem-loop 4 (SL4) of U1snRNA in splice site pairing and novel mediation of this interplay by RNA helicase UAP56. This hypothesis willbe tested via two specific aims: 1) Determine the molecular mechanism(s) whereby SF3A1-dependent splicesite pairing events contribute to spliceosome fidelity and generate normal mRNA profiles and 2) Elucidate theimpact of SF3A1 mutations on its splicing functions and perform a comparative analysis of the influence ofmutations 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 snRNAand 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 profileswill be assessed by siRNA knockdown followed by RNA-seq. Experiments in the second aim are designed todiscover the consequences of SF3A1 mutations on its splicing functions by reconstituted splicing assays invitro and to identify mutation-induced splicing aberrations in human HSPCs by RNA-seq. Hematopoieticdifferentiation assays ex vivo coupled with immunophenotyping will be employed to identify abnormalphenotypic effects of SF3A1 mutations on human HSPCs. The strategy includes comparing the influence ofmutations in SF3A1 with those in SRSF2 and U2AF1 and is expected to reveal molecular and cellularphenotypic defects that underlie abnormal hematopoiesis. Impact: Completion of the proposed research willunravel the network of interactions between core spliceosomal components that govern commitment of anintron to removal and reveal how splicing factor mutations impair splice site pairing and lead to splicingalteration potentially unveiling biochemical interfaces that can be exploited for therapeutic intervention.