Mechanisms of Splice Site Selection in Health and Disease

项目总结 在过去十年中，在对所涉核心组成部分的了解方面取得了重大进展 在成熟剪接体中的剪接催化作用。然而，在对相互作用的了解方面仍然存在重大差距 在剪接体组装的早期步骤中，内含子末端一起配对剪接点。继续缺乏这一点 知识是改善对调节构成成分的机制的理解的一个重大障碍 和选择性剪接来塑造细胞转录组，以及剪接因子中的体细胞突变是如何参与的 在这些早期步骤中，导致髓系恶性肿瘤的发病机制。我们的长期目标是确定 剪接体组装和早期步骤中维持剪接保真度的基本机制 鉴定与髓系疾病中剪接基因突变相关的分子和细胞表型。我们的 中心假设是SF3A1与其伙伴U1小核RNA(SnRNA)的相互作用对 剪接位点配对的功能和SF3A1的突变破坏了这些功能。这一假设一直是 基于我们的初步数据，证明了跨内含子相互作用在 SF3A1和U1单链RNA的茎环4(SL4)在剪接位点配对中的作用及其可能的调节 通过RNA解旋酶UAP56。我们计划通过两个具体目标来检验我们的中心假设：1)确定 依赖于SF3A1的剪接位点配对事件促进剪接体保真度的分子机制(S) 以及2)确定SF3A1突变对其剪接功能的影响 并比较分析SF3A1、U2AF1和SRSF2基因突变对人类造血的影响 干细胞和祖细胞。在第一个目标中，我们将描述SF3A1和UAP56与U1 SnRNA的相互作用 以及通过体外重组剪接方法和接近性- 依赖生物素鉴定(BioID)技术。我们将确定SF3A1和UAP56对细胞的影响 通过siRNA敲除和RNA-seq.在第二个目标中，我们将确定 用体外重组剪接实验鉴定SF3A1突变对其剪接功能的影响 用rna-seq研究突变诱导人HSPC的剪接异常。我们将采用体外造血术 分化试验鉴定SF3A1突变对人HSPC的异常表型效应 免疫表型。我们的策略包括比较髓系疾病SF3A1突变与 SRSF2和U2AF1中的这些基因，预计将揭示潜在的分子和细胞表型缺陷 髓系疾病的发病机制。拟议研究的完成将促进我们对 核心剪接体组件之间的相互作用，这些剪接体组件控制内含子对移除和 剪接因子突变如何损害剪接位点配对导致剪接改变并可能揭示 可用于治疗干预的生化界面。

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 leading to splicing alteration and possibly unveil biochemical interfaces that can be exploited for therapeutic intervention.