Mechanisms of Splice Site Selection in Health and Disease

项目总结 前mRNA剪接的一个关键特征是在早期阶段配对剪接位点的处理步骤 剪接体组装。然而，在支配特定分子相互作用的知识方面仍然存在重大差距。 剪接位点配对，阻碍了对调节结构性和选择性剪接的机制的理解。 重要的是，关于剪接因子的体细胞突变，包括SF3A1，SRSF2，和 U2AF1，介导剪接体组装早期阶段的关键决定，产生髓系恶性肿瘤。 拟议项目的长期目标是确定维持剪接的基本机制 剪接体组装初期的保真度和鉴定分子和细胞表型 与导致骨髓性血液疾病的剪接基因突变有关。中心假说 是SF3A1相互作用吗？SF3A1是一个关键的3-剪接位点蛋白质，桥接到它的5-剪接位点伙伴U1小 核RNA(SnRNA)，在剪接位点配对中起关键作用，而SF3A1的突变破坏了这些 功能。中心假设是从PI实验室的初步数据中得出的，这些数据揭示了 U1单链RNA的SF3A1和茎环4(SL4)在剪接位点配对中的内含子协作和新 RNA解旋酶UAP56对这种相互作用的调节。这一假设将通过两个具体目标进行检验：1) 确定依赖SF3A1的剪接位点配对事件参与的分子机制(S) 剪接体的保真度和产生正常的mRNA图谱，以及2)阐明SF3A1突变对 它的剪接功能，并对SF3A1、U2AF1和 SRSF2对人造血干祖细胞(HSPC)的影响。实验的第一个目的，将勾勒出 SF3A1和UAP56通过In与U1 SnRNA和剪接机械的其他组件相互作用 体外剪接方法和邻近依赖生物素鉴定(BioID)技术。SF3A1的作用 和UAP56在细胞mRNA谱上的作用将通过siRNA敲除和RNA-seq来评估。 第二个目标的实验旨在发现SF3A1突变对其剪接的影响 通过体外剪接试验鉴定突变诱导的人HSPC的剪接异常 RNA序列造血体外分化试验，结合免疫表型，将用于 确定SF3A1突变对人类HSPC的异常表型效应。策略包括比较 SF3A1突变与SRSF2和U2AF1突变的影响有望揭示分子和 细胞表型缺陷是异常造血的基础。影响：拟议工作完成 将解开核心剪接体组件之间的相互作用网络，这些组件支配着 内含子去除并揭示剪接因子突变如何损害剪接位点配对并导致剪接 改变，潜在地揭示了可用于治疗干预的生化界面。

PROJECT SUMMARY A key feature in pre-mRNA splicing is the processing step that pairs splice sites during the early stages of spliceosome assembly. Yet, major gaps remain in the knowledge of specific molecular interactions that govern splice site pairing, impeding understanding of mechanisms that regulate constitutive and alternative splicing. 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 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 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 interactions between SF3A1 and UAP56 with U1 snRNA and other components of the splicing machinery via in vitro splicing methods, 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 in vitro splicing assays, and to identify mutation-induced splicing aberrations in human HSPCs by RNA-seq. Hematopoietic ex vivo differentiation assays, 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 work 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.