Mechanisms of Splice Site Selection in Health and Disease

健康和疾病中剪接位点选择的机制

• 批准号: 10585911
• 负责人: Shalini Sharma
• 金额: $ 31.6万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10585911
• 关键词: 3' Splice Site; 5' Splice Site; Acute Myelocytic Leukemia; Address; Alternative Splicing; Autoimmune Diseases; Binding; Biochemical; Biological Assay; Biological Process; Biotin; Blood Cells; CD34 gene; Chronic Myelomonocytic Leukemia; Complex; Coupled; Cystic Fibrosis; Data; Defect; Disease; Dysmyelopoietic Syndromes; Event; Excision; Foundations; G2/M Arrest; Gene Expression; Gene Mutation; Generations; Goals; Health; Hematopoiesis; Hematopoietic Neoplasms; Hematopoietic stem cells; Human; Immunophenotyping; Impairment; In Vitro; Induced Mutation; Introns; Investigation; Knowledge; Laboratories; Lead; Link; Maintenance; Mediating; Mediation; Messenger RNA; Methodology; Methods; Mission; Molecular; Muscular Dystrophies; Mutation; Myelogenous; Myeloproliferative disease; National Institute of General Medical Sciences; Neurodegenerative Disorders; Nuclear; Pathogenesis; Phenotype; Play; Process; Production; Proliferating; Proteins; Public Health; RNA Helicase; RNA Splice Sites; RNA Splicing; Regulation; Research; Role; SRSF2 gene; Shapes; Site; Small Interfering RNA; Somatic Mutation; Specificity; Spliced Genes; Spliceosome Assembly Pathway; Spliceosomes; Techniques; Testing; Therapeutic Intervention; Transcript; U1 small nuclear RNA; cell transformation; comparative; design; disease diagnosis; experimental study; growth hormone deficiency; hematopoietic differentiation; improved; insight; knock-down; mRNA Precursor; novel; reconstitution; recruit; stem; transcriptome; transcriptome sequencing

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.

项目总结 Pre-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。 RNA剪接位点配对，阻碍了对调节组成和替代RNA的机制的理解 剪接来塑造细胞转录组。重要的是，关于体细胞突变在剪接中是如何发生的，我们知之甚少。 包括SF3A1、SRSF2和U2AF1在内的因子在剪接体早期阶段调节关键决定 组装会产生髓系恶性肿瘤。拟议项目的长期目标是确定 在剪接体组装的初始步骤中保持剪接保真度的基本机制 确定与产生髓系细胞的剪接基因突变相关的分子和细胞表型 血细胞疾病。中心假说是SF3A1的相互作用，SF3A1是一种关键的3剪接位点蛋白，它可以 与其5？剪接位点合作伙伴U1小核RNA(SnRNA)的桥接在剪接位点配对和 SF3A1的突变破坏了这些功能。中心假设是根据以下初步数据得出的 揭示SF3A1与U1茎环4(SL4)之间跨内含子物理合作的Pi实验室 剪接位点配对中的SnRNA以及RNA解旋酶UAP56对这种相互作用的新的调节作用。这一假说将 通过两个特定的目的进行测试：1)确定依赖SF3A1的剪接的分子机制(S) 位点配对事件有助于剪接体的保真度并产生正常的mRNA图谱，以及2)阐明 SF3A1突变对其剪接功能的影响，并进行比较分析 人类造血干细胞和祖细胞(HSPC)上的SF3A1、U2AF1和SRSF2突变。 第一个目标的实验将描绘SF3A1和UAP56与U1 SnRNA之间的相关相互作用 以及通过重组的体外和邻近的剪接方法的剪接机械的其他组件- 依赖生物素鉴定(BioID)技术。SF3A1和UAP56对细胞基因表达谱的影响 将通过siRNA敲除后的RNA-seq进行评估。第二个目标中的实验旨在 通过重组剪接分析发现SF3A1突变对其剪接功能的影响 体外培养，并用RNA-seq方法鉴定突变诱导人HSPC的剪接异常。造血术 分化分析，体外，结合免疫表型将被用来识别异常 SF3A1突变对人HSPC表型的影响。该战略包括比较 SF3A1与SRSF2和U2AF1的突变，有望揭示分子和细胞 导致异常造血的表型缺陷。影响：完成拟议的研究将 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。