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: 10797554
• 负责人: Shalini Sharma
• 金额: $ 11.83万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10797554
• 关键词: Alternative Splicing; Autoimmune Diseases; Biochemical; Biological Assay; Biological Process; Biotin; Catalysis; Cystic Fibrosis; Data; Defect; Disease; Event; Excision; Foundations; Gene Expression; Goals; Health; Hematopoietic Neoplasms; Hematopoietic stem cells; Human; Immunophenotyping; Impairment; In Vitro; Induced Mutation; Introns; Knowledge; Link; Maintenance; Mediation; Messenger RNA; Methods; Mission; Molecular; Muscular Dystrophies; Mutation; Myeloproliferative disease; National Institute of General Medical Sciences; Neurodegenerative Disorders; Pathogenesis; Phenotype; Public Health; RNA Helicase; RNA Splicing; Research; Role; SRSF2 gene; Shapes; Site; Small Interfering RNA; Somatic Mutation; Spliced Genes; Spliceosome Assembly Pathway; Spliceosomes; Techniques; Testing; Therapeutic Intervention; U1 small nuclear RNA; comparative; disease diagnosis; growth hormone deficiency; hematopoietic differentiation; improved; knock-down; mRNA Precursor; reconstitution; stem; transcriptome; transcriptome sequencing

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.

项目摘要 在过去的十年中，在了解参与的核心组成部分方面取得了重大进展， 成熟剪接体中的剪接催化作用。然而，在相互作用的知识方面仍然存在重大差距， 在剪接体组装的早期步骤中，内含子末端在一起以配对剪接位点。持续缺乏这种 知识是一个重要的障碍，以提高对机制的理解，调节组成 以及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如何参与 在这些早期步骤中引起骨髓恶性肿瘤的发病机制。我们的长期目标是确定 在剪接体组装的早期步骤中维持剪接保真度的基本机制， 鉴定与骨髓疾病中剪接基因突变相关的分子和细胞表型。我们 中心假设是SF 3A 1与其配偶体U1小核RNA（snRNA）的相互作用， 在剪接位点配对的功能和SF 3A 1突变破坏这些功能。这一假设一直被 我们的初步数据表明，跨内含子之间的相互作用的作用， SF 3A 1和U1 snRNA的茎环4（SL 4）在剪接位点配对中的作用以及这种相互作用的潜在介导 RNA解旋酶UAP 56我们计划通过两个具体目标来测试我们的中心假设：1）确定 SF 3A 1依赖性剪接位点配对事件有助于剪接体保真度的分子机制 并产生正常的mRNA谱，以及2）确定SF 3A 1突变对其剪接功能的影响 并对SF 3A 1、U2 AF 1和SRSF 2突变对人类造血功能的影响进行比较分析， 干细胞和祖细胞。在第一个目标中，我们将描述SF 3A 1和UAP 56与U1 snRNA的相互作用 和剪接机制的其他组分，通过体外重组剪接方法和邻近- 生物素依赖性鉴定（BioID）技术。我们将确定SF 3A 1和UAP 56对细胞的影响。 通过siRNA敲低随后RNA-seq的mRNA谱。在第二个目标中，我们将确定 通过体外重组剪接试验，研究SF 3A 1突变对其剪接功能的影响，并鉴定 通过RNA-seq在人HSPC中突变诱导的剪接畸变。我们将采用体外造血技术 分化试验，以通过以下方法鉴定SF 3A 1突变对人HSPC的异常表型效应： 免疫分型我们的策略包括比较SF 3A 1髓系疾病突变与 SRSF 2和U2 AF 1中的那些，并有望揭示导致这些疾病的分子和细胞表型缺陷。 髓系疾病发病机制。完成拟议的研究将增进我们对 核心剪接体组件之间的相互作用，控制内含子的承诺，以消除和 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 可以用于治疗干预的生物化学界面。