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; 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，在理解涉及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。但是，主要差距仍然存在于互动的知识中 内含子在剪接体组装的早期步骤中结束，将剪接位点配对。继续缺乏这个 知识是对调节本构的机制的改善理解的重要障碍 和替代剪接以塑造细胞转录组以及剪接因子中的体细胞突变如何涉及 在这些早期步骤中，导致髓样恶性肿瘤发病。我们的长期目标是确定 在剪接组装的早期步骤和 鉴定与髓样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与其伴侣U1小核RNA（SNRNA）的相互作用至关重要 剪接位点配对的功能和SF3A1中的突变破坏了这些功能。这个假设已经 根据我们的初步数据制定，证明 剪接位点配对中U1 snRNA的SF3A1和Stem-Loop 4（SL4）和这种相互作用的潜在调解 由RNA解旋酶UAP56。我们计划通过两个具体目的测试中心假设：1）确定 SF3A1依赖性剪接位点配对事件有助于剪接体忠诚度的分子机制 并产生正常的mRNA轮廓，2）确定SF3A1突变对其剪接函数的影响 并对SF3A1，U2AF1和SRSF2对人造血的突变的影响进行比较分析 茎和祖细胞。在第一个目标中，我们将描述SF3A1和UAP56与U1 snRNA的相互作用 以及通过体外重构剪接方法和接近度的剪接机械组件的其他组件 依赖的生物素鉴定（生物体）技术。我们将确定SF3A1和UAP56对蜂窝的影响 siRNA敲低的mRNA曲线，然后是RNA-Seq。在第二个目标中，我们将确定 SF3A1突变对剪接功能的后果通过体外重构剪接测定并识别 RNA-Seq在人HSPC中突变诱导的剪接畸变。我们将利用体内造血 通过分化测定来确定SF3A1突变对人HSPC的异常表型作用 免疫表型。我们的策略包括将SF3A1中髓样疾病突变的影响与 在SRSF2和U2AF1中 髓样病发病机理。拟议研究的完成将提高我们对 控制内含子去除和 剪接因子突变如何损害剪接位点配对，从而导致剪接改变并可能揭幕 可以利用用于治疗干预的生化界面。