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: 10808389
• 负责人: Shalini Sharma
• 金额: $ 1.02万
• 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/10808389
• 关键词: Alternative Splicing; Autoimmune Diseases; Binding; Biochemical; Biological Assay; Biological Process; Biotin; Coupled; Cystic Fibrosis; Data; Defect; Disease; Event; Excision; Foundations; Gene Expression; Gene Mutation; Goals; Health; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic stem cells; Human; Immunophenotyping; Impairment; In Vitro; Induced Mutation; Introns; Knowledge; Laboratories; Link; Mediating; Mediation; Messenger RNA; Methods; Mission; Molecular; Muscular Dystrophies; Mutation; Myelogenous; Myeloproliferative disease; National Institute of General Medical Sciences; Neurodegenerative Disorders; Pathogenesis; Phenotype; Play; Proteins; Public Health; RNA Helicase; RNA Splicing; Research; Role; SRSF2 gene; Site; Small Interfering RNA; Somatic Mutation; Spliced Genes; Spliceosome Assembly Pathway; Spliceosomes; Techniques; Testing; Therapeutic Intervention; U1 small nuclear RNA; Work; comparative; design; disease diagnosis; experimental study; growth hormone deficiency; improved; knock-down; mRNA Precursor; novel; stem; transcriptome sequencing

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.

项目摘要 前体mRNA剪接的一个关键特征是在剪接的早期阶段配对剪接位点的加工步骤。 剪接体组装。然而，在对决定性的特定分子相互作用的认识方面仍然存在重大差距。 剪接位点配对，阻碍了对组成性剪接和选择性剪接调节机制的理解。 重要的是，对于剪接因子（包括SF 3A 1、SRSF 2和 U2 AF 1在剪接体组装的早期阶段介导关键决定，产生骨髓恶性肿瘤。 该项目的长期目标是确定维持剪接的基本机制 在剪接体组装的初始步骤中的保真度，并鉴定分子和细胞表型 与产生骨髓性血液疾病的剪接基因突变有关。核心假设 SF 3A 1是一个关键3个剪接位点蛋白，它与其5个剪接位点的配偶体U1小分子连接， 核RNA（snRNA）在剪接位点配对中起着至关重要的作用，SF 3A 1的突变破坏了这些作用。 功能协调发展的中心假设来自PI实验室的初步数据，这些数据揭示了交叉- SF 3A 1与U1 snRNA茎环4（SL 4）内含子在剪接位点配对中的协同作用及新的 RNA解旋酶UAP 56介导这种相互作用。这一假设将通过两个具体目标进行检验：1） 确定SF 3A 1依赖性剪接位点配对事件促成 剪接体保真度和产生正常的mRNA谱，和2）阐明SF 3A 1突变对 它的剪接功能，并进行SF 3A 1，U2 AF 1， SRSF 2对人造血干细胞和祖细胞（HSPC）的作用。在第一个实验的目的，将描绘 SF 3A 1和UAP 56与U1 snRNA和剪接机制的其他组分之间的相互作用， 体外剪接方法和邻近依赖性生物素鉴定（BioID）技术。SF 3A 1的作用 和UAP 56对细胞mRNA谱的影响将通过siRNA敲低随后RNA-seq来评估。 第二个目的的实验旨在发现SF 3A 1突变对其剪接的影响 通过体外剪接试验发挥作用，并通过以下方法鉴定人HSPC中突变诱导的剪接畸变 RNA测序将采用造血离体分化测定，结合免疫表型分析， 鉴定SF 3A 1突变对人HSPC的异常表型效应。该战略包括比较 SF 3A 1突变与SRSF 2和U2 AF 1突变的影响，并有望揭示分子和 导致异常造血的细胞表型缺陷。影响：完成拟议工作 将解开核心剪接体组件之间的相互作用网络，这些组件控制着一个人的承诺。 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，可能揭示可用于治疗干预的生化界面。

项目成果

Sequence-specific RNA recognition by an RGG motif connects U1 and U2 snRNP for spliceosome assembly.

• DOI: 10.1073/pnas.2114092119
• 发表时间: 2022-02-08
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: de Vries T;Martelly W;Campagne S;Sabath K;Sarnowski CP;Wong J;Leitner A;Jonas S;Sharma S;Allain FH
• 通讯作者: Allain FH