Regulation and impact of alternative splicing in biology and disease

选择性剪接在生物学和疾病中的调控和影响

• 批准号: 10405870
• 负责人: Klemens J Hertel
• 金额: $ 39.25万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-09 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10405870
• 关键词: 5' Splice Site; Address; Alternative Splicing; Biochemical; Bioinformatics; Biological; Biological Assay; Biology; Cells; Code; Defect; Disease; Elements; Essential Genes; Event; Excision; Exons; Gene Expression; Gene Expression Profile; Generations; Genes; Genetic Diseases; Genetic Transcription; Goals; Growth; Heterogeneous-Nuclear Ribonucleoproteins; Human Genetics; Human Genome; Introns; Kinetics; Knock-in; Knock-out; Label; Lead; Link; Mediating; Messenger RNA; Metabolic; Methods; Modeling; Molecular; Molecular Biology; Mutation; Outcome; Pathway interactions; Positioning Attribute; Process; Proteins; Publishing; RNA; RNA Processing; RNA Splicing; Regulation; Reporter; Repression; Research; Role; Site; Structure; Techniques; Testing; Tissue Differentiation; Transcript; Translating; Translations; U1 Small Nuclear Ribonucleoprotein; Variant; Work; cancer type; cell type; cis acting element; combinatorial; conformer; genetic regulatory protein; human disease; improved; insight; mRNA Precursor; mRNA Stability; novel; premature; programs; radioligand; reconstitution; synthetic biology; therapeutic target; tissue culture; transcriptomics

Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in splicing lead to human genetic disease, and splicing mutations in a number of genes involved in growth control have been implicated in multiple types of cancer. Insights into the basic mechanisms of pre-mRNA splicing and splice site recognition are therefore fundamental to understanding regulated gene expression and human disease. The control of alternative splicing is a highly combinatorial process, where many inputs dictate the splicing outcome for each exon. A critical feature of these regulatory mechanisms is the specific interaction of trans-acting splicing factors with cis-acting RNA elements. We use a highly integrated approach to investigate the molecular mechanisms that regulate pre-mRNA splicing. This includes knockout and knock-in tissue culture models, reconstitution assays using radioligands, transcriptomics, bioinformatics, kinetics, structure- function and biochemical techniques. In the next five years, we aim to address several outstanding challenges in the field, pursuing the following novel research directions. (1) Our demonstration that splicing regulatory proteins display highly position-dependent activities that negatively or positively influence splice site choice changed the way we think about the classical splicing activators (SR proteins) and the classical splicing repressors (hnRNPs). It is now appreciated that the context-dependent activation or repression of U1 snRNP serves as a gateway to allow the abundant U1 snRNP to fulfill its splicing function and its role to protect the pre-mRNA from premature degradation. However, it is not understood how splicing regulators achieve activation or repression of U1snRNP at the 5’ splice site. We aim to dissect the mechanisms of splicing repression by embracing multi-system approaches and by understanding the role of U1 snRNP conformers in mediating spliceosomal assembly. (2) Intron retention is an important alternative splicing pathway that has eluded extensive study. Thus, its regulation is not well-understood. The existence of inefficiently spliced introns within coding exons (exitrons) further highlights the biological importance of understanding when introns are removed efficiently and when they are not. We will decipher the rules of efficient intron removal and investigate the impact of cis-acting elements in this process using synthetic biology approaches. The argument is that the depth of the sequence variation tested in massively parallel reporter assays is far greater than the testing landscape that the human genome offers. Here, we will take advantage of our expertise in experimental molecular biology and bioinformatics. (3) It has become widely appreciated that gene expression events are highly integrated, with evidence suggesting that most pre-mRNA processing occurs co- transcriptionally. Defects in any one of these steps has been linked to disease. However, most published studies evaluate only steady-state levels of gene expression or focus only on a single step. This ignores the dynamics of gene expression steps that collectively contribute to the generation of proteins from mRNAs. Thus, it is unclear how the kinetics of RNA processing and mRNA stability translate into an endpoint gene expression signature. We have established a reliable method to metabolically label nascent RNA, which allows us to track transcripts from synthesis to degradation. Work in this project will probe how steady state mRNA levels are established, how the splicing and translation regulator SRSF1 influences mRNA dynamics and how these processes adjust as a cell undergoes transformation. The goals of our research program are to obtain a better understanding of exon recognition and alternative splicing. The new mechanistic insights will be leveraged to improve strategies to therapeutically target this essential gene expression step.

mRNA 前体剪接是大多数后生动物基因表达所需的基本过程。缺陷于 剪接导致人类遗传病，许多参与生长控制的基因发生剪接突变 与多种癌症有关。深入了解前体 mRNA 剪接的基本机制 因此，剪接位点识别对于理解受调控的基因表达和人类基因表达至关重要。 疾病。选择性剪接的控制是一个高度组合的过程，其中许多输入决定了 每个外显子的剪接结果。这些调节机制的一个关键特征是特定的相互作用 具有顺式作用RNA元件的反式作用剪接因子。我们使用高度集成的方法来调查 调节前体 mRNA 剪接的分子机制。这包括敲除和敲入组织 培养模型、使用放射性配体的重构测定、转录组学、生物信息学、动力学、结构- 功能和生化技术。未来五年，我们的目标是解决几个突出的挑战 在该领域，追求以下新颖的研究方向。 (1) 我们证明拼接监管 蛋白质表现出高度位置依赖性活性，对剪接位点选择产生负面或正面影响 改变了我们对经典剪接激活剂（SR蛋白）和经典剪接的看法 阻遏蛋白（hnRNP）。现在人们认识到 U1 snRNP 的上下文依赖性激活或抑制 作为网关，使丰富的U1 snRNP发挥其剪接功能和保护作用 过早降解的前 mRNA。然而，目前尚不清楚拼接调节器如何实现 U1snRNP 在 5' 剪接位点的激活或抑制。我们的目标是剖析剪接机制 通过采用多系统方法并了解 U1 snRNP 构象异构体在抑制中的作用 介导剪接体组装。 (2)内含子保留是一种重要的选择性剪接途径 逃避了广泛的研究。因此，其监管尚不十分清楚。低效拼接的存在 编码外显子（外显子）内的内含子进一步强调了理解什么时候的生物学重要性 内含子被有效地去除，而当它们没有被去除时。我们将破译高效内含子去除规则 并利用合成生物学方法研究顺式作用元件在此过程中的影响。这 争论的焦点是大规模平行报告基因检测中测试的序列变异的深度要大得多 比人类基因组提供的测试环境更重要。在这里，我们将利用我们的专业知识 实验分子生物学和生物信息学。 (3) 基因表达已被广泛认识 事件是高度整合的，有证据表明大多数前 mRNA 加工同时发生 转录地。这些步骤中任何一个步骤的缺陷都与疾病有关。然而，大多数已发表的 研究仅评估基因表达的稳态水平或仅关注单个步骤。这忽略了 基因表达步骤的动态，共同促进从 mRNA 生成蛋白质。 因此，尚不清楚 RNA 加工动力学和 mRNA 稳定性如何转化为终点基因 表达式签名。我们已经建立了一种可靠的方法来代谢标记新生 RNA，该方法 使我们能够跟踪从合成到降解的转录本。该项目的工作将探讨稳态如何 mRNA水平确定，剪接和翻译调节因子SRSF1如何影响mRNA动态 以及这些过程如何随着细胞的转变而调整。我们研究计划的目标是 更好地了解外显子识别和选择性剪接。新的机制见解将 可用于改进针对这一重要基因表达步骤的治疗策略。