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前剪接是表达大多数后生基因所需的基本过程。缺陷 剪接导致人类遗传疾病，并在许多参与生长控制的基因中剪接突变 在多种类型的癌症中隐含。洞悉前MRNA剪接的基本机制 因此 疾病。替代剪接的控制是一个高度组合过程，许多输入决定了 每个外显子的剪接结果。这些调节机制的关键特征是 带有顺式作用RNA元件的反式剪接因子。我们使用高度集成的方法来调查 调节前MRNA剪接的分子机制。这包括敲除和敲门组织 文化模型，使用放射性配体，转录组学，生物信息学，动力学，结构的重构测定 - 功能和生化技术。在接下来的五年中，我们旨在应对一些杰出的挑战 在该领域，追求以下新颖的研究方向。 （1）我们的剪接调节的演示 蛋白质表现出高度依赖性的活动，对剪接站点选择负面影响 改变了我们思考经典剪接激活剂（SR蛋白）和经典剪接的方式 阻遏物（HNRNP）。现在认为，U1 SNRNP的上下文依赖性激活或表达 充当允许丰富的U1 SNRNP履行其剪接功能的门户及其保护的作用 过早降解的前MRNA。但是，它不了解剪接调节器如何实现 U1SNRNP在5'剪接位点的激活或表达。我们旨在剖析剪接的机制 通过拥抱多系统方法并了解U1 SNRNP构象体中的作用来镇压 介导剪接组件。 （2）内含子保留是具有重要的替代剪接途径 广泛的研究。那是不充分理解的。剪接效率低下的存在 编码外显子内的内含子（出口）进一步强调了理解何时的生物学重要性 内含子被有效地卸下，而没有内含子。我们将破译有效内含子删除的规则 并使用合成生物学方法在此过程中研究了顺式作用元件的影响。 论点是，在大规模平行的记者测定中测试的序列变化的深度要大得多 比人类基因组提供的测试景观。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）基因表达已被广泛理解 事件是高度整合的，有证据表明大多数MRNA处理发生的共同 转录。这些步骤中的任何一项缺陷都与疾病有关。但是，大多数发表 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 基因表达步骤的动力学，共同有助于从mRNA中产生蛋白质。 这是尚不清楚RNA处理和mRNA稳定性的动力学如何转化为终点基因 表达签名。我们已经建立了一种可靠的方法来代谢标记新生的RNA，它 允许我们跟踪从合成到降解的成绩单。在这个项目中工作将探讨如何稳定状态 建立mRNA水平，剪接和翻译调节剂SRSF1如何影响mRNA动力学 以及这些过程如何随着单元的经历而调整。我们的研究计划的目标是 更好地了解外显子识别和替代剪接。新的机械见解将 要杠杆化以改善治疗方法的策略。