Scope and mechanism of coordinated alternative splicing and alternative polyadenylation

协调选择性剪接和选择性多腺苷酸化的范围和机制

• 批准号: 10690936
• 负责人: Pedro Miura
• 金额: $ 28.31万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-16 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10690936
• 关键词: Scope; mechanism; coordinated; alternative; splicing

Project Summary: In metazoan animals there is an incredible diversity of alternative transcript isoforms generated from genes. The alternative parts making up a mature full length mRNA include alternatively selected transcription start sites, 5′ UTRs, protein-coding exons, 3′ UTRs, and polyA sites. We have gained incredible insights into the cellular conditions and protein factors that regulate these alternative choices, but these regulatory events are usually studied in isolation. What is far less understood is how these alternative choices are coordinated together. This is partly because of limitations in the molecular techniques to study full length transcript isoforms. Long- read RNA sequencing technologies present a new tool to study how the alternative parts of a transcript come together. The majority of genes in metazoans undergo alternative polyadenylation to produce alternative length 3′ UTR isoforms. Long 3′ UTR isoforms are enriched in neural tissues, whereas short 3′ UTR isoforms are enriched in proliferating cells and in testis. In Drosophila, we recently identified that long 3′ UTR biogenesis and alternative splicing of the Dscam1 gene are co-regulated by the RNA-binding protein Elav in neurons. We found that these events are required for neuronal function in Drosophila. Our preliminary analysis shows that this coupling of alternative splicing changes to alternative 3′ UTR selection affects other genes in Drosophila. How widespread is the coupling of alternative exon selection to alternative 3′ end formation? Due to limitations in short-read RNA sequencing technologies, the genome-wide scope of such coupling events is unknown. We will employ new long read RNA-sequencing technologies to uncover 3′ UTR-coupled alternative splicing events genome-wide that are regulated by RNA-binding proteins such as Elav. What is the mechanism that coordinates 3′ UTR biogenesis with alternative splicing? In addition to co- regulation of alternative splicing and alternative polyadenylation of Dscam1 by Elav, we found that the presence of the Dscam1 long 3′ UTR was necessary for Elav-regulated alternative splicing. What is the nature of this “at a distance” regulatory event? A possible mechanistic explanation involves looping of the pre-mRNA such that the long 3′ UTR interacts with upstream splice sites to deliver RNA-binding proteins. We will test this pre-mRNA looping model through mapping RNA-RNA interactions, in vivo mini-gene reporter analyses, targeted screens, and RNA-affinity chromatography. This mechanistic investigation will be expanded to other genes and cell types. The proposed research program will identify the rules governing coordinated alternative splicing and alternative polyadenylation. The coordination of RNA processing steps to produce mRNAs with specific protein- coding and regulatory properties is likely important for many cell types and eukaryotic organisms, including humans.

项目总结： 在后生动物中，有令人难以置信的多样性的可供选择的转录异构体，由 基因。组成成熟全长信使核糖核酸的替代部分包括交替选择的转录本 起始位点、5‘端非编码区、蛋白质编码外显子、3’端非编码区和Polya位点。我们已经获得了令人难以置信的对 调节这些替代选择的细胞条件和蛋白质因素，但这些调节事件 通常是在孤立的环境中学习。人们对这些替代选择是如何协调在一起的了解要少得多。 这在一定程度上是因为研究全长转录本亚型的分子技术的局限性。长- Read RNA测序技术为研究转录本的替代部分是如何产生的提供了一种新的工具 在一起。 后生动物中的大多数基因都经历了交替的多聚腺苷酸化，以产生不同长度的3‘ Utr亚型。长3‘非编码区异构体在神经组织中丰富，而短3’非编码区异构体在神经组织中富集性 在增殖细胞和睾丸中。在果蝇中，我们最近发现了长3‘非编码区的生物发生和替代 Dscam1基因的剪接受到神经元中RNA结合蛋白Elav的共同调控。我们发现这些 事件是果蝇神经功能所必需的。我们的初步分析表明，这种耦合 选择性剪接改变为选择性3‘非编码区选择会影响果蝇的其他基因。 选择性外显子选择与选择性3‘末端形成的偶联有多普遍？由于 短读RNA测序技术的局限性，这种偶联事件的全基因组范围是 未知。我们将使用新的长读RNA测序技术来发现3‘非编码区偶联的替代方案 受RNA结合蛋白(如Elav)调控的全基因组剪接事件。 用选择性剪接协调3‘非编码区生物发生的机制是什么？除了共同- Elav对Dscam1选择性剪接和选择性多聚腺苷酸化的调节，我们发现 对于Elav调控的选择性剪接来说，Dscam1长的3‘非编码区是必需的。这个“at”的性质是什么？ 一个“距离”的监管事件？一种可能的机制解释涉及前信使核糖核酸的循环，从而 长3‘非编码区与上游剪接位点相互作用，传递RNA结合蛋白。我们将测试这个前信使核糖核酸 通过绘制RNA-RNA相互作用的循环模型，体内微基因报告分析，靶向筛选， 和RNA亲和层析。这一机制研究将扩展到其他基因和细胞类型。 拟议的研究计划将确定管理协调替代剪接和 交替多聚腺苷。RNA加工步骤的协调，以产生具有特定蛋白质的mRNA- 编码和调节特性可能对许多细胞类型和真核生物很重要，包括 人类。