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.

项目概要： 在后生动物中，存在令人难以置信的多样性的替代转录亚型 组成成熟全长mRNA的替代部分包括替代选择的转录。 我们对起始位点、5' UTR、蛋白质编码外显子、3' UTR 和 PolyA 位点获得了令人难以置信的见解。 调节这些替代选择的细胞条件和蛋白质因素，但这些调节事件是 通常单独研究的是这些替代选择如何协调在一起。 这部分是由于研究全长转录亚型的分子技术的限制。 读取RNA测序技术提供了一种新工具来研究转录本的替代部分是如何产生的 一起。 后生动物中的大多数基因经历选择性多聚腺苷酸化以产生选择性长度 3' 长 3' UTR 亚型在神经组织中富集，而短 3' UTR 亚型在神经组织中富集。 在增殖细胞和睾丸中，我们最近发现了长 3'UTR 的生物发生和替代。 我们发现，Dscam1 基因的剪接受到神经元中 RNA 结合蛋白 Elav 的共同调节。 我们的初步分析表明，果蝇的神经功能需要事件。 替代 3' UTR 选择的替代剪接变化会影响果蝇中的其他基因。 替代外显子选择与替代 3' 末端形成的耦合有多普遍？ 由于短读长 RNA 测序技术的局限性，此类偶联事件的全基因组范围是 我们将采用新的长读长 RNA 测序技术来发现 3'UTR 偶联的替代方案。 全基因组剪接事件由 RNA 结合蛋白（如 Elav）调节。 除了共-之外，协调 3' UTR 生物合成与选择性剪接的机制是什么？ 通过Elav对Dscam1的选择性剪接和选择性多腺苷酸化的调节，我们发现存在 Dscam1 的长 3'UTR 对于 Elav 调控的选择性剪接是必需的，这个“at”的本质是什么？ 一个可能的机制解释涉及前体 mRNA 的循环，使得 长 3' UTR 与上游剪接位点相互作用以传递 RNA 结合蛋白，我们将测试此前 mRNA。 通过绘制 RNA-RNA 相互作用、体内小基因报告分析、靶向筛选来构建循环模型， 这种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协调选择性剪接的规则和 替代性多聚腺苷酸化，协调 RNA 加工步骤以产生具有特定蛋白质的 mRNA。 编码和调控特性可能对许多细胞类型和真核生物很重要，包括 人类。