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: 10797150
• 负责人: Pedro Miura
• 金额: $ 18.43万
• 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/10797150
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Affect; Affinity Chromatography; Alternative Splicing; Animals; Biogenesis; Cell Proliferation; Code; Coupled; Coupling; Drosophila genus; ELAV protein; Event; Exons; Genes; Human; Investigation; Length; Maps; Messenger RNA; Modeling; Molecular; Nature; Neurons; Organism; Poly A; Polyadenylation; Property; Protein Isoforms; Proteins; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Reporter; Research; Site; Techniques; Technology; Testing; Testis; Tissues; Transcript; Transcription Initiation Site; cell type; genome-wide; in vivo; insight; mRNA Precursor; neural; programs; tool; transcriptome sequencing

Project Summary: (as submitted in original application) 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.

项目摘要：（原始申请中提交） 在后生动物中，从 基因。组成成熟全长mRNA的替代零件包括选择转录 启动位点，5'UTR，蛋白质编码外显子，3'UTR和Polya位点。我们对 调节这些替代选择的细胞条件和蛋白质因子，但是这些调节事件是 通常被孤立地研究。远不止理解的是如何将这些替代选择共同协调。 这部分是由于分子技术研究全长转录本的局限性。长的- 读取RNA测序技术提出了一种新工具，用于研究成绩单的替代部分如何出现 一起。 后生动物中的大多数基因经历替代的聚腺苷酸化，产生替代长度3' UTR同工型。长3'UTR同工型富含神经组织，而短3'UTR同工型富含 在增殖细胞和睾丸中。在果蝇中，我们最近确定了3'UTR生物发生和替代 DSCAM1基因的剪接由神经元中的RNA结合蛋白ELAV共同调节。我们发现这些 果蝇中神经元功能需要事件。我们的初步分析表明，这种耦合 替代3'UTR选择的替代剪接会影响果蝇中的其他基因。 替代外显子选择与替代3'末端形成的耦合如何普遍？由于 短读RNA测序技术的局限性，此类耦合事件的全基因组范围是 未知。我们将采用新的长阅读RNA测序技术来发现3'UTR耦合的替代方案 剪接事件全基因组受RNA结合蛋白（例如Elav）调节。 与替代剪接协调3'UTR生物发生的机制是什么？除了共同 通过Elav调节DSCAM1的替代剪接和替代聚烯基化，我们发现存在 DSCAM1长3'utr是伊拉夫调节的替代剪接所必需的。这个“在 距离”调节事件？可能的机械解释涉及循环前mRNA，以至于 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 通过映射RNA-RNA相互作用的循环模型，体内迷你基因报道分析，目标屏幕， 和RNA亲和色谱。该机械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有关协调替代拼接的规则，并 替代聚腺苷酸化。 RNA处理步骤的配位以产生具有特定蛋白质的mRNA 编码和调节性能对于许多细胞类型和真核生物可能很重要，包括 人类。