Scope and mechanism of coordinated alternative splicing and alternative polyadenylation

项目摘要： 在后生动物中，有一个令人难以置信的多样性的替代转录异构体产生的， 基因.构成成熟全长mRNA的替代部分包括替代选择的转录 起始位点、5′ UTR、蛋白编码外显子、3′ UTR和polyA位点。我们已经获得了令人难以置信的洞察力， 调节这些替代选择的细胞条件和蛋白质因子，但这些调节事件是 通常被孤立地研究。人们对这些替代选择是如何协调在一起的了解要少得多。 这部分是由于研究全长转录异构体的分子技术的局限性。长- 阅读RNA测序技术提供了一种新的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交替的多聚腺苷酸化以产生交替的长度3′， UTR同种型。长的3′ UTR异构体在神经组织中富集，而短的3′ UTR异构体在神经组织中富集。 在增殖细胞和睾丸中。在果蝇中，我们最近发现，长3′ UTR的生物发生和替代， Dscam 1基因的剪接受神经元中RNA结合蛋白Elav的共同调节。我们发现这些 事件是果蝇神经元功能所必需的。我们的初步分析表明， 选择性剪接改变选择性3′ UTR影响果蝇中的其他基因。 选择性外显子选择与选择性3′端形成的耦合有多普遍？由于 由于短读段RNA测序技术的局限性，这种偶联事件的全基因组范围是 未知我们将采用新的长读段RNA测序技术来发现3′ UTR偶联的替代方案， 剪接事件全基因组，由RNA结合蛋白如Elav调节。 3′ UTR生物合成与选择性剪接的协调机制是什么？除了Co- Elav对Dscam 1的选择性剪接和选择性多聚腺苷酸化的调节，我们发现， Dscam 1长3′ UTR的缺失是Elav调控的可变剪接所必需的。这是什么性质的“at “远距离”监管事件？一个可能的机制解释涉及前mRNA的循环， 长的3′ UTR与上游剪接位点相互作用以递送RNA结合蛋白。我们将检测这种前mRNA 通过绘制RNA-RNA相互作用，体内小基因报告分析，靶向筛选， 和RNA亲和层析。这种机制的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的选择性剪接的规则， 交替多聚腺苷酸化。RNA加工步骤的协调以产生具有特定蛋白质的mRNA- 编码和调节特性对于许多细胞类型和真核生物可能是重要的，包括 人类

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.