Complex RNA Processing

 描述（由申请人提供）：大多数真核前体mRNA，特别是在后生动物中，被选择性剪接以产生多种mRNA和蛋白质。鉴于选择性剪接在调节基因表达和增强蛋白质组多样性方面的重要性，理解剪接的机制以及选择性剪接是如何被调节的是至关重要的。在这个项目中，我们将研究果蝇中三种不寻常的剪接类型。唐氏综合症细胞粘附分子1（Dscam 1）基因是已知最广泛的可变剪接基因。Dscam 1包含115个外显子，其中95个是选择性剪接的，并有可能产生38，016种不同的mRNA和蛋白质亚型。我们将研究Dscam 1的概率剪接是如何实现的，以及它如何有助于确定细胞身份，这是确定神经布线特异性的关键过程。mdg 4基因的纵向缺失（lola）和修饰基因（mod（mdg 4））是经历反式剪接的基因的最佳例子-通过该过程，来自不同前mRNA的外显子被剪接在一起以产生蛋白质编码mRNA。我们将研究反式剪接是如何发生的，以及同源染色体配对在这一过程中的作用。很长的内含子如何有效地拼接一直是一个谜。17年前，有研究表明，一条长长的果蝇是通过一种叫做递归拼接的过程以渐进的、逐步的方式被移除的。然而，到目前为止，人们还不知道这种现象有多普遍。我们最近在果蝇中发现了近200个递归剪接的例子，它也发生在人类身上。我们将重点阐明这一不寻常过程的机制和功能。我们还将与合作者合作，研究细菌和古细菌如何通过CRISPR途径以及全基因组范围内的决定因子和调节因子或RNA周转来抵抗新遇到的病毒。所有这些问题都将使用我们已经应用或开发的各种各样的尖端方法来解决，包括促进单个神经元解析中的可视化剪接的报告基因、单细胞RNA-Seq纳米孔测序、RNAi或CRISPR筛选、BAC重组工程、果蝇遗传学和计算基因组学。我们还将根据需要或随着该领域技术进步带来的机会，继续开发更多的创新方法来解决这些问题。

 DESCRIPTION (provided by applicant): Most eukaryotic pre-mRNAs, especially in metazoans, are alternatively spliced to generate multiple mRNAs and proteins. Given the importance of alternative splicing in regulating gene expression and enhancing the diversity of the proteome, it is essential to understand the mechanisms of splicing and how alternative splicing is regulated. In this project, we will study three unusual types of splicing in Drosophila The Down Syndrome Cell Adhesion Molecule 1 (Dscam1) gene is the most extensively alternatively spliced gene know. Dscam1 contains 115 exons, 95 of which are alternatively spliced and has the potential to generate 38,016 different mRNA and protein isoforms. We will study how probabilistic splicing of Dscam1 is achieved and how it contributes to determining cell identity, a critical process involved in determining the specificity of neural wiring. The longitudinal lacking (lola) and modifier of mdg4 (mod(mdg4)) genes are the best examples of genes that undergo trans-splicing - a process by which exons from different pre-mRNAs are spliced together to generate a protein-coding mRNAs. We will study how trans-splicing occurs and the role of homologous chromosome pairing in this process. How very long introns are efficiently spliced has long been a mystery. Seventeen years ago, it was shown that one long Drosophila is removed in a progressive, stepwise fashion by a process called recursive splicing. However, until now it was not known how widespread this phenomenon occurred. We recently identified nearly 200 instances of recursive splicing in Drosophila and that it also occurs in humans. We will focus on elucidating the mechanisms and functions of this unusual process. We will also work with collaborators to study how bacteria and archaea become resistant to newly encountered viruses by means of the CRISPR pathway and the determinants and regulators or RNA turnover on a genome-wide scale. All of these problems will be addressed using a wide variety of cutting edge approaches we have applied or developed including reporter genes to facilitate visualizing splicing in single neuron resolution, single cell RNA- Seq nanopore sequencing, RNAi or CRISPR screens, BAC recombineering, Drosophila genetics, and computational genomics. We will also continue to develop additional innovative approaches to address these issues as needed or as opportunities arise due to technical advances in the field.