Complex RNA Processing

 描述(由申请人提供)：大多数真核生物的前mRNAs，特别是后生动物中的前mRNAs，可以交替剪接以产生多个mRNAs和蛋白质。鉴于选择性剪接在调控基因表达和增强蛋白质组多样性方面的重要性，了解选择性剪接的机制以及选择性剪接是如何调控的是至关重要的。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黏附分子1(Dscam1)基因是已知的最广泛的选择性剪接基因。Dscam1包含115个外显子，其中95个外显子可以交替剪接，有可能产生38,016种不同的mRNA和蛋白质亚型。我们将研究Dscam1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，这是确定神经连接特异性的关键过程。MDG4基因的纵向缺失(LOLA)和修饰(moD(MDG4))是经历反式剪接的最好例子-通过这个过程，来自不同前mRNAs的外显子被剪接在一起，产生编码蛋白质的mRNAs。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。长期以来，很长的内含子是如何有效拼接的一直是个谜。17年前，研究表明，通过一种称为递归剪接的过程，一只长果蝇以渐进、循序渐进的方式被移除。然而，到目前为止，还不知道这种现象发生的范围有多广。我们最近在果蝇中发现了近200个递归剪接实例，人类中也存在这种情况。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，研究细菌和古菌如何通过CRISPR途径以及决定因素和调节因子或基因组范围内的RNA周转来对新遇到的病毒产生抗药性。所有这些问题将使用我们已经应用或开发的各种尖端方法来解决，包括报告基因，以促进单个神经元分辨率中的可视化剪接，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。我们还将继续开发更多的创新办法，以便在需要时或在该领域技术进步带来的机会出现时解决这些问题。

 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.