Genomic Analysis of Nucleic Acid Transactions

项目概要（与原申请相同） 大多数真核生物的前mRNA，特别是在后生动物中，是选择性剪接的， 产生多种mRNA和蛋白质。考虑到选择性剪接在调节 基因表达和增强蛋白质组的多样性，这是必要的，以了解 剪接的机制和选择性剪接是如何调节的。在这个项目中，我们将研究 RNA结合蛋白在选择性剪接中的作用，重点是RNA结合蛋白是如何 蛋白质自动和交叉调节其自身和其他RNA结合蛋白基因的剪接。 这项工作将提供新的见解RNA加工的机制，以及如何这些 蛋白质相互调节以实现体内稳态。 许多原核生物编码CRISPR-Cas系统，其是RNA引导的适应性的 免疫系统，保护原核生物免受入侵者，如病毒和 质粒。免疫记忆被编码为短DNA序列，称为“间隔区”， 与入侵者基因组相匹配，并以短重复序列阵列中的散置元件的形式存储 (the CRISPR阵列）。CRISPR阵列被转录并加工成指导RNA， 与Cas核酸酶配对以识别和降解靶核酸（干扰）。新 免疫记忆是在"适应"过程中形成的， 并整合到CRISPR阵列中用于未来的靶向。虽然有大量的 关于入侵核酸的靶向和降解，我们所知甚少， 适应的过程。我们计划进一步描述原核生物中的适应过程， CRISPR-Cas系统。这项工作还将提供深入了解适应机制， 原核生物的免疫系统除了加强我们对基本 原核免疫系统的科学，这项工作有巨大的潜力， 开发可用于基因组编辑应用的新工具。 所有这些项目都将使用我们现有的一般方法类型进行处理 例如剪接报告基因、单细胞RNA-Seq、纳米孔测序、RNAi或 CRISPR筛选和计算基因组学。我们还将继续开发新的 根据需要或在机会出现时， 该领域的技术进步。

Project Summary (as submitted in original application) 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 the roles of RNA binding proteins in alternative splicing, with an emphasis on how RNA binding proteins auto- and cross-regulate the splicing their own and other RNA binding protein genes. This work will provide new insight into the mechanisms of RNA processing and how these proteins regulate one another to achieve homoestasis. Many prokaryotes encode CRISPR-Cas systems which are RNA-guided adaptive immune systems that protects prokaryotic organisms against invaders such as viruses and plasmids. Immune memories are encoded as short DNA sequences, called “spacers”, that match invader genomes and are stored as interspersed elements in an array of short repeats (the CRISPR array). The CRISPR arrays are transcribed and processed into guide RNAs which pair with Cas nucleases to recognize and degrade target nucleic acid (interference). New immune memories are formed during “adaptation” when fragments of invader DNA are acquired and integrated into CRISPR arrays for use in future targeting. While a tremendous amount is known about the targeting and degradation of invading nucleic acids, much less is known about the process of adaptation. We plan to further characterize the adaptation process in prokaryotic CRISPR-Cas systems. This work will also provide insight into the mechanisms of adaptation in the immune systems of prokaryotes. In addition to enhancing our understanding of the basic science of prokaryotic immune systems, there is tremendous potential that this work could lead to the development of new tools that can be used for genome editing applications. All of these projects will be addressed using the types of general approaches we have developed such as splicing reporters, single cell RNA-Seq, nanopore sequencing, RNAi or CRISPR screens, 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.