Genomic Analysis of Nucleic Acid Transactions

项目摘要(与原始申请中提交的相同) 大多数真核生物的前mRNAs，特别是后生动物中的前mRNAs，可以选择性地剪接到 产生多个mRNA和蛋白质。鉴于选择性剪接在调控中的重要性 基因表达和增强蛋白质组的多样性，有必要了解 剪接的机制以及选择性剪接是如何被调控的。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结合如何 蛋白质自动和交叉调节自己和其他RNA结合蛋白基因的剪接。 这项工作将为RNA加工的机制以及这些机制如何 蛋白质之间相互调节以实现同质凝集。 许多原核生物编码RNA引导的适应性CRISPR-CAS系统 保护原核生物免受入侵的免疫系统，如病毒和 质粒。免疫记忆被编码为短DNA序列，称为“间隔区”， 匹配入侵者基因组，并以散布在短重复数组中的元素的形式存储 (CRISPR数组)。CRISPR阵列被转录并加工成引导RNA， 与CaS核酸酶配对，识别和降解目标核酸(干扰)。新的 当获得入侵者DNA片段时，免疫记忆在“适应”过程中形成 并集成到CRISPR阵列中，以用于未来的目标。虽然有大量的 已知入侵核酸的靶向和降解，但对此知之甚少 适应的过程。我们计划在原核生物中进一步描述适应过程 CRISPR-CAS系统。这项工作也将提供对适应机制的洞察 原核生物的免疫系统。除了增进我们对基本知识的理解 原核生物免疫系统的科学，这项工作有巨大的潜力 用于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来解决 发展如剪接记者、单细胞RNA-Seq、纳米孔测序、RNAi或 CRISPR筛选和计算基因组学。我们还将继续开发更多的 根据需要或因以下原因而出现的机会，采用创新方法来解决这些问题 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.