Quantification of HIV-1 Splicing Phenotypes and the Role of RNA Structure and Splice Regulator Elements

 描述（由申请人提供）：HIV-1剪接表型的定量以及RNA结构和剪接调节因子的作用我对RNA二级结构和剪接调节因子在HIV-1表达中作为可变剪接模式决定因素的作用感兴趣。我已经调整了引物ID方法，以创建一个工具，使用深度测序定量HIV RNA剪接模式。这是比较HIV-1转录物的每个大小类别内不同剪接转录物的相对丰度（即，转录物的4kb和1.8kb组内不同转录物的相对量）的直接方法。我将使用更传统的方法（S1核酸酶作图）来比较总剪接与未剪接以及4 kb至1.8 kb的转录物组。我感兴趣的是可能影响剪接模式的决定因素的影响，特别是剪接调控元件和RNA二级结构。我计划研究非特异性破坏二级结构并可能影响剪接模式的基因组缺失，询问控制剪接模式的病毒RNA结构的全局限制是什么。我将与其他研究人员，特别是Blanton Tolbert博士和Alice Telesnitsky博士， 在整个病毒基因组的背景下定义调控位点突变的剪接表型。我还开发了一种工具来识别结构或调控元件受到干扰时可能出现的隐蔽剪接位点。我相信快速量化HIV-1基因表达中复杂剪接模式的能力将增加对RNA结构和功能的理解。

 DESCRIPTION (provided by applicant): Quantification of HIV-1 Splicing Phenotypes and the Role of RNA Structure and Splice Regulator Elements I am interested in the role of RNA secondary structure and splicing regulators as determinants of alternative splicing patterns in the expression of HIV-1. I have adapted the Primer ID method to create a tool that quantifies HIV RNA splicing patterns using deep sequencing. This is a straightforward method to compare the relative abundance of different spliced transcript within each size class of HIV-1 transcripts (i.e. the relative amounts of the different transcripts within the 4 kb and 1.8 kb groups of transcripts). I will use a more traditional method (S1 nuclease mapping) to compare total spliced versus unspliced and 4 kb to 1.8 kb groups of transcripts. I am interested in the effects of determinants likely to affect splicing patterns, particularly splice regulatory elements and RNA secondary structure. I plan to look at deletions across the genome that non-specifically disrupt secondary structure and potentially affect splicing patterns, asking what are the global constraints on viral RNA structure for controlling splicing patterns. I will use this assay in collaboration with other investigators, particularly Dr. Blanton Tolbert and Dr. Alice Telesnitsky, to define the splicing phenotype of regulatory site mutations in the context of the entire viral genome. I have also developed a tool to identify cryptic splice sites that may arise when structure or regulatory elements are perturbed. I believe the ability to rapidly quantify the complex splicing patterns seen in HIV-1 gene expression will add to the current understanding of RNA structure and function.