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

 描述(申请人提供)：HIV-1剪接表型的量化以及RNA结构和剪接调控元件的作用我对RNA二级结构和剪接调控因子作为HIV-1表达中不同剪接模式的决定因素的作用感兴趣。我已经采用了Primer ID方法来创建一个使用深度测序来量化HIV RNA剪接模式的工具。这是一种简单的方法来比较不同剪接的转录本在每种大小类别的HIV-1转录本中的相对丰度(即不同转录本在4kb和1.8kb转录本组中的相对数量)。我将使用一种更传统的方法(S1核酸酶图谱)来比较总剪接和未剪接以及4kb到1.8kb的转录组。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。我计划研究整个基因组中非特异性破坏二级结构并可能影响剪接模式的缺失，询问控制剪接模式的病毒RNA结构的全球约束是什么。我将与其他研究人员合作使用这项测试，特别是布兰顿·托尔伯特博士和爱丽丝·特莱斯尼茨基博士， 在整个病毒基因组的背景下，定义调节位点突变的剪接表型。我还开发了一种工具，可以识别当结构或调控元件受到干扰时可能出现的神秘剪接位点。我相信，能够快速量化在HIV-1基因表达中看到的复杂剪接模式将增加目前对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.