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

HIV-1 剪接表型的定量以及 RNA 结构和剪接调节元件的作用

基本信息

批准号：
9126251
负责人：
Ann L Emery
金额：
$ 3.43万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9126251
关键词：
Accounting Affect Alternative Splicing Amino Acid Sequence Aspergillus Nuclease S1 Asses Binding Sites Biological Assay Biological Models CD4 Positive T Lymphocytes Cell Line Cells Cis-Acting Sequence Collaborations Complex Consensus Sequence Coupled Development Elements Evaluation Exons Gene Expression Genome Growth HIV HIV Genome HIV-1 Human Iodine Label Laboratories Length Location Maps Measures Methods Mutate Mutation Nucleic Acid Regulatory Sequences Pattern Phenotype Point Mutation Process RNA RNA Splicing Regulation Regulatory Element Research Research Personnel Ribonucleic Acid Regulatory Sequences Role Silent Mutation Site Spices Structure System Testing Transcript Viral Genome Virus Diseases Work clinically relevant deep sequencing fitness hnRNP A1 interest mutant prevent protocol development public health relevance reference genome reproductive fitness research study stem tool viral RNA viral fitness

项目摘要

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.

描述（通过应用程序提供）：对HIV-1剪接表型的定量以及RNA结构和剪接调节元素的作用，我对RNA二级结构和剪接调节剂的作用感兴趣，这是HIV-1表达中替代剪接模式的确定因素。我已经适应了底漆ID方法来创建一种使用深层测序量化HIV RNA剪接模式的工具。这是一种直接的方法，可以比较在HIV-1转录本的每个大小类别中不同剪接转录本的相对丰度（即4 kb和1.8 kb的转录本中不同转录本的相对量）。我将使用更传统的方法（S1核酸酶映射）来比较剪接与未贴合的总剪接和4 kb至1.8 kb的转录本。我对可能影响剪接模式的决定剂的影响感兴趣，尤其是剪接调节元素和RNA二级结构。我计划查看基因组中的缺失，这些缺失非特异性破坏二级结构并可能影响剪接模式，询问对控制剪接模式的病毒RNA结构的全局约束。我将与其他研究人员合作使用此评估，尤其是Blanton Tolbert博士和Alice Telesnitsky博士，在整个病毒基因组的背景下定义调节位点突变的剪接表型。我还开发了一种工具来识别当结构或调节元素干扰时可能出现的加密剪接位点。我认为，在HIV-1基因表达中看到的复杂剪接模式快速量化的能力将增加对RNA结构和功能的当前理解。