PROJECT 1

项目1

• 批准号: 8733715
• 负责人: Ming Chen Hammond
• 金额: $ 45.94万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8733715
• 关键词: Alternative Splicing; Binding Sites; Biochemical; Biological Assay; Cells; Chemicals; Data; Data Set; Event; Exons; Gene Expression Profile; Gene Mutation; Genes; Genetic Variation; Goals; Histones; Human; Human Genetics; In Vitro; Individual; Informatin; Link; Maps; Methods; Modeling; Molecular; Molecular Chaperones; Nuclear; Pattern; Phylogenetic Analysis; Play; Positioning Attribute; Proteins; RNA; RNA Binding; RNA Helicase; RNA Polymerase II; RNA Splicing; RNA annealing; Regulatory Element; Relative (related person); Reporter; Role; Site; Structure; System; Systems Biology; Testing; base; genetic variant; genome-wide; helicase; hnRNP A1; human disease; in vivo; mRNA Precursor; network models

Summary Our overall project goal is to systematically link c/s-regulatory elements in pre-mRNAs to RNA structural features that control alternative pre-mRNA splicing in human cells. While the role of pre-mRNA structure in control of splicing events has been studied for individual genes, a comprehensive understanding of the interplay of RNA structure with splicing control—and its relation to genetic variation—is not known for the human transcriptome. Our aims are to: 1) Map nuclear pre-mRNA structure in vivo and in vitro using high-throughput chemical probing methods and test the effects of RNA chaperone proteins on those structures. 2) Determine the positioning of mapped pre-mRNA structures relative to splicing factor binding sites, splice sites and exons, and human genetic variants. 3) Define a role for RNA chaperone proteins in alternative splicing decisions in human cells. 4) Develop and validate RNA maps relating RNA chaperone binding, RNA structure, and pre-mRNA splicing events, and validate roles of predicted structures in alternative splicing. These models will incorporate biochemical data, splicing reporter assays, as well as phylogenetic information and human genetic variation. Our efforts will initially focus on the genome-wide mapping of single-stranded RNA regions detected by chemical probing of pre-mRNA structure, as influenced by two known RNA chaperone proteins, the hnRNP protein hnRNPA1 and the p68/DDX5 RNA helicase. The RNA binding and chaperone activities (RNA annealing or unwinding) of hnRNP A1 and p68/DDX5 helicase will be correlated to global RNA structural data and alternative splicing patterns. We want to define instances where RNA structure plays a role in alternative splicing decisions and then integrate genome-wide data sets into these models. Our ultimate goal will be to generate systems level network models for how RNA structure and RNA chaperones, human genetic variation and histone marks, and RNA polymerase II distribution control alternative pre-mRNA splicing to inform both mechanistic aspects and the underlying molecular basis of human disease gene mutations.

概括 我们的总体项目目标是系统地将前体 mRNA 中的顺式/顺式调节元件与控制人类细胞中选择性前体 mRNA 剪接的 RNA 结构特征联系起来。虽然已经针对单个基因研究了前 mRNA 结构在控制剪接事件中的作用，但对于人类转录组，对 RNA 结构与剪接控制的相互作用及其与遗传变异的关系的全面了解尚不清楚。我们的目标是： 1) 使用高通量化学探测方法绘制体内和体外核前 mRNA 结构图，并测试 RNA 伴侣蛋白对这些结构的影响。 2) 确定映射的前mRNA结构相对于剪接因子结合位点、剪接位点和外显子以及人类遗传变异的定位。 3) 定义 RNA 伴侣蛋白在人类细胞选择性剪接决策中的作用。 4) 开发和验证与RNA伴侣结合、RNA结构和前mRNA剪接事件相关的RNA图谱，并验证预测结构在选择性剪接中的作用。这些模型将结合生化数据、剪接报告分析以及系统发育信息和人类遗传变异。 我们的工作最初将集中于通过化学探测前 mRNA 结构检测到的单链 RNA 区域的全基因组图谱，这些区域受到两种已知的 RNA 伴侣蛋白（hnRNP 蛋白 hnRNPA1 和 p68/DDX5 RNA 解旋酶）的影响。 hnRNP A1 和 p68/DDX5 解旋酶的 RNA 结合和伴侣活性（RNA 退火或解旋）将与全局 RNA 结构数据和选择性剪接模式相关。我们想要定义 RNA 结构在选择性剪接决策中发挥作用的实例，然后将全基因组数据集整合到这些模型中。我们的最终目标是生成系统级网络模型，用于了解 RNA 结构和 RNA 伴侣、人类遗传变异和组蛋白标记以及 RNA 聚合酶 II 分布如何控制替代的前 mRNA 剪接，以告知人类疾病基因突变的机制方面和潜在的分子基础。