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MRNA中的C/S调节元素与RNA结构特征联系起来，以控制人类细胞中的替代性MRNA剪接。虽然已经研究了单个基因的MRNA结构在控制剪接事件中的作用，但对RNA结构与剪接控制的相互作用的全面了解及其与遗传变异的关系 - 以人类转录组而闻名。我们的目标是： 1）使用高通量化学探测方法在体内和体外绘制核前-MRNA结构，并测试RNA链酮蛋白对这些结构的影响。 2）确定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映射前MRNA结构的定位。 3）定义RNA链酮蛋白在人类细胞中替代剪接决策中的作用。 4）开发和验证与RNA链结合，RNA结构和前MRNA剪接事件的RNA图，并验证预测结构在替代剪接中的作用。这些模型将结合生化数据，剪接报告基因测定以及系统发育信息和人类遗传变异。 我们的努力最初将集中在通过两种已知的RNA链酮蛋白HNRNP蛋白HNRNPA1和p68/ddx5 RNA Helicase的HNRNP蛋白HNRNPA1和DDX5 RNA旋转酶的影响，从而将通过MRNA结构的化学探测检测到的单链RNA区域的全基因组映射。 HNRNP A1和P68/DDX5解旋酶的RNA结合和链酮活性（RNA退火或放松）将与全局RNA结构数据和替代剪接模式相关。我们要定义RNA结构在替代剪接决策中起作用的实例，然后将全基因组数据集整合到这些模型中。我们的最终目标是为RNA结构和RNA链酮，人遗传变异和组蛋白标记以及RNA聚合酶II分布控制替代pre-MRNA剪接如何为机械方面和人类疾病基因突变的基本分子基础提供信息，以生成系统级网络模型。