Comprhensive analysis of functional RNA elements encoded in the human genome

人类基因组中编码的功能性RNA元件的综合分析

基本信息

批准号：
8719767
负责人：
Brenton R. Graveley
金额：
$ 228.14万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-21 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8719767
关键词：
Base Sequence Binding Binding Sites Bioinformatics Biological Biological Assay Biology Cataloging Catalogs Cell Line Cells ChIP-seq Chromatin Collection Communities Computer software Consensus Coupled Coupling Defect Disease Elements Epitopes Functional RNA Gene Expression Profile Genetic Transcription Genome Genomic DNA Genomics Goals HeLa S3 Human Human Biology Human Cell Line Human Genome In Vitro Indium Individual Informatics Investigation Knowledge Maps Measures Messenger RNA Metabolism Nuclear Nuclear RNA Nucleotides Pattern Polyadenylation Protein Binding Proteins RNA RNA Editing RNA Sequences RNA Splicing RNA Stability RNA-Binding Proteins RNA-Protein Interaction Regulation Resolution Resources Ribosomes Role Series Subcellular Fractions Subfamily lentivirinae Surveys Tissues Translations base cell type design genome-wide human disease insight research study small hairpin RNA stable cell line transcriptome sequencing

项目摘要

The goal of this proposal is to comprehensively characterize the functional sequence elements encoded in the human genome that are recognized by 250 RNA binding proteins (RBPs) in two cell lines. To do this, we will generate stable HeLa-S3 and GM 12878 cell lines expressing epitope-tagged RBPs and determine the sub cellular localization pattern of each RBP. These cells will be used to perform CLIP-Seq assays to define genome-wide, and at single-nucleotide resolution, the RNA sequence elements recognized by 250 RBPs. The RNA sequence elements identified will be validated using sequence-based in vitro binding assays. Furthermore, ChlP-Seq will be performed for all nuclear localized RBPs to determine the regions of the genome and chromatin that each RBP associates with. These binding assays will be supplemented with functional assays in RBP-depleted cells that will be critical for assigning functions to the identified binding sites. These assays include RNA-Seq of total cellular RNA and RNA purified from various cellular fractions, ribosomal footprint profiling, and Gro-Seq. Together, these assays will provide functional information regarding the roles of each RBP in splicing, cleavage and polyadenylation, RNA stability, RNA editing, translation, RNA localization, and transcription. Bioinformatic analysis will be performed, largely using software generated by our group, to quantitate all assays and to associate functions to the sequence elements identified in the binding assays. Together, these experiments will provide a comprehensive and in depth measure of the functions of approximately half of the human RBPs and the functional sequence elements that they interact with. This project will fill a major gap in the catalog of functional elements encoded in the human genome that are being characterized by the ENCODE consortium. The product of this project will be a unique and valuable community resource that will push the field forward in new and exciting ways and will almost certainly create new paradigms regarding the functions of RBPs and RNA-protein networks in human biology and disease.

该提案的目的是全面地表征人类基因组中编码的功能序列元件，这些序列元素在两个细胞系中被250个RNA结合蛋白（RBP）识别。为此，我们将生成稳定的HELA-S3和GM 12878细胞系，以表达表位标记的RBP，并确定每个RBP的亚细胞定位模式。这些细胞将用于执行夹子seq分析以定义全基因组，并且在单核苷酸分辨率下，由250 rbps识别的RNA序列元件。确定的RNA序列元件将使用基于序列的体外结合测定法进行验证。此外，将对所有核局部RBP进行CHLP-SEQ，以确定每个RBP与之相关的基因组和染色质区域。这些结合测定将补充RBP缺失的细胞中的功能测定，这对于将功能分配给已识别的结合位点至关重要。这些测定包括从各种细胞级分，核糖体足迹分析和gro-seq纯化的总细胞RNA和RNA的RNA-SEQ。总之，这些分析将提供有关每个RBP在剪接，裂解和聚腺苷酸化，RNA稳定性，RNA编辑，翻译，RNA定位和转录的作用的功能信息。将在很大程度上进行生物信息学分析使用我们组生成的软件来量化所有测定法，并将功能与绑定测定中确定的序列元素相关联。总之，这些实验将提供大约一半人RBP的功能以及与其相互作用的功能序列元素的全面和深度衡量。该项目将填补人类基因组中用编码财团表征的功能元素目录中的主要空白。该项目的产物将是一种独特而有价值的社区资源，它将以新的和令人兴奋的方式推动该领域的前进，并且几乎可以肯定会创建有关RBP和RNA-蛋白网络在人类生物学和疾病中的功能的新范式。