Comprhensive analysis of functional RNA elements encoded in the human genome

本提案的目标是全面表征人类基因组编码的功能序列元件，这些元件在两种细胞系中被250种RNA结合蛋白（rbp）识别。为此，我们将生成稳定的表达表位标记RBP的HeLa-S3和GM 12878细胞系，并确定每个RBP的亚细胞定位模式。这些细胞将被用于进行CLIP-Seq测定，以确定全基因组和单核苷酸分辨率，250个rbp识别的RNA序列元件。鉴定的RNA序列元件将使用基于序列的体外结合试验进行验证。此外，将对所有核定位RBP进行ChlP-Seq，以确定每个RBP与基因组和染色质相关的区域。这些结合试验将辅以rbp缺失细胞的功能试验，这将是为鉴定的结合位点分配功能的关键。这些检测包括对细胞总RNA和从各种细胞组分中纯化的RNA进行RNA测序，核糖体足迹分析和Gro-Seq。总之，这些检测将提供关于每个RBP在剪接、切割和聚腺苷化、RNA稳定性、RNA编辑、翻译、RNA定位和转录中的作用的功能信息。将主要进行生物信息学分析

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.