Comprhensive analysis of functional RNA elements encoded in the human genome

该提案的目标是全面表征人类基因组中编码的功能序列元件，这些元件被两种细胞系中的250种RNA结合蛋白（RBP）识别。为此，我们将产生表达表位标记的RBP的稳定的HeLa-S3和GM 12878细胞系，并确定每个RBP的亚细胞定位模式。这些细胞将用于进行CLIP-Seq分析，以确定全基因组范围内的单核苷酸分辨率，由250个RBP识别的RNA序列元件。将使用基于序列的体外结合试验验证鉴定的RNA序列元件。此外，将对所有核定位的RBP进行ChIP-Seq，以确定每个RBP所关联的基因组和染色质的区域。这些结合试验将补充RBP耗竭细胞中的功能试验，这对于将功能分配给已鉴定的结合位点至关重要。这些测定包括总细胞RNA和从各种细胞级分纯化的RNA的RNA-Seq、核糖体足迹分析和Gro-Seq。总之，这些测定将提供关于每种RBP在剪接、切割和聚腺苷酸化、RNA稳定性、RNA编辑、翻译、RNA定位和转录中的作用的功能信息。生物信息学分析将主要 使用我们小组生成的软件，对所有的分析进行定量，并将功能与结合分析中鉴定的序列元件相关联。总之，这些实验将提供一个全面和深入的措施，约一半的人类RBPs的功能和功能序列元件，他们相互作用。该项目将填补人类基因组中编码的功能元件目录中的一个主要空白，ENCODE财团正在对其进行表征。该项目的产品将是一个独特而有价值的社区资源，将以新的和令人兴奋的方式推动该领域的发展，几乎肯定会创造关于RBP和RNA蛋白质网络在人类生物学和疾病中的功能的新范式。

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.