Comprhensive analysis of functional RNA elements encoded in the human genome

这项建议的目标是全面表征人类基因组中编码的功能序列元件，这些元件可由两个细胞系中的250个RNA结合蛋白(RBPs)识别。为此，我们将建立稳定的HeLa-S3和GM 12878细胞系，表达表位标记的RBPs，并确定每个RBP的亚细胞定位模式。这些细胞将被用来进行CLIP-SEQ分析，以确定全基因组，并在单核苷酸分辨率下，由250个限制性商业惯例识别的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分析进行验证。此外，将对所有核定位限制性商业惯例进行ChlP-Seq，以确定每个限制性商业惯例所关联的基因组和染色质区域。这些结合分析将辅以RBP耗竭细胞中的功能分析，这对于将功能分配给已确定的结合部位至关重要。这些分析包括细胞总RNA和从不同细胞组分纯化的RNA的RNA-Seq、核糖体足迹图谱和Gro-Seq。总之，这些分析将提供关于每个RBP在剪接、切割和多聚腺苷基化、RNA稳定性、RNA编辑、翻译、RNA定位和转录中所起作用的功能信息。将进行生物信息学分析，主要是 使用我们小组生成的软件，对所有检测进行量化，并将功能与结合检测中确定的序列元件相关联。总之，这些实验将对大约一半的人类限制性商业惯例及其相互作用的功能序列元件的功能提供全面和深入的测量。这个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，这些功能元件是由ENCODE财团描述的。该项目的成果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，并几乎肯定会创建关于限制性商业惯例和核糖核酸-蛋白质网络在人类生物学和疾病中的功能的新范例。

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.