Genomic analysis of RNA binding protein target specificity

描述(申请人提供)：人类转录组的复杂性远远超过先前的预期。转录多样性可以通过转录后RNA加工反应以及广泛的基因间转录来扩大。调节蛋白质编码基因和非编码基因间转录本表达的顺式作用RNA元件的基因组组织尚不清楚。这种缺陷不仅阻碍了我们对储存在人类基因组中的信息是如何利用的理解，而且还削弱了基因表达的转录后控制与细胞增殖、分化甚至人类疾病等生物过程之间的联系。这项提议的目标是阐明由RNA结合蛋白协调的转录后网络。为了实现这一目标，我们将在全基因组范围内鉴定由一系列系统发育保守的基本RNA结合蛋白识别的顺式作用RNA元件。我们的应用主要集中在富含丝氨酸和精氨酸的前mRNA剪接因子家族(SR蛋白)上。生化方法将被用来在保持完整细胞中RNA-蛋白质相互作用的生理背景的条件下纯化SR蛋白质-RNA复合体。复制的RNA分子将通过两种独立的高通量方法直接鉴定，焦磷酸测序和与平铺基因组微阵列的杂交。我们的R01应用程序将解决以下特定目标：(1)全面识别SR蛋白家族识别的顺式作用RNA元件。(2)确定SR蛋白质识别的顺式作用RNA元件的基因组图谱。(3)从功能上验证SR蛋白顺式作用的RNA元件。

DESCRIPTION (provided by applicant): The complexity of human transcriptome is far greater than previously anticipated. Transcript diversity can be expanded through post-transcriptional RNA processing reactions as well as extensive inter-genic transcription. The genomic organization of cisacting RNA elements regulating expression of protein coding genes and non-coding inter-genic transcripts is poorly defined. This deficiency not only hampers our understanding of how the information stored within the human genome is utilized, but also weakens the connections between post-transcriptional control of gene expression and biological processes such as cell proliferation, differentiation and even human disease. The goal of this proposal is to illuminate post-transcriptional networks coordinated by RNA binding proteins. To achieve this goal we will identify cis-acting RNA elements recognized by a complete family of phylogenetically conserved, essential RNA binding proteins on a genome-wide scale. Our application focuses on the Serine and Arginine-rich family of pre-mRNA splicing factors (SR proteins). Biochemical methods will be employed to purify SR protein-RNA complexes under conditions that preserve the physiological context of RNA-protein interactions in the intact cell. Copurifying RNA molecules will be directly identified by two independent high throughput methods, pyrosequencing and hybridization to tiled genomic microarrays. Our R01 application will address the following specific aims: (1) Comprehensively identify cis-acting RNA elements recognized by the SR protein family. (2) Determine the genomic landscape of cis-acting RNA elements recognized by SR proteins. (3) Functionally validate SR protein cis-acting RNA elements.