Genomic analysis of RNA binding protein target specificity

描述（由申请人提供）：人类转录组的复杂性远远大于先前预期的。转录多样性可以通过转录后的RNA加工反应以及广泛的基因间转录来扩展。调节蛋白质编码基因和非编码基因间转录物表达的顺作用RNA元件的基因组组织尚不清楚。这一缺陷不仅阻碍了我们对人类基因组中存储的信息如何被利用的理解，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。这一建议的目标是阐明由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被一个完整的系统发育保守的、必需的RNA结合蛋白家族识别的顺式作用RNA元件。我们的应用侧重于富含丝氨酸和精氨酸的pre-mRNA剪接因子家族（SR蛋白）。在保留完整细胞中rna -蛋白相互作用的生理背景的条件下，将采用生化方法纯化SR蛋白- rna复合物。Copurifying RNA分子将直接鉴定两种独立的高通量方法，焦磷酸测序和杂交到平铺基因组微阵列。我们的R01申请将解决以下具体目标：(1)全面鉴定SR蛋白家族识别的顺式作用RNA元件。(2)确定SR蛋白识别的顺式作用RNA元件的基因组图谱。(3)功能验证SR蛋白顺式作用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.