Comprehensive functional analysis of the SR protein family of RNA-binding proteins in vivo

SR蛋白是一个由7个保守的、必需的RNA结合蛋白组成的家族，涉及mRNA前剪接、mRNA输出和翻译。由于其内源性RNA靶点未知，SR蛋白的功能仍然是神秘的。我们建立了gfp标记的SR蛋白的生理表达，并表明它们与pre-mRNA共转录和剪接后的特定mrna相关（Sapra et al., 2009）。SR蛋白带着特定的mRNA货物离开细胞核。因此，现在可以在体内检测到SR蛋白与mrna在基因表达的各个阶段的相互作用。SR蛋白的内源性pre-mRNA和mRNA靶点将在循环和分化的神经细胞中得到鉴定。重要的是，将确定每个SR蛋白在体内直接结合的RNA中的特定序列，映射到转录组，并与SR蛋白定位的基因组模式进行比较。我们将使用“模型基因”系统分析SR蛋白和同源结合位点在选择性剪接中的活性。这些数据集的整合将揭示SR蛋白RNA靶点的动态和特异性，并为理解SR蛋白如何调节基因表达提供一个框架。

The SR proteins are a family of seven conserved, essential RNA binding proteins, implicated in pre-mRNA splicing, mRNA export, and translation. Because their endogenous RNA targets are unknown, SR protein functions remain mysterious. We established physiological expression of GFP-tagged versions of SR proteins and showed that each associates with pre-mRNA co-transcriptionally and with specific mRNAs after splicing (Sapra et al., 2009). SR proteins exit the nucleus with specific mRNA cargoes. Thus, SR protein interactions with mRNAs at all stages of gene expression can now be detected in vivo. Endogenous pre-mRNA and mRNA targets of SR proteins will be identified in cycling and differentiated neural cells. Importantly, the specific sequences in RNA directly bound by each SR protein in vivo will be determined, mapped to the transcriptome, and compared to genomic patterns of SR protein localization. We will assay the activities of SR proteins and cognate binding sites in alternative splicing, using a “model gene” system. Integration of these datasets will reveal the dynamics and specificity of SR protein RNA targets and provide a framework for understanding how SR proteins regulate gene expression.