Comprehensive Maps of U1 snRNP Binding to Nascent RNA in Human Cells

项目总结/摘要 我们建议开发新的方法，用于蛋白质和RNP相互作用的全球识别与新生 未剪接的RNA我们将使用这些方法来产生剪接体U1 snRNP结合的综合图谱 在人类转录组中。U1在前体mRNA剪接中起作用，在抑制早产儿中起作用。 切割/聚腺苷酸化，以及RNA的核保留。然而，关于其结合位点的信息非常 有限的，以及这些网站如何不同的U1的不同功能是不理解的。未剪接内含子 我们最近发现，在染色质区室中， 这些因素之间的相互作用在其他地方是看不到的。我们开发了分离蛋白质和RNP的方法， 允许鉴定与U2 snRNP相互作用的新的调节蛋白，并分离U2结合的 包含HEK 293细胞的内含子分支点的前mRNA片段。我们把这个方法叫做 Fractionation/immunopurification/RNAse protection（FIRP），并找到U2/pre-mRNA相互作用的FIRP图谱 比以前的映射分支点的方法更全面。我们现在建议采用FIRP 来表征U1 snRNP与前mRNA的相互作用。我们将优化材料的提取， 染色质，以获得与前mRNA结合的U1 snRNP复合物。我们将开发新的抗U1抗体， 允许对所有类型的细胞进行5 ′剪接位点结合分析。这些方法将同时应用于FIRP检测 的U1 snRNP结合和iCLIP分析的U1蛋白接触染色质相关的RNA。通过 在全球范围内表征U1结合位点，并开发分析其在不同环境中相互作用的方法。 细胞、调控环境和遗传背景，我们可以检查5'剪接位点的过程， 以新的广度和精度获得认可。

PROJECT SUMMARY/ABSTRACT We propose to develop new methods for the global identification of protein and RNP interactions with nascent unspliced RNA. We will use these methods to produce comprehensive maps of spliceosomal U1 snRNP binding across the human transcriptome. U1 functions in pre-mRNA splicing, in the suppression of premature cleavage/polyadenylation, and in the nuclear retention of RNA. However, information on its binding sites is very limited, and how these sites differ for the different functions of U1 is not understood. Unspliced introns in nascent RNA fractionate with the chromatin, and we recently showed that within the chromatin compartment, splicing factors engage in interactions not seen elsewhere. We developed methods to isolate proteins and RNP’s that allowed identification of new regulatory proteins interacting with the U2 snRNP, and the isolation of U2-bound pre-mRNA fragments encompassing the intronic branch points of HEK293 cells. We call this method fractionation/immunopurification/RNAse protection (FIRP) and find the FIRP maps of U2/pre-mRNA interactions to be more comprehensive than previous approaches for mapping branchpoints. We now propose to adapt FIRP to characterizing interactions of the U1 snRNP with pre-mRNA. We will optimize the extraction of material from chromatin to obtain U1 snRNP complexes bound to pre-mRNA. We will develop new anti-U1 antibodies that allow 5’ splice site binding analysis across all types of cells. These methods will be applied both to FIRP assays of U1 snRNP binding and to iCLIP analyses of U1 protein contacts on chromatin-associated RNA. By characterizing U1 binding sites on a global scale and developing methods for assaying its interactions in different cells, regulatory environments and genetic backgrounds, we can examine the processes of 5’ splice site recognition with new breadth and precision.