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

项目摘要/摘要 我们建议开发新的方法来全球鉴定蛋白质和RNP与新生的相互作用 未剪接的RNA。我们将使用这些方法来制作剪接体U1 SnRNP结合的全面图谱 在人类转录组中。U1在前-mRNA剪接中的作用，在抑制早产中的作用 裂解/多聚腺苷化，以及在RNA的核保留中。然而，有关其结合位点的信息非常多。 有限，而且这些位点对于U1的不同功能有何不同尚不清楚。新生中的未剪接内含子 RNA与染色质分离，我们最近发现在染色质隔间内，剪接 各种因素相互作用，这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相互作用的新调节蛋白，并分离U2结合蛋白 HEK293细胞内含子分支点的前-mRNA片段。我们称此方法为 分级/免疫纯化/核糖核酸酶保护(FIRP)和寻找U2/Pre-mRNA相互作用的FIRP图谱 比以前的绘制分支点的方法更全面。我们现在建议修改FIRP 目的：研究U1SnRNP与Pre-mRNA的相互作用。我们将优化从 染色质获得与Pre-mRNA结合的U1SnRNP复合体。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。这些方法都将应用于FIRP分析 并对染色质相关RNA上的U1蛋白接触进行了iCLIP分析。通过 全球范围内U1结合位点的特征及其在不同物种中相互作用的分析方法 细胞、调控环境和遗传背景，我们可以研究5‘剪接位点的过程 识别具有新的广度和精确度。

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.