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

U1 snRNP 与人类细胞中新生 RNA 结合的综合图谱

• 批准号: 10507429
• 负责人: Douglas L Black
• 金额: $ 42.9万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-12 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10507429
• 关键词: 5' Splice Site; Affinity; Antibodies; Binding; Binding Sites; Biological Assay; Cells; Chromatin; Complex; Data; Disease model; Environment; Fractionation; Frequencies; Gene Expression; Genetic; Genome; Goals; Human; Immunoprecipitation; Introns; Maps; Medical Genetics; Methods; Mutation; Nuclear; Pathologic; Patients; Play; Polyadenylation; Procedures; Process; Proteins; RNA; RNA Splicing; RNase protection assay; Reaction; Ribonucleases; Role; Site; System; Tissues; U1 Small Nuclear Ribonucleoprotein; U2 Small Nuclear Ribonucleoprotein; Variant; cell type; crosslink; genetic regulatory protein; human disease; in vivo; insight; mRNA Precursor; premature; tool; transcriptome

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.

项目摘要/摘要 我们建议开发新方法，用于全局鉴定蛋白质和RNP与新生的相互作用 未填充的RNA。我们将使用这些方法来生成剪接型U1 SNRNP绑定的综合图 跨越人类的转录组。 U1在前MRNA剪接中起作用，在过早的抑制中 切割/聚腺苷酸化，以及RNA的核保留率。但是，有关其绑定站点的信息非常 限制，这些站点在U1的不同功能方面有何不同。新生未贴 用染色质分级RNA分数，我们最近表明，在染色质室内，剪接 因素参与其他地方看不到的互动。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，并隔离U2结合 前MRNA片段包含HEK293细胞的内含子分支点。我们称这种方法 分馏/免疫功能/RNase保护（FIRP），并找到U2/PRE-MRNA相互作用的FIRP图 比以前的映射分支点的方法更全面。我们现在建议适应FIRP 表征U1 SNRNP与前MRNA的相互作用。我们将优化从中提取的材料 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 在所有类型的细胞中允许5'剪接位点结合分析。这些方法将两者都应用于FIRP测定 U1 SNRNP结合和与染色质相关RNA上U1蛋白接触的ICLIP分析。经过 在全球范围内表征U1绑定位点，并开发在不同的相互作用的方法 细胞，调节环境和遗传背景，我们可以检查5'剪接位点的过程 具有新的广度和精确度的认可。