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.

项目概要/摘要 我们建议开发新方法来全面鉴定蛋白质和 RNP 与新生蛋白的相互作用。 我们将使用这些方法来生成剪接体 U1 snRNP 结合的综合图谱。 U1 在前体 mRNA 剪接中发挥作用，抑制早熟。 然而，关于其结合位点的信息非常多。 有限，并且这些位点对于U1的不同功能有何不同尚不清楚。 RNA 与染色质分离，我们最近表明，在染色质区室中，剪接 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 片段我们称这种方法为包含。 分级分离/免疫纯化/RNAse 保护 (FIRP) 并查找 U2/pre-mRNA 相互作用的 FIRP 图 比以前的分支点映射方法更全面，我们现在建议采用 FIRP。 为了表征 U1 snRNP 与前 mRNA 的相互作用，我们将优化从中提取材料。 染色质以获得与前体 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。这些方法将应用于 FIRP 测定。 U1 snRNP 结合和 iCLIP 分析 U1 蛋白与染色质相关 RNA 的接触。 在全球范围内表征 U1 结合位点并开发分析其在不同环境下相互作用的方法 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。