Profiling the locations of U1 snRNP binding across the nuclear human and Drosophila transcriptomes.

分析 U1 snRNP 在人类核转录组和果蝇核转录组中的结合位置。

基本信息

批准号：
9789352
负责人：
DONALD C RIO
金额：
$ 23.55万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-20 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9789352
关键词：
5&apos Splice Site Address Affect Affinity Alternative Splicing Architecture Binding Binding Sites Biochemical Bioinformatics Biological Assay Cell Nucleus Cells Chromatin Complementary DNA Complex Computing Methodologies Development Disease Drosophila genus Elements Event Excision Gene Expression Genetic Transcription Goals Human Introns Investigation KH Domain Knowledge Lead Link Location Malignant Neoplasms Maps Mediating Messenger RNA Methods Molecular Mutation Nerve Degeneration Nuclear Nuclease Protection Assays Oligonucleotides Pattern Phosphorus Play Poly A Polyadenylation Positioning Attribute Post-Transcriptional RNA Processing Post-Transcriptional Regulation Process Protein Structure Initiative Proteins RNA RNA Binding RNA Polymerase II RNA Processing RNA Splicing Regulatory Pathway Repression Research Ribonucleases Ribonucleoproteins Role Site Small Nuclear Ribonucleoproteins Small RNA Spinal Muscular Atrophy Splice-Site Mutation Spliceosome Assembly Pathway Spliceosomes Tertiary Protein Structure Therapeutic Time Transcript Transcriptional Silencer Elements U1 Small Nuclear Ribonucleoprotein Untranslated RNA Work bioinformatics tool bone genome-wide insight mRNA Precursor novel novel therapeutic intervention premature promoter small molecule transcriptome

项目摘要

PROJECT SUMMARY / ABSTRACT Profiling the locations of U1 snRNP binding across the nuclear human and Drosophila transcriptomes. D. Rio – P.I. Spliceosomal U1 snRNP functions in the nucleus to influence both pre-mRNA alternative splicing and polyadenylation site usage, which are two key gene expression mechanisms. The goal of this proposal is to develop a method to comprehensively map the locations of U1 snRNP across the nuclear transcriptomes of Drosophila and human cells and systematically categorize the function of the spliceosomal U1 snRNP at specific sites in modulating global pre-mRNA splicing and polyadenylation patterns. In order to address this question we will: 1) Develop a genome-wide mapping and profiling method for characterizing the binding sites of U1 snRNP across the nuclear transcriptomes of human and Drosophila cells. To do this, we will develop a novel and highly-specific two-step immunoaffinity selection strategy in combination with an RNase T1 nuclease protection assay to characterize the widespread targeting of U1 snRNP (a complex of 10 proteins and one non-coding, small RNA) to intron 5' splice sites, premature cleavage and polyadenylation (PCPA) sites and splicing silencer elements. We will develop novel computational methods to extensively and accurately profile significant U1 snRNP binding sites; 2) Categorize and define U1 snRNP binding sites as bone fide or cryptic 5' splice sites, telescripting sites or splicing silencer elements. For this purpose we will use state-of-the-art cDNA sequencing after perturbation of U1 snRNP activity in human and Drosophila cells. We will use the accurate U1 snRNP profile maps generated in Aim 1 to correlate with altered pre-mRNA splicing patterns, polyadenylation events and splicing control elements transcriptome-wide. Mapping U1 snRNP binding sites to the nuclear transcriptome will link pre-mRNA splicing patterns, splicing silencer elements and PCPA sites to decode the function of U1 snRNP-mediated post-transcriptional regulation at specific binding sites. These new methods will be transformative by allowing predictions about where and how U1 snRNP binding to nuclear pre-mRNA affects constitutive splicing, alternative splicing, surveillance by premature transcript cleavage and polyadenylation (PCPA) and alternative polyadenylation, all of which are profoundly perturbed in many disease states. The proposed research will reveal for the first time a bona-fide transcriptome-wide map of U1 snRNP binding to nuclear pre-mRNAs and allow the definition of molecular function of the U1 snRNP- mediated post-transcriptional regulatory pathways in both RNA surveillance and RNA processing of the human and Drosophila transcriptomes. This information has the potential to allow the development of new therapeutic strategies to treat disease through investigation and manipulation of U1 snRNP function.

项目概要/摘要分析 U1 snRNP 在核人和果蝇中的结合位置转录组。 D. Rio – P.I. 剪接体 U1 snRNP 在细胞核中发挥作用，影响前 mRNA 的选择性剪接和多腺苷酸化位点的使用，这是两个关键的基因表达机制。建议开发一种方法来全面绘制 U1 snRNP 的位置图果蝇和人类细胞的核转录组，并对核转录组的功能进行系统分类特定位点的剪接体 U1 snRNP 调节整体前 mRNA 剪接为了解决这个问题，我们将：1）开发全基因组范围的模式。用于表征 U1 snRNP 跨区域结合位点的作图和分析方法为此，我们将开发一种新型的人类和果蝇细胞的核转录组。与 RNase T1 核酸酶结合的高度特异性两步免疫亲和选择策略保护测定来表征 U1 snRNP（10 种蛋白质和一种非编码小 RNA）连接到内含子 5' 剪接位点、过早切割和聚腺苷酸化 (PCPA) 我们将开发新的计算方法来主要和拼接消音器元件。准确分析重要的 U1 snRNP 结合位点；2) 对 U1 snRNP 结合进行分类和定义位点如真实或神秘的 5' 剪接位点、转录位点或剪接消音器元件。为此，我们将在扰动 U1 snRNP 活性后使用最先进的 cDNA 测序我们将使用目标 1 中生成的准确的 U1 snRNP 配置图。与改变的前 mRNA 剪接模式、聚腺苷酸化事件和剪接控制相关将 U1 snRNP 结合位点映射到核转录组将链接元件。 mRNA前体剪接模式、剪接沉默元件和PCPA位点以解码U1的功能这些新方法将在特定结合位点介导 snRNP 介导的转录后调节。通过预测 U1 snRNP 与核前 mRNA 结合的位置和方式，实现变革影响组成型剪接、选择性剪接、过早转录本切割的监测和聚腺苷酸化（PCPA）和替代性聚腺苷酸化，所有这些都在许多疾病状态。拟议的研究将首次揭示 U1 的真实转录组范围图谱 snRNP 与核前 mRNA 结合并允许定义 U1 snRNP-的分子功能介导的RNA监视和RNA加工中的转录后调节途径该信息有可能允许人类和果蝇转录组。通过研究和操纵来开发新的治疗策略来治疗疾病 U1 snRNP 功能。