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。

• 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
• 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' 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

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.

项目总结/摘要 分析U1 snRNP在人和果蝇细胞核中的结合位置 转录组 D. Rio - P.I. 剪接体U1 snRNP在细胞核中发挥作用，影响前体mRNA选择性剪接 和多聚腺苷酸化位点的使用，这是两个关键的基因表达机制。这个目标 建议是开发一种方法来全面绘制U1 snRNP的位置， 果蝇和人类细胞的核转录组和系统分类的功能， 剪接体U1 snRNP在特定位点调节整体前mRNA剪接， 多聚腺苷酸化模式。为了解决这个问题，我们将：1）开发全基因组 用于表征U1 snRNP跨细胞的结合位点的作图和分析方法 人和果蝇细胞的核转录组。为了做到这一点，我们将开发一个新的， 与RNase T1核酸酶组合高特异性两步免疫亲和选择策略 保护试验，以表征U1 snRNP（10种蛋白质的复合物， 一个非编码的小RNA）到内含子5'剪接位点，过早切割和多聚腺苷酸化（PCPA） 站点和拼接消音器元件。我们将开发新颖的计算方法来广泛和 准确描述显著的U1 snRNP结合位点; 2）分类和定义U1 snRNP结合 作为真正或隐蔽的5 ′剪接位点、剪接位点或剪接沉默元件的位点。 为此，我们将在干扰U1 snRNP活性后使用最先进的cDNA测序 在人类和果蝇细胞中。我们将使用Aim 1中生成的准确U1 snRNP配置文件图 与改变的前mRNA剪接模式、多聚腺苷酸化事件和剪接控制相关 转录组范围内的元素。将U1 snRNP结合位点定位到核转录组将连接 前体mRNA剪接模式、剪接沉默元件和PCPA位点，以解码U1的功能 snRNP介导的转录后调节在特定的结合位点。这些新方法将 通过预测U1 snRNP在何处以及如何与核前体mRNA结合来实现变革 影响组成性剪接、选择性剪接、通过过早转录物切割的监视， 多聚腺苷酸化（PCPA）和替代性多聚腺苷酸化，所有这些都是深刻的扰动， 许多疾病状态。 这项拟议中的研究将首次揭示U1的真正转录组范围的图谱。 snRNP与核前mRNA结合，并允许定义U1 snRNP的分子功能。 介导的转录后调节途径在RNA监视和RNA加工中的作用 人类和果蝇的转录组。这些信息有可能使 开发新的治疗策略，通过研究和操纵 U1 snRNP功能。