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

项目摘要/摘要 人和果蝇核内U1核糖核酸核糖核蛋白结合位置的研究 抄本。 D.里约热内卢-P.I. 剪接体U1 SnRNP在细胞核中的作用影响前mRNA的选择性剪接 和多聚腺苷酸化位点利用，这是两种关键的基因表达机制。这样做的目的是 建议开发一种方法来全面绘制U1 SNRNP在整个 果蝇和人类细胞的核转录本，并对其功能进行系统分类 剪接体U1SnRNP在调控全球Pre-mRNA剪接中的作用 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 U1-SnRNP跨膜结合位点的作图和图谱分析方法 人和果蝇细胞的核转录本。为了做到这一点，我们将开发一部小说和 结合RNaseT1核酸酶的高特异性两步免疫亲和选择策略 保护试验以表征U1SnRNP(由10种蛋白质和 一个非编码的小RNA)到内含子5‘剪接点，过早切割和多聚腺苷酸化(PCPA) 位置和拼接消音器元件。我们将开发新的计算方法，以广泛和 准确描述重要的U1短链RNP结合位点；2)分类和定义U1短链RNP结合 如骨性或神秘的5‘剪接位点、远端转录位点或剪接消音器元件。 为此，我们将使用最先进的cdna测序技术，在干扰了U1的nRNP活性之后。 在人类和果蝇细胞中。我们将不会使用在AIM 1中生成的更准确的U1 SnRNP剖面图 与改变的前信使核糖核酸剪接模式、多聚腺苷化事件和剪接控制相关 转录组中的所有元素。将U1 SnRNP结合位点映射到核转录组将链接 前mRNA剪接模式、剪接沉默元件和PCPA位点对U1功能的解码 SNRNP介导的特定结合位点的转录后调控。这些新方法将是 通过允许预测U1 SnRNP在哪里以及如何与核Pre-mRNA结合来实现变革性 影响组成性剪接、选择性剪接、通过过早转录切割和 多聚腺苷酸化(PCPA)和交替多聚腺苷酸化，所有这些都深深地扰乱了 许多疾病状态。 这项拟议中的研究将首次揭示U1真实的转录组范围的图谱 SnRNP与核前mRNAs结合，并允许定义U1 SnRNP-的分子功能 介导的转录后调控通路在RNA监视和RNA加工中的作用 人类和果蝇的转录本。此信息有可能允许 通过调查和操作治疗疾病的新治疗策略的发展 U1 SNRNP函数。

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.