Exploring hidden determinants of splicing with genome-targeted proximity labeling

项目总结/摘要 绝大多数人类基因是多外显子的，并进行选择性剪接以产生多个外显子。 产生的转录物和蛋白质的种类成倍增加。在像癌症这样的疾病中， 总体异常，与正常组织高度不同，内含子保留水平增加，替代 剪接和从头剪接点的使用。RNA生物学中一个基本的未回答的问题是， 包括拼接代码的许多层被解释以产生给定的拼接结果，以及这些层如何被解释以产生给定的拼接结果。 过程在人类疾病中变得失调。发生在 DNA和那些作用于RNA的分子表明，染色质状态尤其可能参与了协调 选择性剪接我们已经观察到剪接报告基因构建体的随机基因组整合导致 在对应于每种可能的剪接结果的细胞群体中，这表明了基因组背景在细胞中的作用。 整合位点对剪接有很大影响。然而，主要由于技术限制， 关于染色质和RNA剪接之间相互调节的确切性质，仍有许多未知之处 以及可能涉及的潜在因素。该提案的目标是应对当前的挑战 阻碍从头发现，并制定公正的实验方法，以揭示隐藏的决定因素， 染色质水平的剪接调控。我们的方法旨在将剪接结果与 相关基因组区域的无偏蛋白质组学分析，以识别所有潜在的染色质 参与实施特定剪接模式的特征。我们还建议设计新的系统 功能上验证表观基因组在剪接调控中的直接作用，并随意调节剪接。我们 这些发现对表观遗传学和RNA生物学都有意义，特别是它们之间的相互作用 影响基因表达的各种过程。它也有可能揭示 在人类癌症中观察到的广泛表观遗传和剪接失调的原因和后果。 重要的是，我们的方法不仅限于剪接的研究，还将使无偏见的发现， 参与其他基本过程的机制。

PROJECT SUMMARY/ABSTRACT The vast majority of human genes are multi-exonic and undergo alternative splicing to generate a several- fold increase in the variety of transcripts and proteins that are produced. In diseases like cancer, splicing is often globally aberrant and highly distinct from normal tissues, with increased levels of intron retention, alternative splicing and usage of de novo splicing junctions. A fundamental unanswered question in RNA biology is how the many layers that comprise the splicing code are interpreted to produce a given splicing outcome, and how these processes become dysregulated in human diseases. The extensive crosstalk between processes occurring on the DNA and those that act on the RNA suggest the chromatin state in particular may participate in orchestrating alternative splicing. We have observed that random genomic integration of a splicing reporter construct results in populations of cells corresponding to each possible splicing outcome, suggesting the genomic context at the site of integration exerts a powerful influence on splicing. However, due primarily to technological limitations, much remains unknown about the precise nature of the regulatory interplay between chromatin and RNA splicing and the potential factors that may be involved. The goal of this proposal is to address the current challenges impeding de novo discovery and develop unbiased experimental approaches to reveal hidden determinants of splicing regulation at the chromatin level. Our approach is designed to directly connect splicing outcome with unbiased proteomic profiling of the associated genomic region, in order to identify all of the potential chromatin features that are involved in enforcing a particular pattern of splicing. We also propose to engineer new systems to functionally validate a direct role for the epigenome in splicing regulation and to modulate splicing at will. Our findings have implications for both epigenetics and RNA biology, and in particular, how their interactions influence the various processes involved in gene expression. It also has the potential to shed insight into the causes and consequences of the widespread epigenetic and splicing dysregulation observed in human cancer. Importantly, our approach is not limited to the study of splicing and will also enable unbiased discovery of the mechanisms involved in other fundamental processes.