Genome-wide Identification of Coordinated Post-transcriptional Gene Regulation by RNA Binding Proteins

项目总结/摘要 RNA结合蛋白（RBP）是基因表达的主要控制者， 转录基因调控（PTGR），并已被牵连在许多发病 疾病，包括神经退行性疾病和癌症进展;然而， 在全基因组范围内协调控制PTGR仍然知之甚少。根据 根据最近对RNA相互作用组的调查，人类基因组估计编码约1500个 RBP，其中约600个特异性结合mRNA。新开发的下一代应用 测序工具，如PAR-CLIP，使RBP在全基因组上的定位成为可能。 规模，揭示了单个RBP可以结合高达50%的表达mRNA。鉴于 RBP的多样性及其针对转录组重要部分的倾向，a 单个转录物通常被多个RBP结合。因此，PTGR的最终结果是 这取决于与转录物结合的RBP的组合控制。有趣的是，RBP 靶向相同转录本的基因并不总是彼此独立地起作用。几 RBP协同或竞争性结合mRNA以控制PTGR的实例， 在文献中有报道。然而，这些研究仅通过 限制性商业惯例很少，因此是对其活动的高度集中的观察。现在仍不知道 RBP如何在全基因组范围内协调PTGR的组合控制。考虑到他们 与许多疾病的临床相关性，迫切需要确定调节 RBP在基因组水平上的相互作用。如果没有这些信息， 疾病状态和PTGR机制之间的联系将是不完整的。目标1 每一个公开可用的和内部生成的PAR-CLIP数据集都将用于识别限制性商业惯例 共同靶向相似的mRNA组或结合沿mRNA的共定位位点沿着。数据分析将 使用已建立的PAR-CLIP分析管道结合自定义 分析脚本。在目标2中，协调组合PTGR的机制将是 通过RNA-seq、免疫共沉淀和原位杂交确定候选RBP对 共定位实验。第一对待表征的RBP，即胃L1和CDK 1， 已经通过我们对公开的PAR-CLIP数据的初步分析确定。 这些结果将为分析PTGR的协调作用提供一个框架， 限制性商业惯例，从而为研究该机制的研究人员提供关键知识， 临床重要基因的调控系统。

PROJECT SUMMARY/ABSTRACT RNA binding proteins (RBPs) are major controllers of gene expression through post- transcriptional gene regulation (PTGR) and have been implicated in the onset of numerous disorders, including neurodegenerative diseases and cancer progression; however, the coordinated control of PTGR on a genome-wide scale remains poorly understood. According to recent surveys of the RNA interactome, the human genome is estimated to encode ~1500 RBPs, of which ~600 specifically bind mRNAs. Application of newly developed next-generation sequencing tools, such as PAR-CLIP, that enable the mapping of RBPs on a genome-wide scale, revealed that a single RBP can bind up to 50% of expressed mRNAs. Given the multiplicity of RBPs and their propensity to target significant fractions of the transcriptome, a single transcript is often bound by multiple RBPs. As such, the final outcome for PTGR is dependent on the combinatorial control of the RBPs bound to a transcript. Interestingly, RBPs targeting the same transcript do not always act independently from each other. Several instances of RBPs either cooperatively or competitively binding mRNAs to control PTGR have been reported in the literature. However, these studies identified coordinated PTGR by only a few RBPs and is therefore a highly focused observation of their activity. It remains unknown how RBPs coordinate combinatorial control of PTGR on a genome-wide scale. Considering their clinical relevance to numerous diseases, there is a critical need to identify regulatory interactions of RBPs on a genomic scale. Without such information, scientific understanding of the link between the disease states and mechanisms of PTGR will be incomplete. In Aim 1 every publicly available and in-house generated PAR-CLIP dataset will be used to identify RBPs that co-target similar sets of mRNAs or bind co-localized sites along mRNAs. Data analysis will be carried out using an established PAR-CLIP analysis pipeline in combination with custom analytical scripts. In Aim 2 the mechanism of coordinated combinatorial PTGR will be determined for candidate pairs of RBPs through RNA-seq, co-immunoprecipitation and in situ co-localization experiments. The first pair of RBPs to be characterized, METTL1 and CDK1, were already identified through our preliminary analysis of publicly available PAR-CLIP data. These results will provide a framework for the analysis of PTGR by the coordinated action of RBPs, thereby providing critical knowledge to researchers studying the mechanism or regulatory system of clinically important genes.