Posttranscriptional regulation by mRNA-binding shuttling and transport proteins

描述(申请人提供)：所有的信使核糖核酸分子都受到转录后基因调控(PTGR)的影响，包括剪接、切割和多聚腺苷化、编辑、运输、稳定性和翻译的序列依赖的调节。近年来，范式的转变 在我们对PTGR的理解中发生了变化，这是由发现调节RNA和广泛的RNA加工引发的，包括替代的聚腺苷酸化和剪接。这些进展导致人们意识到，几乎每个人类基因都受到一定程度的PTGR的影响。因此，了解靶RNA识别的机制以及编码在人类基因组中的数百种mRNA结合蛋白的功能是一项开放的挑战，需要多学科团队的共同努力。最近引入的深度测序技术使得精确定位RNA结合蛋白(RBPs)与其在人类细胞中的RNA靶点之间的相互作用位点的新方法的发展成为可能。现在才有可能解决限制性商业惯例和核糖核蛋白颗粒(RNPs)与mRNA分子结合的相互依赖和冗余，并在生物发育或正常和疾病状态的背景下评估它们对基因调控的贡献。这项应用的广泛目标是在序列、结构和功能水平上识别和表征mRNA结合蛋白的相互作用网络，特别是运输和穿梭--这是一种关键的PTGR机制，很少受到关注。一个驱动力的概念是，更大的类似染色质的RNP包装是否有助于运输和翻译调节。具体目标包括：(1)通过光激活-核糖核苷增强的交联和免疫沉淀(PAR-CLIP)技术(PAR-CLIP)为核质运输和穿梭蛋白质的综合小组确定目标RNA位点，包括所有蛋白质文库的深度测序和计算分析。(2)跨文库对转录本上结合位点的集成注释，使用概率图形建模方法从异质数据中识别流行的位点配置。PAR-CLIP数据辅以表达以及序列和RNA结构特征，将使识别组合和冗余调控的靶标成为可能。(3)开发实验系统，用于评估转运蛋白相互作用网络的扰动的表型结果，并阐明其在正常和疾病状态下的作用。(4)进行生物物理和结构研究，以证实生化和计算结果。天然的和设计的代表RNA配体的RNA识别元件(RRE)将通过与它们各自的重组蛋白共结晶来强调获得更大的RNP结构。因此，拟议的工作将建立第一个将蛋白质-RNA相互作用的全球图谱与RNA运输联系起来的理论和实验模型，为未来对PTGR的系统范围研究奠定基础。 与公共卫生相关：在mRNA水平上控制基因表达发挥着比先前预期更大的作用。这一建议旨在解决数十个穿梭于细胞核和细胞质之间的mRNA结合蛋白以及参与核质运输的蛋白质的共同贡献。这一方法将识别mRNAs中的许多重要调控区域，并发现mRNAs和RNA结合蛋白在疾病状态下特别容易受到突变和放松调控的影响。

DESCRIPTION (provided by applicant): All mRNA molecules are subject to posttranscriptional gene regulation (PTGR) involving sequence-dependent modulation of splicing, cleavage and polyadenylation, editing, transport, stability, and translation. In recent years, a paradigm shift has occurred in our understanding of PTGR, triggered by discoveries of regulatory RNAs and extensive RNA processing including alternative polyadenylation and splicing. These developments have led to the realization that virtually every human gene is subject to some degree of PTGR. Understanding the mechanisms of target RNA recognition, and the function of the hundreds of mRNA-binding proteins encod- ed in the human genome, is therefore an open challenge that requires the joint effort of multidisciplinary teams. The recent introduction of deep sequencing technologies has enabled the development of new methods for precisely mapping interaction sites between RNA-binding proteins (RBPs) and their RNA target sites in human cells. It is only now possible to resolve interdependencies and redundancies of binding of RBPs and ribonucleoprotein particles (RNPs) to mRNA molecules and evaluate their contribution to gene regulation in the context of organismal development or normal and disease states. The broad aim of this application is to identify and characterize the interaction networ of mRNA-binding proteins at the sequence, structural and functional level, with a particular focus on transport and shuttling - crucial PTGR mechanisms that have received little attention. A driving concept is whether larger "chromatin"- like packaging of RNPs facilitates transport and translational regulation. The specific aims include: (1) Global identification of target RNA site for a comprehensive panel of nucleocytoplasmic transport and shuttling proteins by Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR- CLIP) involving deep sequencing of libraries for all proteins and computational analysis. (2) Integrated annotation of binding sites on transcripts across libraries, using a probabilistic graphical modeling approach to identify prevalent site configurations from heterogenenous data. PAR-CLIP data, complemented by expression as well as sequence and RNA structural features, will allow for the identification of targets for combinatorial and redundant regulation. (3) Development of experimental systems for assessment of the phenotypic outcomes of the perturbation of the transporter interaction network and elucidation of its role in normal and disease states. (4) Perform biophysical and structural studies substantiating the biochemical and computational findings. Natural, as well as designed RNA-recognition element (RRE)-representing RNA ligands, will by co-crystallized with their respective recombinant proteins placing emphasis on obtaining larger RNP structures. The proposed work will thus establish the first theoretical and experimental models that relate global maps of protein-RNA interactions to RNA transport, setting the stage for future systems-wide studies of PTGR. PUBLIC HEALTH RELEVANCE: The control of gene expression at the mRNA level plays a larger role than previously anticipated. This proposal aims to resolve the joint contribution of dozens of mRNA-binding proteins shuttling between nucleus and cytoplasm, as well of proteins participating in nucleocytoplasmic transport. This approach will identify numerous important regulatory regions in mRNAs, as well as uncover mRNAs and RNA-binding proteins particularly vulnerable to mutation and deregulation in disease states.