Dissecting the role of shuttling mRNA binding proteins in nucleocytoplasmic RNA transport

所有的mRNA分子都受到转录后基因调控(PTGR)的影响，包括序列依赖的剪接、切割和聚腺苷酸化、编辑、运输、稳定性和翻译。最近引入的深度测序技术使人们能够开发新的方法来广泛地定位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。因此，现在有可能解决限制性商业惯例和核糖核蛋白颗粒(RNP)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这些相互作用对基因调控的贡献。揭示RNA-RBP相互作用的(冗余)序列元件和调控重要性，对于解释基因组深度测序日益发现的调控RNA区域和非编码转录本中的人类遗传变异将是至关重要的。 研究mRBPs在核质运输中的灵感来自于我们意外的观察到，Exportin 5(XPO5)是一种核粘附素类型的转运蛋白，以前被认为是专门负责前microRNAs的核质运输，它大约与2000多个mRNAs结合。8,000个确定的结合位点驻留在预测形成稳定茎环的区域。这些结果表明，描绘核质转运体的RNA结合特性将对RNA转运体的冗余和特异性产生重要的见解。 该项目的主要目标是在序列和功能水平上识别和表征mRNA结合运输蛋白及其RNA靶标的相互作用网络，并建立将这些特性与RNA运输过程和PTGR联系起来的实验模型。我们将系统地研究人类转运素/核粘附素/Exportin/Ran结合蛋白(KAP)和核输出因子TAP(NXF)蛋白的成员，同时识别在货物装卸过程中重要的运输适配器或因子。我们将通过三个具体目标解决这一具有挑战性的问题： 目的1：全面鉴定核质转运蛋白的靶RNA位点。将识别由人KAP和NXF蛋白运输和调节的RNA靶标，并通过可光激活的核糖核苷增强的交联和免疫沉淀(PAR-CLIP)来确定靶点和结合元件。跨文库转录本上结合位点的综合注释将允许识别组合和冗余调控的靶标。 目的2：鉴定与核质转运蛋白相互作用并参与RNA亚群转运的适配RNA结合蛋白。与转运蛋白相互作用的未知限制性商业惯例将通过银染凝胶的靶向质谱学或基于SILAC的蛋白质组学进行全球鉴定。新发现的蛋白质的RNA靶标和结合位点将由PAR-CLIP全局确定。 目的3：建立检测系统以询问转运蛋白相互作用网络，并阐明其在正常和疾病状态下的作用。将产生报告RNA，以评估限制性商业惯例在核质运输中的具体作用。此外，在患病和健康的FFPE组织中，RNA-FISH将显示丰富的转运的mRNAs。现有的疾病相关表达和基因组变异数据将被生物信息挖掘，具体考虑到限制性商业惯例中的冗余和本研究发现的mRNAs中的多个位点出现，以确定可用于开发报告RNAs的mRNA靶标。

All mRNA molecules are subject to posttranscriptional gene regulation (PTGR) involving sequence-dependent modulation of splicing, cleavage and polyadenylation, editing, transport, stability, and translation. The recent introduction of deep sequencing technologies has enabled the development of new methods for broadly mapping interaction sites between RNA-binding proteins (RBPs) and their RNA target sites in human cells. Therefore, it is now possible to resolve interdependencies and redundancies of binding of RBPs and ribonucleoprotein particles (RNPs) to mRNA molecules and evaluate the contribution of these interactions to gene regulation in the context of organismal development or normal and disease states. Uncovering the (redundant) sequence elements and the regulatory importance of RNA-RBP interactions will be critical to interpret human genetic variation in regulatory RNA regions and non-coding transcripts increasingly uncovered by genome-wide deep sequencing. The inspiration to study mRBPs in nucleocytoplasmic transport came from our unexpected observation that exportin 5 (XPO5), a karyopherin-type transporter previously thought to be exclusively responsible for the nucleocytoplasmic transport of pre-microRNAs, bound more than 2,000 mRNAs at approx. 8,000 defined binding sites residing in regions predicted to form stable stem-loops. These results indicate that delineating the RNA binding properties of the nucleocytoplasmic transporters will yield important insights in the redundancies and specificities of RNA transport. The major goals of this project are to identify and characterize the interaction network of mRNA-binding transport proteins and their RNA targets at a sequence and functional level and to establish experimental models that relate these features to RNA transport processes and PTGR. We will systematically study the members of the human transportin/karyopherin/exportin/Ran-binding proteins (KAP), and the nuclear export factor TAP (NXF) proteins, and at the same time identify transport adapters or factors important during cargo loading and release. We will address this challenging problem through three specific aims: Aim 1: Comprehensive identification of target RNA sites for nucleocytoplasmic transport proteins. RNA targets transported and regulated by human KAP and NXF proteins will be identified and the target sites and binding elements will be determined by Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP). Integrated annotation of binding sites on transcripts across libraries will allow for the identification of targets for combinatorial and redundant regulation. Aim 2: Identification of adapter RNA binding proteins interacting with nucleocytoplasmic transporters and involved with the transport of subsets of RNA. Unknown RBPs interacting with transporters will be identified either by targeted mass-spectrometry from silver-stained gels, or globally by SILAC based proteomics. The RNA targets and binding sites for newly identified proteins will be globally determined by PAR-CLIP. Aim 3: Development of assay systems to interrogate the transporter interaction network and elucidation of its role in normal and disease states. Reporter RNAs will be generated to assess the specific role of RBPs in nucleocytoplasmic transport. In addition, abundant transported mRNAs will be visualized by RNA-FISH in diseased and healthy FFPE tissue. Existing disease-relevant expression and genome variation data will be bioinformatically mined with specific consideration of the redundancies in RBPs and multiple site occurrences in mRNAs uncovered by this study to identify mRNA targets that can be used to develop reporter RNAs.