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

所有mRNA分子都受到转录后基因调控（PTGR）的影响，涉及剪接、切割和聚腺苷酸化、编辑、转运、稳定性和翻译的序列依赖性调节。最近引入的深度测序技术使得能够开发用于广泛绘制RNA结合蛋白（RBP）与其RNA靶位点之间相互作用位点的新方法。因此，现在有可能解决RBP和核糖核蛋白颗粒（RNP）与mRNA分子结合的相互依赖性和冗余性，并评估这些相互作用对生物体发育或正常和疾病状态背景下基因调控的贡献。揭示（冗余）序列元件和RNA-RBP相互作用的调控重要性对于解释调控RNA区域和非编码转录物中的人类遗传变异至关重要，这些变异越来越多地被全基因组深度测序发现。 研究核质转运中mRBP的灵感来自于我们意外的观察，即输出蛋白5（XPO 5），一种以前被认为专门负责前体microRNA核质转运的核质转运蛋白型转运蛋白，在大约2,000个mRNA中结合超过2,000个mRNA。8，000个确定的结合位点位于预测形成稳定茎环的区域。这些结果表明，描绘的RNA结合特性的核质转运蛋白将产生重要的见解的冗余和特异性的RNA运输。 该项目的主要目标是在序列和功能水平上识别和表征mRNA结合转运蛋白及其RNA靶点的相互作用网络，并建立将这些特征与RNA转运过程和PTGR联系起来的实验模型。我们将系统地研究人类转运蛋白/核转运蛋白/输出蛋白/Ran结合蛋白（KAP）和核输出因子TAP（NXF）蛋白的成员，同时确定在货物装载和释放过程中重要的转运适配器或因子。我们将通过三个具体目标来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。将鉴定由人KAP和NXF蛋白转运和调节的RNA靶标，并通过光活化核糖核苷增强交联和免疫沉淀（PAR-CLIP）确定靶位点和结合元件。跨文库的转录本上的结合位点的综合注释将允许鉴定用于组合和冗余调节的靶标。 目标二：与核质转运蛋白相互作用并参与RNA亚类转运的衔接RNA结合蛋白的鉴定。与转运蛋白相互作用的未知RBP将通过银染凝胶的靶向质谱法或基于SILAC的蛋白质组学进行全面鉴定。新鉴定的蛋白质的RNA靶标和结合位点将通过PAR-CLIP进行全局确定。 目的3：开发检测系统，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。将生成报告RNA以评估RBP在核质转运中的特定作用。此外，通过RNA-FISH可以在患病和健康的FFPE组织中观察到丰富的转运mRNA。现有的疾病相关表达和基因组变异数据将进行生物信息学挖掘，特别考虑RBP中的冗余和本研究发现的mRNA中的多个位点的出现，以确定可用于开发报告RNA的mRNA靶点。

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.