GRPE: Understanding the role in RNA metabolism and applications for biotechnology

我们的研究计划，题为：GRPE：了解在RNA代谢和生物技术应用中的作用将研究基因组RNA包装，mRNA稳定性，避免无义介导的RNA衰变（NMD）途径的机制，最重要的是，以改善慢病毒基因或shRNA的交付，即世界各地的细胞生物学家常用的系统。显然，NSERC发现补助金无法支持本文概述的所有项目，但Arts博士正在与Western，麦吉尔，哥伦比亚，UCLA，俄亥俄州州立大学，华盛顿大学，斯特拉斯堡的CNRS和美国国家实验室的专家团队合作，以支持特定项目并为NSERC支持的研究生提供高级培训。GRPE的缺失不影响病毒产生，但引起vgRNA包装减少>50倍和病毒感染性下降2个对数（Chamanian等人Cell Host and Microbe 2013）。该计划的项目1将涉及猫（FIV），猴（SIV），马（EIAV）和人类慢病毒（HIV-1和-2）中GRPE样元件的鉴定。 通过使用各种缺失和点突变，我们将研究GRPE在vgRNA包装，病毒mRNA翻译和核糖体移码，病毒RNA稳定性和NMD途径降解中的作用。通过鉴定各种不同慢病毒中的GRPE样元件，我们可以对来自RNAseq和转录组分析的哺乳动物mRNA进行序列比对，以鉴定可能影响mRNA稳定性和代谢的推定GRPE同源物。在GRPE的线性映射之后，项目2将确定这些GRPE功能所需的二级和三级GRPE结构。我们已经提出了三个模型的高阶GRPE RNA相互作用，将使用修改后的SHAPE，Pb++足迹，RNA NMR和小角X射线散射（SAXS）分析进行测试。慢病毒Gag蛋白从全长mRNA翻译并在Gag的内部mRNA终止密码子处终止可以激活NMD途径，这是由UPF 1蛋白在长的3 '非翻译区（3' UTR）上的积累触发的，随后降解该vgRNA/mRNA。在项目3中，我们将首先调节已知的翻译组分（PABA，eRF 1，eRF 3，CBP 20，CBP 80，eEF 2，eIF 4 E/F等），以检查对核糖体移码（RFS'ing），Gag-Pol翻译，vgRNA降解和vgRNA稳定性/降解的影响。然后，我们将利用RNA下拉和质谱的组合来识别与GRPE相互作用的新的RNA结合蛋白。假定的相互作用蛋白将进行敲低、显性阴性和敲除分析。这些蛋白质也将在含有GRPE的慢病毒RNA上具有或不具有慢病毒Gag蛋白的情况下被足迹标记（项目2）。 最后，项目4将确定GRPE对慢病毒基因递送载体的影响。

Our program of study, entitled:GRPE: Understanding the role in RNA metabolism and applications for biotechnology will study the mechanisms involved in genomic RNA packaging, mRNA stability, avoidance of the nonsense mediated RNA decay (NMD) pathway, and most importantly, to improve lentiviral gene or shRNA delivery, i.e. a system commonly used by cell biologists around the world. Obviously, the NSERC Discovery Grant cannot support all the projects outlined herein but Dr. Arts is collaborating with a team experts at Western, McGill, Columbia, UCLA, Ohio State Univ, Univ of Washington, CNRS at Strasbourg, and US National laboratories to support specific projects and to provide advanced training for the NSERC supported graduate students.***Deletion of the GRPE does not impact virus production but caused a >50 fold reduction in vgRNA packaging and a two log drop in virus infectivity (Chamanian et al. Cell Host and Microbe 2013). Project 1 of this program will involve identification of GRPE-like element in feline (FIV), simian (SIV), equine (EIAV), and human lentiviruses (HIV-1 and -2). By using various deletion and point mutations, we will then examine the role of GRPE in vgRNA packaging, viral mRNA translation and ribosomal frameshifting, viral RNA stability and degradation by NMD pathways. With identification of GRPE-like elements in various diverse lentiviruses, we can then perform sequence alignments on mammalian mRNA derived from RNAseq and transcriptome analyses to identify putative GRPE homologues that may impact mRNA stability and metabolism. Following the linear mapping of the GRPE, Project 2 will determine both secondary and tertiary GRPE structures necessary for these GRPE functions. We have proposed three models for higher order GRPE RNA interactions which will be tested using modified SHAPE, Pb++ footprinting, RNA NMR, and small-angle X-ray scattering (SAXS) analyses. Translation of the lentiviral Gag protein from the full length mRNA and termination at an internal mRNA stop codon for Gag could activate the NMD pathway, triggered by the accumulation of UPF1 protein on the long 3'untranslated region (3'UTR) and subsequently degrade this vgRNA/mRNA. In Project 3, we will first modulate the known components of translation (PABA, eRF1, eRF3, CBP20, CBP80, eEF2, eIF4E/F, and others) to examine the impact on ribosomal frameshifting (RFS'ing), Gag-Pol translation, vgRNA encapsidation, and vgRNA stability/degradation. We will then utilize a combination of RNA pulldowns and mass spectrometry to identify new RNA binding proteins interacting with the GRPE. Putative interacting proteins will be subject to knockdown, dominant negative, and knockout analyses. These proteins will also be footprinted (Project 2) with or without lentiviral Gag proteins on the GRPE-containing lentiviral RNA. Finally, Project 4 will determine the impact of GRPE on lentiviral gene delivery vectors.**