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

我们的研究计划题为：GRPE：了解RNA代谢中的作用和生物技术的应用，将研究基因组RNA包装、mRNA稳定性、避免无意义介导的RNA衰退(NMD)途径，以及最重要的是，改善慢病毒基因或shRNA传递的机制，即世界各地的细胞生物学家常用的系统。显然，NSERC发现拨款不能支持这里列出的所有项目，但艺术博士正在与西方、麦吉尔、哥伦比亚大学、加州大学洛杉矶分校、俄亥俄州立大学、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，支持特定的项目，并为NSERC支持的研究生提供高级培训。*删除GRPE不会影响病毒生产，但会导致vgRNA包装减少50倍，病毒传染性下降两倍(Chamanian等人)。细胞宿主和微生物(2013)。该计划的项目1将涉及鉴定猫(FIV)、猿猴(SIV)、马(EIAV)和人类慢病毒(HIV-1和HIV-2)中的GRPE样分子。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。随着各种慢病毒中GRPE类似元件的鉴定，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，以确定可能影响mRNA稳定性和新陈代谢的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。我们提出了三个高阶GRPE RNA相互作用的模型，将使用改进的形状、铅++足迹、RNA核磁共振和小角X射线散射(SAXS)分析进行验证。慢病毒Gag蛋白从全长mRNA翻译而来，并终止于GAG的内部mRNA终止密码子，可以激活NMD途径，该途径由UPF1蛋白在长3‘非翻译区(3’UTR)上的积累触发，并随后降解该vgRNA/mRNA。在项目3中，我们将首先调制已知的翻译成分(PABA、eRF1、eRF3、CBP20、CBP80、eEF2、eIF4E/F等)，以研究其对核糖体移码(RFS‘ing)、Gag-Pol翻译、vgRNA封装和vgRNA稳定性/降解的影响。然后，我们将利用RNA下拉和质谱分析相结合的方法来鉴定与GRPE相互作用的新的RNA结合蛋白。推测相互作用的蛋白质将受到击倒、显性阴性和敲除分析的影响。这些蛋白质也将在含有GRPE的慢病毒RNA上带有或不带有慢病毒Gag蛋白的足迹(项目2)。最后，项目4将确定GRPE对慢病毒基因传递载体的影响。

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.**