Grant: Discovery Grants Program - Individual

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.**