A quick and simplified method (QuickRibo-mRNA) for isolation of ribosome protected mRNA fragments for translatome identification

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The goal of this proposal is to establish and validate a novel method for the isolation of ribosome protected mRNA footprints for translatome identification. Translatome or Ribosome profiling (RP), a deep sequencing technique of ribosome-protected mRNA footprints, is a powerful method that gives a “snapshot” of actively translating mRNA. Revolutionizing the capacity to capture every step of mRNA translation at sub-codon resolution, it potentially allows one to monitor the rate and location of mRNA translation in the cell and annotate the new protein coding sequences of genomes. RP, therefore, affords a vast opportunity to explore and model complex cellular processes in real time. This methodology has been mined extensively in numerous diseases including cancer, immunology and neurology. RP identified several new micro-peptides and protein from COVID mRNA, further expanding its potential as a novel target-identification method. In this context, RP has emerged as a potent method to identify mRNA frameshifting and novel ORFs, and providing valuable insights for understanding functional elements of the genome, disease pathogenesis and evaluation of impact treatment in the case of mRNA therapeutics and cancer therapy in a gene and the tissues specific manner. However, RP applications are stunted and limited to specialized labs because it requires expertise, specialized instruments and time-consuming - taking up to ~5 days on an average. In turn, this affects the cost, quality of the final data and thereby obscuring the mRNA translational landscape. Given the power and potential for widespread use, there is an urgent and unmet need for alternative/improved RP technology similar to RNA purification methods which are less time consuming, free of artifacts and inexpensive. In Phase-I application, we proposed two specific aims to refine the QuickRibo-mRNA method, validate and compare it with current RP methods to establish its efficacy. In aim-1 we will establish and validate the QuickRibo-mRNA methodology in various conditions and cell types in obtaining uniform ribosome protected mRNA fragments with improved percent read mapping to coding sequences. In aim-2 we will compare the efficacy of QuickRibo-mRNA with known ribosome profiling technologies. We believe that phase-I proposal will provide a benchmark for phase II, which can streamline the QuickRibo-mRNA method for performing ribosome profiling with high reproducibility and efficiency. In the end RP will be done proficiently in the simplest way with less time, instrumentation and expertise to identify gene “expression with reading frames and translation efficiency” before making any conclusions entirely based on RNA-gene “expression profile”.