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