Abstract

While standard RNA -Seq experiments can be used to monitor the abundance of every messenger RNA (mRNA) species in a cell, this is only an inaccurate proxy for actual levels of protein synthesis, since different mRNAs can have strikingly different translation efficiencies. Further, mRNA translation efficiency can vary in response to environmental and developmental stimuli. In contrast, Ribosome Profiling (Ribo-Seq) provides a direct measurement of protein synthesis. Ribosome profiling relies on the fact that a translating ribosome protects ~30 nucleotides of mRNA. An RNA nuclease is used to digest unprotected RNA , the 30-nt ribosome-protected fragments (RPFs) are purified for deep sequencing, and the sequenced fragments are mapped back to the transcriptome, to give a global snapshot of protein synthesis in the cell. Ribosome profiling has proven to be increasingly valuable in studies of the translation process, for example, in the discovery of novel translated open reading frames (ORFs), determination of elongation rates, and identification of sites of ribosome pausing. It also has broad application in the analysis of global protein synthesis and has been exploited in studies of infectious diseases, cell growth, differentiation and development, mitochondrial gene expression, and cell stress. In collaboration with the Brierley lab (http://www.path.cam.ac.uk/directory/ian-brierley) we have been applying Ribo-Seq and parallel RNA -seq to a variety of virus species to study the kinetics of virus gene expression, unusual transcriptional and translational mechanisms employed by viruses, and host responses to virus infection at both the transcriptional and translational levels.