Beginners guide to ribosome profiling

Abstract

To synthesize proteins, cells must first transcribe an mRNA which specifies the sequence of amino acids, the building blocks of proteins. The next step involves translation of this mRNA into protein using the cell’s protein-synthesizing machinery called ribosomes. Many gene expression studies rely solely on RNA-seq, which provides information of relative abundances of mRNAs in a cell; however, RNA-seq data ignore gene regulation at the translational level. Developed by Nicholas Ingolia and Jonathan Weissman, ribosome profiling (Ribo-seq) is a technique that provides a genome-wide view of in vivo translation. Ribo-seq is based on the principle that a translating ribosome protects a short stretch of mRNA within its structure. Once ribosomes are ‘frozen’ in the act of translation using translation elongation inhibitors, RNA-digesting enzymes known as RNases can be added to destroy any mRNA that is unprotected by the arrested ribosomes. After RNase digestion, ribosomes are enriched and the ribosome-protected mRNA is then isolated and converted into Illumina-compatible cDNA libraries. These ribosome-protected mRNA fragments are commonly called RPFs or ribosome footprints. Mapping these sequenced RPFs to the transcriptome provides a ‘snapshot’ of translation that reveals the positions and densities of ribosomes on individual mRNAs transcriptome-wide. This snapshot can help determine which proteins were being synthesized in the cell at the time of the experiment. Ribo-seq enables the identification of alternative mRNA translation start sites, the confirmation of annotated open reading frames (ORFs) as well as upstream (uORFs) that may be involved in the regulation of translation, the distribution of ribosomes on an mRNA and the rate at which ribosomes decode codons