Absolute calibration of ribosome profiling assesses the dynamics of ribosomal flux on transcripts

Abstract

AbstractRibosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool to unveil the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate means to determine overall ribosome numbers in transcripts. Here, we overcome these hurdles through the development of “Ribo-FilterOut”, which is based on the separation of footprints from ribosome subunits by ultrafiltration, and “Ribo-Calibration”, which relies on external spike-ins of stoichiometry-defined mRNA-ribosome complexes. A combination of these approaches measures the absolute numbers of ribosomes on transcripts, the translation initiation rate, and the numbers of translation events on a transcript before its decay, all in a genome-wide manner. Moreover, our method revealed the allocations of ribosomes under heat shock stress, during aging, and across cell types. Our strategy transforms the ribosome profiling technique from relative to absolute quantification of translation.