Active Ribosome Profiling with RiboLace

Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution. [Display omitted] •RiboLace isolates ribosomes in active translation by antibody-free and tag-free pull-down•RiboLace works reliably with low amounts of input material in vitro and in vivo•RiboLace provides positional data of active ribosomes with nucleotide resolution•RiboLace estimates translation levels and predicts protein levels with accuracy Clamer et al. present RiboLace, a method for isolating active ribosomes and associated proteins, intact mRNAs, or ribosome-protected fragments. RiboLace accurately quantifies translation levels, providing positional data of active ribosomes with nucleotide resolution. Requiring lower input than current ribosome profiling protocols, RiboLace can be used with challenging biological samples.