iTP-Seq: a scalable profiling method to study context-dependent translational events in vitro

Uneven translation rates resulting from the mRNA context, nascent amino acid sequence, or a variety of extraneous factors are key to controlling the expression level, folding, and regulation of the proteome. Although ribosome profiling (Ribo-Seq) is the method of choice to provide genome-wide information on protein synthesis in vivo, tools to systematically study this process in vitro are also needed. To this end, we developed inverse toeprinting coupled to next-generation sequencing (iTP-seq), a scalable profiling method that generates ribosome-protected mRNA fragments without a priori knowledge of the translated sequences (Ref. 1: Seip et al. 2018, Life Science Alliance). Protected mRNA fragments obtained by iTP-Seq consist of the entire coding region upstream of stalled ribosomes, making it possible to work with random or focused transcript libraries rather than sequenced genomes onto which short mRNA footprints must be mapped. We have used iTP-seq to investigate the context dependence of ribosome-targeting antibiotics (Ref. 2: Beckert et al. 2021, Nature Communications; Ref 3: Leroy et al. 2023, Nature Chemical Biology). More generally, iTP-Seq could be used to study any ribosome-related process in which the mRNA sequence or context impact the rate of translation. Here, we present a reproducible protocol for iTP-Seq, which substantially reduces the time and effort required to generate sequencing libraries. This protocol consists in 12 main steps: DNA library amplification, in vitro transcription, 5'end mRNA biotinylation, in vitro translation, RNase R digestion, retention on straptavidin-coated beads, linker ligation, reverse transcription, second-strand syntesis, restriction enzyme digestion, PCR amplification and NGS-adapters addition. The time required to complete a single round of iTP-seq is approximately 10 days (Figure 1: Method overview).