Effect of SRI-41315 on translation termination in rabbit reticulocyte lysate-based in vitro translation system
Translation termination is an essential cellular process that is also of therapeutic interest for diseases that manifest from premature stop codons. In eukaryotes, translation termination requires eRF1, which recognizes stop codons, catalyzes the release of nascent proteins fom ribosomes, and facilitates ribosome recycling. The small molecule SRI-41315 triggers eRF1 degradation and enhances translational readthrough of premature stop codons. SRI-41315 promotes retention of eRF1 on ribosomes and leads to a higher frequency of translation termination at near-cognate stop codons. In this study, the systematic effect of SRI-41315 on translation termination at cognate and near-cognate stop codons is evaluated using a rabbit reticulocyte lysate-based in vitro translation system with endogenous transcript as well as reporter transcripts containing defined near-cognate stop codon. Overall design: In vitro translation (IVT) reactions in rabbit reticulocyte lysate (RRL) were subject to ribosome profiling to investigate termination level at cognate and near-cognate stop codons. All IVT reactions contained endogenous transcripts as well as reporter transcripts containing cognate (UGA) or near-cognate (UGG or UGG_QW) stop codon as internal control to establish a baseline level of translation termination in the absence of SRI-41315. In SRI-41315 treatment groups (UGA_SRI, UGG_SRI, or UGG_QW_SRI), SRI-41315 was added to the IVT reactions to investigate its effect on translation termination on endogenous and reporter transcripts. The eRF1-AAQ mutant is known to trap terminating ribosomes in place and therefore was used as positive control (UGG_AAQ). Each condition contains two biological replicates using independently assembled IVT reactions.