Extended Data Fig. 2: SRI-41315 traps ubiquitylated eRF1 on ribosomes.
From: The eRF1 degrader SRI-41315 acts as a molecular glue at the ribosomal decoding center
a, RNF14 levels in different mammalian lysates. Two-fold dilutions of rabbit reticulocyte lysate (RRL) or HEK293T cell lysate analyzed by SDS-PAGE and immunoblotting. Note: low levels of RNF14 in RRL relative to GCN1 and ribosomal proteins, representative of 2 replicates with similar results. b, RNF14 mediates eRF1 ubiquitylation with SRI-41315. In vitro translation reactions containing 10 µM His-tagged ubiquitin without or with 50 nM wildtype (WT) or catalytically inactive (C417A) recombinant RNF14 (rRNF14) and 100 µM SRI-41315 were analyzed directly (total) or after denaturing pulldowns of His-tagged ubiquitin (His-Ub PD) by SDS-PAGE and immunoblotting. Note: WT but not C417A rRNF14 enhances SRI-41315-dependent ubiquitylation of eRF1, representative of 3 replicates with similar results. Residual eRF1 ubiquitylation is likely due to endogenous RNF14 in RRL. c, Titration of SRI-41315 into in vitro translation reactions containing 50 nM recombinant RNF14 analyzed directly (total) or after His-Ub PD by SDS-PAGE and immunoblotting, representative of 3 replicates with similar results. d, eRF1 ubiquitylation is slower than translation. Timecourses assaying eRF1 ubiquitylation in the presence of 50 nM recombinant RNF14, 10 µM His-tagged ubiquitin, and 25 µM SRI-41315 (top) compared to timecourses of radiolabeled nascent protein (NC) synthesis (bottom) in in vitro translation reactions, representative of 2 replicates with similar results. e, SRI-41315 traps eRF1 on ribosomes. Translation reactions as in Fig. 1c were size fractionated over 10-50% sucrose gradients. A total (T) sample and eleven fractions collected from the top of each gradient were analyzed directly or after His-Ub PD by SDS-PAGE and immunoblotting for eRF1. Note: SRI-41315 retains both unmodified and ubiquitylated eRF1 in ribosomal fractions, representative of 3 replicates with similar results.
