Fig. 1. In vitro reconstitution of translational arrest and ZNF598-mediated ubiquitination.

A Schematic representation of mRNA templates used in RRL in vitro translation reactions. All constructs encode the N-terminal part of XBP1u (1–193), which is appended (X) by either the XBP1u (194–261) stalling sequence, a poly(A) stretch, a stable RNA stem-loop sequence or a self-cleaving ribozyme (Rz). All constructs contain an N-terminal His₆ and PA-tag for affinity purification and antibody detection. B Schematic overview of the in vitro stalling and ubiquitination assays. C–E In vitro translation of staller mRNAs from A. The free peptide and the peptidyl-tRNA (pep-tRNA) arrest products were detected by Western blotting using an anti-PA antibody. We obtained essentially the same results as two independent experiments. C Purified RNCs were treated with RNase A to verify the peptidyl-tRNA arrest products. D, E RNCs purified from in vitro translation reactions using all mRNA templates (A) but XBP1u (1–235) is used as a control and were subjected to ultracentrifugation through sucrose density gradients and individual gradient fractions were visualized by Western blotting using the anti-PA antibody. F In vitro ubiquitination by ZNF598 of RNCs stalled on mRNAs from A. RNCs from in vitro translation reactions with or without recombinantly purified 3FLAG-ZNF598 were purified and eS10 and uS10 ubiquitination was visualized by Western blotting. We obtained essentially the same results in at least three independent experiments.