Fig. 4

Xrn1-dependence for translation is specific and mediated by the interaction with eIF4G. a Rat1∆NLS fully replaces Xrn1 in mRNA decay and cell proliferation. xrn1∆ cells expressing BMV RNA2 under GAL1 promoter and either WT Xrn1, Rat1∆NLS or an empty plasmid were grown in galactose. Left, transcription of RNA2 was shut-off upon glucose addition and BMV RNA2 stability was determined by monitoring RNA2 levels by northern blot analysis at various time-points post-glucose addition. A representative example out of three replicates is shown. Right, growth curves in galactose media. Results represent averages obtained from three replicates. b Rat1∆NLS does not replace Xrn1 in RNA2 translation. Western blot (upper panel) and northern blot (lower panel) analysis showing steady-state levels of viral protein 2a and viral RNA2 in xrn1∆ cells expressing Xrn1 or Rat1∆NLS. Asterisk points at a non-specific band. Quantifications are relative to xrn1∆ transformed with WT Xrn1 plasmid. Results represent averages of n = 3 biological replicates. c Xrn1 interacts with eIF4G. Western blot analysis of immunoprecipitation assays. Xrn1-FLAG and Rat1∆NLS-FLAG proteins were expressed in yeast strains expressing either eIF4G-GFP, eIF4A-GFP, or eIF4E-GFP fusion proteins. As a control, the functionality of GFP-fused strains was assessed (Supplementary Fig. 5). Immunoprecipitations were carried out with GFP-trap beads with extracts treated ( + ) or not treated (−) with RNase A. Expression levels of eIF4G, eIF4A, and eIF4A were detected by anti-GFP antibody. d Expression of Rat1∆NLS-XC rescues BMV RNA2 translation. Western blot (upper panel) and northern blot (lower panel) analysis. Results represent averages of three replicates. Expression levels of flag-tagged Xrn1, Rat1∆NLS, and Rat1∆NLS-XC were analyzed by western blot (Supplementary Fig. 6). e Interaction with eIF4G studied by co-immunoprecipitation analyses. Source data are provided as a Source Data file