The UBA domain of Mex67 contributes to its mRNA export activity. (A) Subcellular localization of poly(A)+ RNA was analyzed by FISH by using oligodT Cy3 as probe in Mex67–3HA or in mex67ΔUBA-3HA shuffle strains grown overnight at 23°C and then shifted to 37°C for 1 h. (B) GAL1 mRNA was analyzed by FISH by using a specific probe in Mex67–3HA or mex67ΔUBA-3HA shuffle strains after a 30-min induction in galactose at 23°C. (C) Cells expressing Lex or Lex-UBA Mex67 under the control of a galactose-inducible promoter were grown overnight in galactose containing medium at 23°C and then shifted to 37°C for 2 h before FISH analysis by using oligodT Cy3 as probe.

The UBA domain of Mex67 interacts with Hpr1. (A) Two-hybrid analysis of the Lex-Mex67 baits vs. Gal4-Hpr1-derived preys. (B) Purified recombinant UBA-Mex67 and GST-Hpr1 fusion proteins were mixed (input) and complexes were analyzed after purification on glutathione-Sepharose beads (bound). (C) In vivo Mex67/THO complex interaction was analyzed by coimmunoprecipitating HA-tagged Hpr1 with anti-Mex67 antibodies or alternatively HA-tagged Thp2 with anti-Mex67 antibodies in Thp2-HA (HPR1) or Δhpr1/Thp2-HA (Δhpr1) strains. Preimmune serum (IP mock) was used as negative control.

Ubiquitylation of Hpr1 is required for proper mRNA export and cotranscriptional recruitment of Mex67. (A) Mutation Y338A within the Rsp5-binding site of Hpr1 slowed down the turnover of Hpr1. (B) GAL1 mRNA localization was analyzed by FISH by using a specific probe in Hpr1wt or hpr1Y338A mutant strains after a 30-min induction in 2% galactose at 30°C. (C) The association of Mex67 with the 5′, middle, 3′, and 3′UTR regions of the GAL10 gene was analyzed by ChIP by using anti-Mex67, anti-CTD, anti-Hpr1, or anti-HA antibodies and extracts from Hpr1wt/Thp2-HA or hpr1Y338A/Thp2-HA cells induced with 2% galactose for 1 h at 25°C. Values correspond to the mean of three independent experiments.

The UBA domain of Mex67 contributes to its cotranscriptional recruitment. (A) ChIP experiments on GAL10 gene were performed with extracts prepared from Mex67–3HA or mex67ΔUBA-3HA strains shifted to galactose for 2 h by using the indicated antibodies. (B) ChIP experiments on the PMA1 gene were performed with extracts prepared from Mex67–3HA or mex67ΔUBA-3HA strains by using antibodies against HA or CTD. Each immunoprecipitation was repeated at least from three different extracts. Error bars correspond to standard deviations.

The UBA domain of Mex67 interacts with polyubiquitin chains and prevents proteasome-mediated degradation of Hpr1. (A) Pull-down assays using the indicated GST fusion recombinant proteins and extracts from Δerg6 cells +/− MG132. (B) The stability of Hpr1-HA was analyzed in cells (YFS 1748) transformed with a pLex10-UBA-Mex67 or a pLex10-UBA-Rad23 plasmid and collected before (0) or after a shift at 37°C for 15 min in the absence (−CX) or presence (+CX) of cycloheximide (Left) or alternatively in Mex67–3HA or in mex67ΔUBA-3HA shuffle strains shifted to 37°C in the presence of cycloheximide for different incubation times (Right). (C) Ni-Purified 6His-ubiquitin conjugated forms of Hpr1-HA from cim3.1 cells transformed with pYEp96–6His-Ub and a p426ADH-Lex-UBA-Mex67 or a p426ADH-Lex-UBA-Rad23 plasmid and shifted to 37°C during 4 h. The purified material was examined by Western blotting with an anti-HA antibody.