Cotranscriptional Recruitment to the mRNA Export Receptor Mex67p Contributes to Nuclear Pore Anchoring of Activated Genes

Mex67p is required for transcription-induced GAL10 gene relocation. (A) The GAL10 gene tagged with LacO was localized in wild-type (black bars) and mex67-5 (gray bars) cells also expressing LacI-GFP and Nup49-GFP. Cells were grown from an OD₆₀₀ of 0.1 to 0.5 at 25°C in synthetic complete (SC) medium containing 2% raffinose and shifted to 2% glucose or 2% galactose for 2 h. Cells were then either kept at 25°C or shifted to 37°C for 15 min (15′) or 30 min (30′). GAL10 gene localization was defined as described in the legend to Fig. 1. (B) Northern blot analysis of GAL10 mRNA in wild-type and mex67-5 cells grown as described above for panel A and shifted to 37°C for the indicated times. The GAL10 mRNA signal was normalized to 18S rRNA and expressed as a percentage of the wild-type value before the shift to 37°C. (C) Association of TATA binding protein, RNA polymerase II, and Mex67p with galactose-induced GAL10 in wild-type and mex67-5 cells at 25°C and 37°C. Cross-linked and sonicated extracts were immunoprecipitated with antibodies against TBP, PolII, or Mex67p. Coprecipitating DNA was amplified by real-time PCR with primers specific for the GAL10 promoter (prom), 5′, middle (mid), 3′, 3′UTR, and a nontranscribed intergenic region (int), as indicated. The relative enrichment of the GAL10 gene segments in each ChIP was expressed as the increase with respect to the nontranscribed intergenic region value, arbitrarily set to 1. Values correspond to the means of three independent experiments. Bars correspond to standard deviations. (D) Western blot analysis of Mex67p levels in wild-type and mex67-5 extracts from cells used for ChIP. TBP was used as an internal loading control.

Mex67p is required for transcription-induced HSP104 gene relocation. (A) The HSP104 gene tagged with LacO was localized in wild-type (black bars) and mex67-5 (gray bars) cells expressing LacI-GFP and Nup49-GFP. Cells were grown in YEPD at 25°C and scored before and after a 30-min treatment with 10% ethanol (10% EtOH 30′) as described in the legend to Fig. 1. (B) Northern blot of HSP104 mRNAs in wild-type and mex67-5 strains grown at 25°C, shifted to 37°C, or treated with 10% ethanol for 30 min. HSP104 mRNA levels were normalized to 18S rRNA values and expressed as a percentage of the value for the wild type incubated with ethanol for 30 min at 25°C. (C) ChIP analysis of TBP, RNA PolII, and Mex67p association with HSP104. Extracts were prepared from wild-type or mex67-5 cells induced with 10% ethanol for 30 min at 25°C and immunoprecipitated with antibodies against TBP, RNA PolII, or Mex67p as indicated. Coprecipitating DNA was amplified with primers specific for HSP104 promoter (prom), 5′, middle (mid), 3′, and a nontranscribed intergenic region (int). The relative enrichment of the HSP104 gene segments was calculated as described in the legend to Fig. 2C. Values correspond to the means of three independent experiments. (D) Western blot analysis of Mex67p and TBP levels in wild-type and mex67-5 extracts from cells used for ChIP. (E) Genomically tagged MEX67-GFP and mex67-5-GFP strains were grown in YEPD at 25°C, incubated with 10% ethanol, or shifted to 37°C for 30 min and immediately examined with a fluorescence microscope.

The cotranscriptional recruitment of Mex67p is RNA independent. Strain FSY1651 expressing HA-tagged Sub2p was induced for 2 hours with 2% galactose. Cross-linked and sonicated extracts were immunoprecipitated with antibodies against HA, RNA PolII, or Mex67p. Coprecipitating DNA was purified from beads before or after RNase treatment and quantified by real-time PCR with primers specific for GAL10 as described in the legend to Fig. 2C. Values correspond to the means of three independent immunoprecipitation experiments.

The promoter, but not the protein-coding region or 3′UTR, is required for transcription-induced GAL2 gene anchoring. (A) Diagram of GAL2 genomic deletions. The positions− 200 and −550 are relative to GAL2 ATG. The UAS lies between −350 and −550, the TATA box lies between −100 and −200, and transcription initiation was mapped at −97 (22). The deleted 3′UTR lies between the GAL2 stop codon and position+ 400 relative to the stop codon. The LoxP sequence is 106 bp long and results from the Cre-LoxP recombination procedure. (B) Localization of the GAL2 locus tagged with LacO repeats in wild-type, Δgal2, Δ3′UTR, Δgal2-3′UTR, Δprom-gal2, and ΔUAS-3′UTR cells grown in YEP medium containing 2% glucose or 2% galactose to an OD₆₀₀ of 0.5. (C) ChIP analysis of TBP binding at the GAL2 promoter (position −181 to −106 encompassing the TATA box) in the wild type and the indicated mutant strains induced for 2 h with 2% galactose. The relative enrichment of the GAL2 promoter segment in each ChIP was expressed as the enrichment with respect to the nontranscribed intergenic region (Int) value, set to 1. Values are the means of three independent experiments.