Cotranscriptional recruitment to the mRNA export receptor Mex67p contributes to nuclear pore anchoring of activated genes

Abstract

Transcription activation of some Saccharomyces cerevisiae genes is paralleled by their repositioning to the nuclear periphery, but the mechanism underlying gene anchoring is poorly defined. We show that the nuclear pore complex-associated Mlp1p and the shuttling mRNA export receptor Mex67p contribute to the stable association of the activated GAL10 and HSP104 genes with the nuclear periphery. However, we find no obligatory link between gene positioning and gene expression. Furthermore, gene anchoring correlates with the cotranscriptional recruitment of Mex67p to transcribing genes. Notably, the association of Mex67p with chromatin is not mediated by RNA. Interestingly, a mutant GAL2 gene lacking the coding region is still able to recruit Mex67p upon transcriptional activation and to relocate to the nuclear periphery. Together these data suggest that, at least for GAL2, nascent messenger ribonucleoprotein does not play a major role in gene anchoring and that the early recruitment of Mex67p contributes to gene repositioning by virtue of an RNA-independent process.

FIG. 1.

Mlp1p is required for efficient transcription-induced GAL10 and HSP104 gene relocation. (A) Yeast cells with GAL10 tagged with LacO repeats and expressing LacI-GFP and Nup49-GFP. Binding of LacI-GFP to LacO gives rise to the intense bright spot, whereas Nup49-GFP marks the nuclear periphery. A spot was scored as peripheral when located in the most peripheral 33% cross-sectional area of the nucleus (see Materials and Methods). (B) Localization of GAL10 or HSP104 tagged with LacO repeats in the wild-type (black bars) or Δmlp1 (gray bars) cells. To ensure isogenicity, WT and Δmlp1 strains were obtained by transforming the Δmlp1 strain with a plasmid expressing Mlp1p or an empty vector, respectively. GAL10-LacO cells were grown at 25°C in selective medium containing 2% raffinose to an OD₆₀₀ of 0.5 and shifted to 2% glucose or 2% galactose for 2.5 h. HSP104-LacO cells were grown at 25°C in YEPD to an OD₆₀₀ of 0.5 and examined before or after a 30-min treatment with 10% ethanol (10% EtOH 30′). A population of live cells was photographed with a fluorescence microscope. In each cell, the position of the GAL10 or HSP104 gene spot was defined with respect to the nuclear periphery. Graph bars represent the percentage of cells with the indicated tagged gene positioned at the periphery. Error bars were derived from three independent experiments; n is the total number of cells counted for each strain and condition. The broken line at 33% marks a random distribution; based on a proportional test, two distributions were considered as significantly different when P < 0.05. (C) Northern blot analysis of GAL10 mRNA in Δmlp1 and WT cells after induction with galactose (Gal) at 25°C for the indicated times. The GAL10 mRNA signal was normalized to 18S rRNA used as internal control and expressed as a percentage of the WT level, after 20 min in galactose at 25°C.

FIG. 2.

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.

FIG. 3.

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.

FIG. 4.

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.

FIG. 5.

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.

FIG. 6.

The Δgal2 mutant gene encodes a short unstable transcript but recruits wild-type levels of RNA PolII and Mex67p. (A) Northern blot analysis of total RNA from the wild type (lane 1) and indicated mutant strains (lanes 2 to 4) with probes diagrammed at the top and spanning the GAL2 protein-coding region (GAL2 probe [left panel]) or the GAL2 3′UTR region (3′UTR probe [right panel]). For quantification, the GAL2 RNA signals were normalized to endogenous actin mRNA levels. The 3′UTR probe weakly hybridized to the GAL2 mRNA produced in the Δ3′UTR strain, as both the probe and the transcript produced by this mutant extend beyond the 3′UTR-deleted region. This band was not quantified, as it is only partially complementary to the probe. NA, not applicable. (B) ChIP analyses of TBP, RNA PolII and Mex67p on wild-type GAL2 and the mutant Δgal2 gene. Extracts from galactose-induced cultures were immunoprecipitated with antibodies against TBP, PolII, and Mex67p. Coprecipitating DNA segments (diagrammed as short bars on top) were quantified by real-time PCR with primers specific for the GAL2 promoter (positions −181 to −106), LoxP (106 bp derived from Cre-Lox recombination), 3′UTR1 (positions +145 to +257) and 3′UTR2 (positions +238 to +328). The 3′UTR was numbered positively starting from the GAL2 stop codon, and 3′ end formation is predicted to occur around +250 (A). The relative enrichment of DNA segments in each ChIP was expressed as the enrichment with respect to the nontranscribed intergenic (int) value, set to 1. Values are means of three independent experiments.