Rrd1 isomerizes RNA polymerase II in response to rapamycin

Abstract

Background: In Saccharomyces cerevisiae, the immunosuppressant rapamycin engenders a profound modification in the transcriptional profile leading to growth arrest. Mutants devoid of Rrd1, a protein possessing in vitro peptidyl prolyl cis/trans isomerase activity, display striking resistance to the drug, although how Rrd1 activity is linked to the biological responses has not been elucidated.

Results: We now provide evidence that Rrd1 is associated with the chromatin and it interacts with RNA polymerase II. Circular dichroism revealed that Rrd1 mediates structural changes onto the C-terminal domain (CTD) of the large subunit of RNA polymerase II (Rpb1) in response to rapamycin, although this appears to be independent of the overall phosphorylation status of the CTD. In vitro experiments, showed that recombinant Rrd1 directly isomerizes purified GST-CTD and that it releases RNA polymerase II from the chromatin. Consistent with this, we demonstrated that Rrd1 is required to alter RNA polymerase II occupancy on rapamycin responsive genes.

Conclusion: We propose as a mechanism, that upon rapamycin exposure Rrd1 isomerizes Rpb1 to promote its dissociation from the chromatin in order to modulate transcription.

Figure 1

Rrd1 is associated with the chromatin and interacts with Rpb1. A) Rrd1 is bound to chromatin. Whole cell extract (WCE), soluble (SOL) and chromatin (CHR) fractions were derived (see Methods) from the parent cells expressing either Rrd1-MYC, Swe1-MYC, Rad52-MYC or Apn1-MYC and the distribution of the MYC-tagged proteins was examined by Western blots. The data is representative of two independent analyses. B) Rpb1 pull-down of Rrd1. The 8WG16 antibodies were used to immunoprecipitate extracts from untreated (-) and rapamycin-treated (+) (200 ng/ml for 30 min) cells expressing either Rrd1-MYC or Swe1-MYC. The presence of Rrd1 in the immunoprecipitates was determined by Western blotting. C) Specificity of Rpb1 pull-down of GFP tagged proteins. The 8WG16 antibodies were used to immunoprecipitate extracts from cells expressing either of the following GFP tagged proteins: Imp2, Rrd1, Rad52 or Sit4. The presence of the GFP-tagged proteins in the immunoprecipitates was detected by Western blotting using GFP antibody.

Figure 2

Analysis of the GST-CTD and its interaction with Rrd1. A) Comparison of the expression and phosphorylation status of the GST-CTD between parent and rrd1Δ mutant cells following rapamycin exposure. The indicated cells expressing GST-CTD were treated with (+) and without (-) rapamycin (200 ng/ml for 30 min) and total protein extracts were probed for Ser-2 phosphorylation (H5) or Ser-5 phosphorylation (H14). The membranes were stripped and reprobed with anti-GST antibody. B-D) Retention of Rrd1-MYC by GST-CTD affinity beads. B) The input (5% of the total amount of protein extracts added to the beads) of parent cells expressing Yap8-MYC and Rrd1-MYC from the endogenous locus. Western blot analysis was done using anti-MYC antibody. C) and D) Total protein extracts derived from the parent or parent expressing either Yap8-MYC or Rrd1-MYC were incubated with the empty beads or beads containing either GST-CTD or GST-Apn1 (see Methods). The beads were then washed and an aliquot examined for retention of the MYC tagged proteins using anti-GST antibodies (C) or anti-GST antibodies (D). Results shown are representative of two independent experiments.

Figure 3

rrd1Δ mutants are unable to induce conformational changes to the GST-CTD in response to rapamycin. A) Silver stained gel of purified GST and GST-CTD. The indicated strains carrying either the GST (lanes 1 and 2) or GST-CTD expressing plasmid (lanes 3-6) were untreated (-) or treated (+) with rapamycin (RAP) (200 ng/ml for 30 min). B) Far-UV circular dichroism (CD) spectral analysis of purified GST-CTD. The purified GST-CTD (0.45 μM) was derived from the parent strain (triangle) or rrd1Δ mutant (circle) that were untreated (opened symbol) or treated (closed symbol) with rapamycin. C) Far-UV CD spectral analysis of purified GST-CTD. The purified GST-CTD (0.45 μM) was derived from the rrd1Δ mutant carrying the empty vector (circle) or the pRRD1 plasmid (triangle) that were untreated (opened symbol) or treated (closed symbol) with rapamycin. D) CD analysis of purified GST (0.76 μM) derived from untreated (opened symbol) and rapamycin treated (closed symbol) parent cells as above. Results shown are the averages of two independent experiments. E) Limited proteolysis of purified GST-CTD derived from parent cells untreated or treated with rapamycin. The purified GST-CTD was subjected to partial chymotrypsin digestion and analyzed by silver staining. Results shown are representative of two independent experiments.

Figure 4

4-NQO, but not MMS, induces structural changes onto the GST-CTD. A and B) CD analysis of the purified GST-CTD derived from exponentially growing parent (triangle) and rrd1Δ mutant (circle) that were untreated (opened symbol) or treated (closed symbol) with either 4-NQO (2 μg/ml 30 min) panel A or MMS (1% for 60 min) panel B.

Figure 5

Purified recombinant Rrd1 alters the structure of purified GST-CTD in vitro. A) Silver stained gel of purified recombinant HIS-Rrd1 from E. coli expression system (see Methods). Lanes 1-2 and 3-4 are elution samples from two independent purifications obtained directly from Talon affinity column; lane 5, molecular weight standard. B) Equimolar amounts (4.5 μM) of purified GST-CTD derived from the rrd1Δ mutant and the purified recombinant HIS-Rrd1 (triangle) were incubated at 30°C in phosphate buffer in the absence (opened symbol) and presence (closed symbol) of Mg²⁺/ATP. The resulting GST-CTD was re-purified free of the recombinant HIS-Rrd1 and subjected to CD analysis as in Figure 3. The result is the average of two independent experiments.

Figure 6

Comparison of RNA pol II occupancy at the indicated target genes in the parent and rrd1Δ mutant strain in response to rapamycin treatment. Cells were untreated or treated with 200 ng/ml rapamycin for 30 min and Rpb1 localization was analyzed by ChIP assay (see Methods). Primer locations are indicated below the diagram. The respective input normalized IP amounts were quantified relative to the ACT1 gene using the ΔΔCT method. Results are shown as the average of three independent experiments. Error bars represent standard deviation and the P-values compare untreated vs. treated.

Figure 7

Purified recombinant Rrd1 dissociates Rpb1 from the chromatin in vitro. Increasing amounts of purified HIS-Rrd1 were added to the chromatin fraction isolated from rrd1Δ mutant strain expressing Apn1-MYC and incubated at 30°C for 1 h in phosphate buffer. Chromatin was recovered from the buffer and both fractions were analyzed by Western blotting probed with 4H8 (against Rpb1) and anti-MYC antibodies. Apn1-MYC was used as loading control. Result shown is representative of at least three experiments.