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Rrd1p, an RNA polymerase II-specific prolyl isomerase and activator of phosphoprotein phosphatase, promotes transcription independently of rapamycin response.

Sen R, Malik S, Frankland-Searby S, Uprety B, Lahudkar S, Bhaumik SR - Nucleic Acids Res. (2014)

Bottom Line: Similarly, inducible, but rapamycin-responsive, non-GAL genes such as CTT1, STL1 and CUP1 are also regulated by Rrd1p.Consistently, transcription of the constitutively active genes is not changed in the Δrrd1 strain.Taken together, our results demonstrate a new function of Rrd1p in stimulation of initial rounds of transcription, but not steady-state/constitutive transcription, of both rapamycin-responsive and non-responsive genes independently of rapamycin treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA.

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Rrd1p facilitates transcription of GAL7 and GAL10 following 90 min transcriptional induction. (A) Rrd1p associates with the coding sequences of GAL7 and GAL10. Yeast strain expressing Myc-tagged Rrd1p was grown, crosslinked and immunoprecipitated as in Figure 1F. The ChIP signal at the ORF was set to 100, and the ChIP signal at the promoter was normalized with respect to 100. (B and C) ChIP analysis for the association of RNA polymerase II with the GAL7 and GAL10 core promoters and coding sequences in the wild-type and Δrrd1 strains following 90 min transcriptional induction in YPG. (D) RT-PCR analysis of GAL7, GAL10 and ADH1 mRNA levels in the wild-type and Δrrd1 strains following 90 min transcriptional induction in YPG. (E) RT-PCR analysis of GAL7, GAL10 and ACT1 mRNA levels in the presence and absence of rapamycin. Yeast cells were grown in YPR up to an OD600 of 0.9, transferred to YPG for 60 min, and then treated with 100 nM rapamycin (Sigma) for next 30 min prior to harvesting for RNA analysis. (F and G) ChIP analysis for the recruitment of TBP and Myc-tagged Rad3p to the core promoters of GAL7 and GAL10 following 90 min transcriptional induction in YPG. ChIP experiments were carried out as in Figure 2B. (H) RT-PCR analysis of GAL7 and GAL10 mRNA levels in the wild-type and Δrrd1 strains following continuous growth in YPG.
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Figure 5: Rrd1p facilitates transcription of GAL7 and GAL10 following 90 min transcriptional induction. (A) Rrd1p associates with the coding sequences of GAL7 and GAL10. Yeast strain expressing Myc-tagged Rrd1p was grown, crosslinked and immunoprecipitated as in Figure 1F. The ChIP signal at the ORF was set to 100, and the ChIP signal at the promoter was normalized with respect to 100. (B and C) ChIP analysis for the association of RNA polymerase II with the GAL7 and GAL10 core promoters and coding sequences in the wild-type and Δrrd1 strains following 90 min transcriptional induction in YPG. (D) RT-PCR analysis of GAL7, GAL10 and ADH1 mRNA levels in the wild-type and Δrrd1 strains following 90 min transcriptional induction in YPG. (E) RT-PCR analysis of GAL7, GAL10 and ACT1 mRNA levels in the presence and absence of rapamycin. Yeast cells were grown in YPR up to an OD600 of 0.9, transferred to YPG for 60 min, and then treated with 100 nM rapamycin (Sigma) for next 30 min prior to harvesting for RNA analysis. (F and G) ChIP analysis for the recruitment of TBP and Myc-tagged Rad3p to the core promoters of GAL7 and GAL10 following 90 min transcriptional induction in YPG. ChIP experiments were carried out as in Figure 2B. (H) RT-PCR analysis of GAL7 and GAL10 mRNA levels in the wild-type and Δrrd1 strains following continuous growth in YPG.

Mentions: We have shown above that Rrd1p associates with GAL1 and promotes its transcription. We next analyzed whether Rrd1p also associates with other GAL genes such as GAL7 and GAL10 to promote their transcription. In this direction, we first analyzed the association of Rrd1p with the core promoters and coding sequences of GAL7 and GAL10 following 90 min transcriptional induction. Like the results at GAL1, we find that Rrd1p predominantly associates with the coding sequences of GAL7 and GAL10 (Figure 5A, Supplementary Figure S1). Subsequently, we analyzed the association of RNA polymerase II with the core promoters and coding sequences of GAL7 and GAL10 following 90 min transcriptional induction in the Δrrd1 and wild-type strains. We find that Rrd1p promotes the association of RNA polymerase II with GAL7 and GAL10 (Figure 5B and C). Consistently, transcription of GAL7 and GAL10 was significantly decreased in the absence of Rrd1p (Figure 5D). Further, we show that transcription of GAL7 and GAL10 is not regulated by rapamycin (Figure 5E). Thus, our results demonstrate that Rrd1p promotes transcription of rapamycin non-responsive GAL7 and GAL10 genes following 90 min transcriptional induction independently of rapamycin treatment (or TOR pathway). Furthermore, similar to the results at GAL1, we find that Rrd1p promotes the recruitment of the PIC components such as TBP and TFIIH (Rad3p) to the core promoters of GAL7 and GAL10 (Figure 5F and G), hence supporting the role of Rrd1p in stimulation of the PIC formation (and hence transcriptional initiation). However, the defect in formation of the PIC at GAL7 and GAL10 in the absence of Rrd1p is much less than the defect in RNA polymerase II association with the coding sequence. These results indicate that Rrd1p facilitates transcriptional elongation, in additional to its role in transcriptional initiation.


Rrd1p, an RNA polymerase II-specific prolyl isomerase and activator of phosphoprotein phosphatase, promotes transcription independently of rapamycin response.

Sen R, Malik S, Frankland-Searby S, Uprety B, Lahudkar S, Bhaumik SR - Nucleic Acids Res. (2014)

Rrd1p facilitates transcription of GAL7 and GAL10 following 90 min transcriptional induction. (A) Rrd1p associates with the coding sequences of GAL7 and GAL10. Yeast strain expressing Myc-tagged Rrd1p was grown, crosslinked and immunoprecipitated as in Figure 1F. The ChIP signal at the ORF was set to 100, and the ChIP signal at the promoter was normalized with respect to 100. (B and C) ChIP analysis for the association of RNA polymerase II with the GAL7 and GAL10 core promoters and coding sequences in the wild-type and Δrrd1 strains following 90 min transcriptional induction in YPG. (D) RT-PCR analysis of GAL7, GAL10 and ADH1 mRNA levels in the wild-type and Δrrd1 strains following 90 min transcriptional induction in YPG. (E) RT-PCR analysis of GAL7, GAL10 and ACT1 mRNA levels in the presence and absence of rapamycin. Yeast cells were grown in YPR up to an OD600 of 0.9, transferred to YPG for 60 min, and then treated with 100 nM rapamycin (Sigma) for next 30 min prior to harvesting for RNA analysis. (F and G) ChIP analysis for the recruitment of TBP and Myc-tagged Rad3p to the core promoters of GAL7 and GAL10 following 90 min transcriptional induction in YPG. ChIP experiments were carried out as in Figure 2B. (H) RT-PCR analysis of GAL7 and GAL10 mRNA levels in the wild-type and Δrrd1 strains following continuous growth in YPG.
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Figure 5: Rrd1p facilitates transcription of GAL7 and GAL10 following 90 min transcriptional induction. (A) Rrd1p associates with the coding sequences of GAL7 and GAL10. Yeast strain expressing Myc-tagged Rrd1p was grown, crosslinked and immunoprecipitated as in Figure 1F. The ChIP signal at the ORF was set to 100, and the ChIP signal at the promoter was normalized with respect to 100. (B and C) ChIP analysis for the association of RNA polymerase II with the GAL7 and GAL10 core promoters and coding sequences in the wild-type and Δrrd1 strains following 90 min transcriptional induction in YPG. (D) RT-PCR analysis of GAL7, GAL10 and ADH1 mRNA levels in the wild-type and Δrrd1 strains following 90 min transcriptional induction in YPG. (E) RT-PCR analysis of GAL7, GAL10 and ACT1 mRNA levels in the presence and absence of rapamycin. Yeast cells were grown in YPR up to an OD600 of 0.9, transferred to YPG for 60 min, and then treated with 100 nM rapamycin (Sigma) for next 30 min prior to harvesting for RNA analysis. (F and G) ChIP analysis for the recruitment of TBP and Myc-tagged Rad3p to the core promoters of GAL7 and GAL10 following 90 min transcriptional induction in YPG. ChIP experiments were carried out as in Figure 2B. (H) RT-PCR analysis of GAL7 and GAL10 mRNA levels in the wild-type and Δrrd1 strains following continuous growth in YPG.
Mentions: We have shown above that Rrd1p associates with GAL1 and promotes its transcription. We next analyzed whether Rrd1p also associates with other GAL genes such as GAL7 and GAL10 to promote their transcription. In this direction, we first analyzed the association of Rrd1p with the core promoters and coding sequences of GAL7 and GAL10 following 90 min transcriptional induction. Like the results at GAL1, we find that Rrd1p predominantly associates with the coding sequences of GAL7 and GAL10 (Figure 5A, Supplementary Figure S1). Subsequently, we analyzed the association of RNA polymerase II with the core promoters and coding sequences of GAL7 and GAL10 following 90 min transcriptional induction in the Δrrd1 and wild-type strains. We find that Rrd1p promotes the association of RNA polymerase II with GAL7 and GAL10 (Figure 5B and C). Consistently, transcription of GAL7 and GAL10 was significantly decreased in the absence of Rrd1p (Figure 5D). Further, we show that transcription of GAL7 and GAL10 is not regulated by rapamycin (Figure 5E). Thus, our results demonstrate that Rrd1p promotes transcription of rapamycin non-responsive GAL7 and GAL10 genes following 90 min transcriptional induction independently of rapamycin treatment (or TOR pathway). Furthermore, similar to the results at GAL1, we find that Rrd1p promotes the recruitment of the PIC components such as TBP and TFIIH (Rad3p) to the core promoters of GAL7 and GAL10 (Figure 5F and G), hence supporting the role of Rrd1p in stimulation of the PIC formation (and hence transcriptional initiation). However, the defect in formation of the PIC at GAL7 and GAL10 in the absence of Rrd1p is much less than the defect in RNA polymerase II association with the coding sequence. These results indicate that Rrd1p facilitates transcriptional elongation, in additional to its role in transcriptional initiation.

Bottom Line: Similarly, inducible, but rapamycin-responsive, non-GAL genes such as CTT1, STL1 and CUP1 are also regulated by Rrd1p.Consistently, transcription of the constitutively active genes is not changed in the Δrrd1 strain.Taken together, our results demonstrate a new function of Rrd1p in stimulation of initial rounds of transcription, but not steady-state/constitutive transcription, of both rapamycin-responsive and non-responsive genes independently of rapamycin treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901, USA.

Show MeSH
Related in: MedlinePlus