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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 has no effect on the steady-state level of GAL1 transcription. (A) RT-PCR analysis of GAL1 mRNA levels in the wild-type and Δrrd1 strains following continuous growth in YPG. (B) ChIP analysis for the association of RNA polymerase II with the GAL1 coding sequence following continuous growth in YPG. (C) ChIP analysis of RNA polymerase II association with the GAL1 coding sequence at different time points (20, 40 and 60 min) following transcriptional induction. Maximum ChIP signal was set to 100, and other ChIP signals were normalized with respect to 100. The normalized ChIP signal (represented as normalized occupancy) is plotted in the form of a histogram. (D) ChIP analysis of RNA polymerase II association with the GAL1 coding sequence following 2, 4 and 6 h transcriptional induction in YPG. The ChIP signal for wild-type strain was set to 100, and the ChIP signal for the Δrrd1 strain was normalized with respect to 100. (E) Growth analysis of the wild-type and Δrrd1 strains in solid YPG medium. (F and G) ChIP analysis of histone H2B level at the GAL1 coding sequence in the wild-type and Δrrd1 strains expressing Flag-tagged histone H2B. Yeast cells were grown and crosslinked as in panel C, and immunoprecipitation was performed using an anti-Flag antibody (F1804, Sigma) against Flag-tagged histone H2B.
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Figure 3: Rrd1p has no effect on the steady-state level of GAL1 transcription. (A) RT-PCR analysis of GAL1 mRNA levels in the wild-type and Δrrd1 strains following continuous growth in YPG. (B) ChIP analysis for the association of RNA polymerase II with the GAL1 coding sequence following continuous growth in YPG. (C) ChIP analysis of RNA polymerase II association with the GAL1 coding sequence at different time points (20, 40 and 60 min) following transcriptional induction. Maximum ChIP signal was set to 100, and other ChIP signals were normalized with respect to 100. The normalized ChIP signal (represented as normalized occupancy) is plotted in the form of a histogram. (D) ChIP analysis of RNA polymerase II association with the GAL1 coding sequence following 2, 4 and 6 h transcriptional induction in YPG. The ChIP signal for wild-type strain was set to 100, and the ChIP signal for the Δrrd1 strain was normalized with respect to 100. (E) Growth analysis of the wild-type and Δrrd1 strains in solid YPG medium. (F and G) ChIP analysis of histone H2B level at the GAL1 coding sequence in the wild-type and Δrrd1 strains expressing Flag-tagged histone H2B. Yeast cells were grown and crosslinked as in panel C, and immunoprecipitation was performed using an anti-Flag antibody (F1804, Sigma) against Flag-tagged histone H2B.

Mentions: Our above results at GAL1 reveal that Rrd1p promotes the PIC formation, association of elongating RNA polymerase II, and hence transcription following 90 min transcriptional induction. We next asked whether Rrd1p has any effect on GAL1 transcription when the steady-state is reached after a long induction in galactose-containing growth medium. To address this, we have continuously grown both the wild-type and Δrrd1 strains in galactose-containing growth medium up to an OD600 of 1.0 prior to crosslinking/harvesting, and then performed RT-PCR and ChIP analyses. We found that transcription of GAL1 in the Δrrd1 strain reached the wild-type level when the steady-state is reached after a long transcriptional induction (Figure 3A). Consistently, the level of RNA polymerase II at the GAL1 coding sequence in the Δrrd1 strain was almost same as that of the wild-type equivalent following long transcriptional induction (Figure 3B). Further, we performed the kinetic analysis for the association of RNA polymerase II with GAL1, and found that Rrd1p has significant stimulatory effects on RNA polymerase II association with GAL1 during initial stages of transcriptional induction, but not after long induction (Figure 3C and D). Thus, Rrd1p promotes the initial rounds of GAL1 transcription, but has no effect on steady-state transcription. Therefore, the growth defect of the Δrrd1 strain was not observed in the solid medium containing galactose (Figure 3E). Furthermore, we find that the role of Rrd1p in stimulation of RNA polymerase II association with GAL1 (and hence transcription) is correlated with facilitated nucleosomal disassembly as the eviction of histone H2B from GAL1 is impaired in the Δrrd1 strain following transcriptional induction (Figure 3F and G).


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 has no effect on the steady-state level of GAL1 transcription. (A) RT-PCR analysis of GAL1 mRNA levels in the wild-type and Δrrd1 strains following continuous growth in YPG. (B) ChIP analysis for the association of RNA polymerase II with the GAL1 coding sequence following continuous growth in YPG. (C) ChIP analysis of RNA polymerase II association with the GAL1 coding sequence at different time points (20, 40 and 60 min) following transcriptional induction. Maximum ChIP signal was set to 100, and other ChIP signals were normalized with respect to 100. The normalized ChIP signal (represented as normalized occupancy) is plotted in the form of a histogram. (D) ChIP analysis of RNA polymerase II association with the GAL1 coding sequence following 2, 4 and 6 h transcriptional induction in YPG. The ChIP signal for wild-type strain was set to 100, and the ChIP signal for the Δrrd1 strain was normalized with respect to 100. (E) Growth analysis of the wild-type and Δrrd1 strains in solid YPG medium. (F and G) ChIP analysis of histone H2B level at the GAL1 coding sequence in the wild-type and Δrrd1 strains expressing Flag-tagged histone H2B. Yeast cells were grown and crosslinked as in panel C, and immunoprecipitation was performed using an anti-Flag antibody (F1804, Sigma) against Flag-tagged histone H2B.
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Figure 3: Rrd1p has no effect on the steady-state level of GAL1 transcription. (A) RT-PCR analysis of GAL1 mRNA levels in the wild-type and Δrrd1 strains following continuous growth in YPG. (B) ChIP analysis for the association of RNA polymerase II with the GAL1 coding sequence following continuous growth in YPG. (C) ChIP analysis of RNA polymerase II association with the GAL1 coding sequence at different time points (20, 40 and 60 min) following transcriptional induction. Maximum ChIP signal was set to 100, and other ChIP signals were normalized with respect to 100. The normalized ChIP signal (represented as normalized occupancy) is plotted in the form of a histogram. (D) ChIP analysis of RNA polymerase II association with the GAL1 coding sequence following 2, 4 and 6 h transcriptional induction in YPG. The ChIP signal for wild-type strain was set to 100, and the ChIP signal for the Δrrd1 strain was normalized with respect to 100. (E) Growth analysis of the wild-type and Δrrd1 strains in solid YPG medium. (F and G) ChIP analysis of histone H2B level at the GAL1 coding sequence in the wild-type and Δrrd1 strains expressing Flag-tagged histone H2B. Yeast cells were grown and crosslinked as in panel C, and immunoprecipitation was performed using an anti-Flag antibody (F1804, Sigma) against Flag-tagged histone H2B.
Mentions: Our above results at GAL1 reveal that Rrd1p promotes the PIC formation, association of elongating RNA polymerase II, and hence transcription following 90 min transcriptional induction. We next asked whether Rrd1p has any effect on GAL1 transcription when the steady-state is reached after a long induction in galactose-containing growth medium. To address this, we have continuously grown both the wild-type and Δrrd1 strains in galactose-containing growth medium up to an OD600 of 1.0 prior to crosslinking/harvesting, and then performed RT-PCR and ChIP analyses. We found that transcription of GAL1 in the Δrrd1 strain reached the wild-type level when the steady-state is reached after a long transcriptional induction (Figure 3A). Consistently, the level of RNA polymerase II at the GAL1 coding sequence in the Δrrd1 strain was almost same as that of the wild-type equivalent following long transcriptional induction (Figure 3B). Further, we performed the kinetic analysis for the association of RNA polymerase II with GAL1, and found that Rrd1p has significant stimulatory effects on RNA polymerase II association with GAL1 during initial stages of transcriptional induction, but not after long induction (Figure 3C and D). Thus, Rrd1p promotes the initial rounds of GAL1 transcription, but has no effect on steady-state transcription. Therefore, the growth defect of the Δrrd1 strain was not observed in the solid medium containing galactose (Figure 3E). Furthermore, we find that the role of Rrd1p in stimulation of RNA polymerase II association with GAL1 (and hence transcription) is correlated with facilitated nucleosomal disassembly as the eviction of histone H2B from GAL1 is impaired in the Δrrd1 strain following transcriptional induction (Figure 3F and G).

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