Limits...
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.

Show MeSH

Related in: MedlinePlus

Rrd1p associates with the coding sequence of a rapamycin non-responsive GAL1 gene, and promotes the association of RNA polymerase II following 90 min transcriptional induction. (A) Schematic diagram showing the locations of different primer pairs at GAL1 for the ChIP analysis. The numbers are presented with respect to the position of the first nucleotide of the initiation codon (+1). (B) Analysis of Rpb1p association with the GAL1 coding sequence in the wild-type and Δrrd1 strains. Both wild-type and Δrrd1 strains expressing Myc-tagged Rpb1p were grown in YPR at 30°C up to an OD600 of 0.9, and then switched to YPG for 90 min prior to formaldehyde-based in vivo cross-linking. Immunoprecipitation was carried out using an anti-Myc antibody (9E10; Santa Cruz Biotechnology, Inc.) against Myc-tagged Rpb1p. Immunoprecipitated-DNA was analyzed by PCR using the primer pair targeted to the coding sequence of GAL1. The ratio of immunoprecipitate over the input in the autoradiogram (i.e. ChIP signal) was measured. The ChIP signal of the wild-type strain was set to 100, and the ChIP signal of the mutant strain was normalized with respect to 100. The normalized ChIP signal (represented as normalized occupancy) is plotted in the form of a histogram. (C) Western blot analysis under similar growth conditions as in panel B. (D) Growth analysis of the wild-type and Δrrd1 strains after switching to YPG from YPR (i.e. during 90 min induction time period). (E) RT-PCR analysis of the GAL1 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) Rrd1p associates with the coding sequence of GAL1. Yeast strain expressing Myc-tagged Rrd1p was grown as in panel B. Immunoprecipitation was carried out using an anti-Myc antibody against Myc-tagged Rrd1p. Immunoprecipitated-DNA was analyzed by PCR using the primer pairs targeted to the UAS, core promoter and two different locations (ORF and ORF1) of the coding sequence of GAL1. 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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150799&req=5

Figure 1: Rrd1p associates with the coding sequence of a rapamycin non-responsive GAL1 gene, and promotes the association of RNA polymerase II following 90 min transcriptional induction. (A) Schematic diagram showing the locations of different primer pairs at GAL1 for the ChIP analysis. The numbers are presented with respect to the position of the first nucleotide of the initiation codon (+1). (B) Analysis of Rpb1p association with the GAL1 coding sequence in the wild-type and Δrrd1 strains. Both wild-type and Δrrd1 strains expressing Myc-tagged Rpb1p were grown in YPR at 30°C up to an OD600 of 0.9, and then switched to YPG for 90 min prior to formaldehyde-based in vivo cross-linking. Immunoprecipitation was carried out using an anti-Myc antibody (9E10; Santa Cruz Biotechnology, Inc.) against Myc-tagged Rpb1p. Immunoprecipitated-DNA was analyzed by PCR using the primer pair targeted to the coding sequence of GAL1. The ratio of immunoprecipitate over the input in the autoradiogram (i.e. ChIP signal) was measured. The ChIP signal of the wild-type strain was set to 100, and the ChIP signal of the mutant strain was normalized with respect to 100. The normalized ChIP signal (represented as normalized occupancy) is plotted in the form of a histogram. (C) Western blot analysis under similar growth conditions as in panel B. (D) Growth analysis of the wild-type and Δrrd1 strains after switching to YPG from YPR (i.e. during 90 min induction time period). (E) RT-PCR analysis of the GAL1 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) Rrd1p associates with the coding sequence of GAL1. Yeast strain expressing Myc-tagged Rrd1p was grown as in panel B. Immunoprecipitation was carried out using an anti-Myc antibody against Myc-tagged Rrd1p. Immunoprecipitated-DNA was analyzed by PCR using the primer pairs targeted to the UAS, core promoter and two different locations (ORF and ORF1) of the coding sequence of GAL1. 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.

Mentions: Since Rrd1p interacts with RNA polymerase II (9), and is involved in regulation of proline isomerization (9) and phosphorylation of CTD of RNA polymerase II (10), it may regulate the association of RNA polymerase II with the coding sequence of active gene, and hence transcriptional elongation. However, previous studies (10) have demonstrated the dispensability of Rrd1p in regulation of transcription of rapamycin-responsive as well as non-responsive genes in the absence of rapamycin treatment under vegetative growth conditions. It is quite possible that Rrd1p promotes initial rounds/cycles of transcription via its RNA polymerase II-specific prolyl isomerase activity independently of rapamycin response, and the function of Rrd1p in transcription becomes minimal (or absent) when the steady-state is reached. To test this possibility, we analyzed the association of RNA polymerase II with the coding sequence of a galactose-inducible GAL1 gene following transcriptional induction in the absence of rapamycin treatment. In this direction, we grew both the wild-type and Δrrd1 strains in raffinose-containing growth medium (non-inducing) up to an OD600 of 0.9, and then switched to galactose-containing growth medium for 90 min prior to formaldehyde-based in vivo crosslinking. Using crosslinked cells, we performed the ChIP experiments to analyze the level of RNA polymerase II (Rpb1p) at the GAL1 coding sequence. We found that the association of RNA polymerase II with the GAL1 coding sequence was dramatically impaired in the Δrrd1 strain in the absence of rapamycin treatment (Figure 1A and B). Such reduction in the association of RNA polymerase II with the GAL1 coding sequence could be due to decreased stability of Rpb1p in the Δrrd1 strain. To test this possibility, we analyzed the global levels of Rpb1p in the wild-type and Δrrd1 strains, using the western blot assay. We found that the global level of Rpb1p was not changed in the Δrrd1 strain as compared to the wild-type equivalent (Figure 1C). The level of actin was monitored as a loading control, and its level was not altered in the Δrrd1 strain (Figure 1C). Thus, reduced association of RNA polymerase II with the GAL1 coding sequence following 90 min transcriptional induction in the Δrrd1 strain was not due to an impaired stability of Rpb1p. However, it is quite possible that the Δrrd1 strain grew slowly in galactose-containing growth medium during 90 min transcriptional induction, leading to a less number of the Δrrd1 cells and consequently decreased level of RNA polymerase II at GAL1. To test this possibility, we measured OD600 of the wild-type and Δrrd1 strains under the growth conditions used in the above ChIP experiments. We found that OD600 of the Δrrd1 strain was less than that of the wild-type strain at 90 min following the switch of the growth medium containing raffinose to galactose (Figure 1D). Thus, the relatively smaller number of the Δrrd1 cells following 90 min transcriptional induction in the ChIP experiments might have led to decreased amount of immunoprecipitated-DNA in comparison to the wild-type equivalent. However, we rule out this possibility as we normalized the immunoprecipitated-DNA signal with respect to its input DNA (referred to as ChIP signal), and then compared the ChIP signal of the Δrrd1 strain with that of the wild-type equivalent. Thus, our results support that Rrd1p promotes the association of RNA polymerase II with the GAL1 coding sequence. Further, we found that transcription of GAL1 was not regulated by rapamycin (Figure 1E), consistent with previous studies (10). Transcription of ACT1 was analyzed as a control (Figure 1E), since ACT1 is not responsive to rapamycin (8,10). Taken together, our results (Figure 1B–E) demonstrate that Rrd1p promotes the association of RNA polymerase II with the coding sequence of a rapamycin non-responsive GAL1 gene following 90 min transcriptional induction in the absence of rapamycin treatment. Consistently, we observed predominant association of Rrd1p with the coding sequence of GAL1 (Figure 1A and F, Supplementary Figure S1).


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 associates with the coding sequence of a rapamycin non-responsive GAL1 gene, and promotes the association of RNA polymerase II following 90 min transcriptional induction. (A) Schematic diagram showing the locations of different primer pairs at GAL1 for the ChIP analysis. The numbers are presented with respect to the position of the first nucleotide of the initiation codon (+1). (B) Analysis of Rpb1p association with the GAL1 coding sequence in the wild-type and Δrrd1 strains. Both wild-type and Δrrd1 strains expressing Myc-tagged Rpb1p were grown in YPR at 30°C up to an OD600 of 0.9, and then switched to YPG for 90 min prior to formaldehyde-based in vivo cross-linking. Immunoprecipitation was carried out using an anti-Myc antibody (9E10; Santa Cruz Biotechnology, Inc.) against Myc-tagged Rpb1p. Immunoprecipitated-DNA was analyzed by PCR using the primer pair targeted to the coding sequence of GAL1. The ratio of immunoprecipitate over the input in the autoradiogram (i.e. ChIP signal) was measured. The ChIP signal of the wild-type strain was set to 100, and the ChIP signal of the mutant strain was normalized with respect to 100. The normalized ChIP signal (represented as normalized occupancy) is plotted in the form of a histogram. (C) Western blot analysis under similar growth conditions as in panel B. (D) Growth analysis of the wild-type and Δrrd1 strains after switching to YPG from YPR (i.e. during 90 min induction time period). (E) RT-PCR analysis of the GAL1 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) Rrd1p associates with the coding sequence of GAL1. Yeast strain expressing Myc-tagged Rrd1p was grown as in panel B. Immunoprecipitation was carried out using an anti-Myc antibody against Myc-tagged Rrd1p. Immunoprecipitated-DNA was analyzed by PCR using the primer pairs targeted to the UAS, core promoter and two different locations (ORF and ORF1) of the coding sequence of GAL1. 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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150799&req=5

Figure 1: Rrd1p associates with the coding sequence of a rapamycin non-responsive GAL1 gene, and promotes the association of RNA polymerase II following 90 min transcriptional induction. (A) Schematic diagram showing the locations of different primer pairs at GAL1 for the ChIP analysis. The numbers are presented with respect to the position of the first nucleotide of the initiation codon (+1). (B) Analysis of Rpb1p association with the GAL1 coding sequence in the wild-type and Δrrd1 strains. Both wild-type and Δrrd1 strains expressing Myc-tagged Rpb1p were grown in YPR at 30°C up to an OD600 of 0.9, and then switched to YPG for 90 min prior to formaldehyde-based in vivo cross-linking. Immunoprecipitation was carried out using an anti-Myc antibody (9E10; Santa Cruz Biotechnology, Inc.) against Myc-tagged Rpb1p. Immunoprecipitated-DNA was analyzed by PCR using the primer pair targeted to the coding sequence of GAL1. The ratio of immunoprecipitate over the input in the autoradiogram (i.e. ChIP signal) was measured. The ChIP signal of the wild-type strain was set to 100, and the ChIP signal of the mutant strain was normalized with respect to 100. The normalized ChIP signal (represented as normalized occupancy) is plotted in the form of a histogram. (C) Western blot analysis under similar growth conditions as in panel B. (D) Growth analysis of the wild-type and Δrrd1 strains after switching to YPG from YPR (i.e. during 90 min induction time period). (E) RT-PCR analysis of the GAL1 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) Rrd1p associates with the coding sequence of GAL1. Yeast strain expressing Myc-tagged Rrd1p was grown as in panel B. Immunoprecipitation was carried out using an anti-Myc antibody against Myc-tagged Rrd1p. Immunoprecipitated-DNA was analyzed by PCR using the primer pairs targeted to the UAS, core promoter and two different locations (ORF and ORF1) of the coding sequence of GAL1. 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.
Mentions: Since Rrd1p interacts with RNA polymerase II (9), and is involved in regulation of proline isomerization (9) and phosphorylation of CTD of RNA polymerase II (10), it may regulate the association of RNA polymerase II with the coding sequence of active gene, and hence transcriptional elongation. However, previous studies (10) have demonstrated the dispensability of Rrd1p in regulation of transcription of rapamycin-responsive as well as non-responsive genes in the absence of rapamycin treatment under vegetative growth conditions. It is quite possible that Rrd1p promotes initial rounds/cycles of transcription via its RNA polymerase II-specific prolyl isomerase activity independently of rapamycin response, and the function of Rrd1p in transcription becomes minimal (or absent) when the steady-state is reached. To test this possibility, we analyzed the association of RNA polymerase II with the coding sequence of a galactose-inducible GAL1 gene following transcriptional induction in the absence of rapamycin treatment. In this direction, we grew both the wild-type and Δrrd1 strains in raffinose-containing growth medium (non-inducing) up to an OD600 of 0.9, and then switched to galactose-containing growth medium for 90 min prior to formaldehyde-based in vivo crosslinking. Using crosslinked cells, we performed the ChIP experiments to analyze the level of RNA polymerase II (Rpb1p) at the GAL1 coding sequence. We found that the association of RNA polymerase II with the GAL1 coding sequence was dramatically impaired in the Δrrd1 strain in the absence of rapamycin treatment (Figure 1A and B). Such reduction in the association of RNA polymerase II with the GAL1 coding sequence could be due to decreased stability of Rpb1p in the Δrrd1 strain. To test this possibility, we analyzed the global levels of Rpb1p in the wild-type and Δrrd1 strains, using the western blot assay. We found that the global level of Rpb1p was not changed in the Δrrd1 strain as compared to the wild-type equivalent (Figure 1C). The level of actin was monitored as a loading control, and its level was not altered in the Δrrd1 strain (Figure 1C). Thus, reduced association of RNA polymerase II with the GAL1 coding sequence following 90 min transcriptional induction in the Δrrd1 strain was not due to an impaired stability of Rpb1p. However, it is quite possible that the Δrrd1 strain grew slowly in galactose-containing growth medium during 90 min transcriptional induction, leading to a less number of the Δrrd1 cells and consequently decreased level of RNA polymerase II at GAL1. To test this possibility, we measured OD600 of the wild-type and Δrrd1 strains under the growth conditions used in the above ChIP experiments. We found that OD600 of the Δrrd1 strain was less than that of the wild-type strain at 90 min following the switch of the growth medium containing raffinose to galactose (Figure 1D). Thus, the relatively smaller number of the Δrrd1 cells following 90 min transcriptional induction in the ChIP experiments might have led to decreased amount of immunoprecipitated-DNA in comparison to the wild-type equivalent. However, we rule out this possibility as we normalized the immunoprecipitated-DNA signal with respect to its input DNA (referred to as ChIP signal), and then compared the ChIP signal of the Δrrd1 strain with that of the wild-type equivalent. Thus, our results support that Rrd1p promotes the association of RNA polymerase II with the GAL1 coding sequence. Further, we found that transcription of GAL1 was not regulated by rapamycin (Figure 1E), consistent with previous studies (10). Transcription of ACT1 was analyzed as a control (Figure 1E), since ACT1 is not responsive to rapamycin (8,10). Taken together, our results (Figure 1B–E) demonstrate that Rrd1p promotes the association of RNA polymerase II with the coding sequence of a rapamycin non-responsive GAL1 gene following 90 min transcriptional induction in the absence of rapamycin treatment. Consistently, we observed predominant association of Rrd1p with the coding sequence of GAL1 (Figure 1A and F, Supplementary Figure S1).

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