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Regulation of rDNA transcription by proto-oncogene PELP1.

Gonugunta VK, Nair BC, Rajhans R, Sareddy GR, Nair SS, Vadlamudi RK - PLoS ONE (2011)

Bottom Line: Using pharmacological compounds and CDK site mutants of PELP1, we found that CDK's activity plays an important role on PELP1 nucleolar localization.Depletion of PELP1 by siRNA decreased the expression of pre-rRNA.Collectively, our results suggest that proto-oncogene PELP1 plays a vital role in rDNA transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, and Cancer Therapy and Research Center, University of Texas Health Science Center, San Antonio, Texas, United States of America.

ABSTRACT

Background: Proline-, glutamic acid-, and leucine-rich protein (PELP1) is a novel nuclear receptor coregulator with a multitude of functions. PELP1 serves as a scaffolding protein that couples various signaling complexes with nuclear receptors and participates as a transcriptional coregulator. Recent data suggest that PELP1 expression is deregulated in hormonal cancers, and that PELP1 functions as a proto-oncogene; however, the mechanism by which PELP1 promotes oncogenesis remains elusive.

Methodology/principal findings: Using pharmacological inhibitors, confocal microscopy and biochemical assays, we demonstrated that PELP1 is localized in the nucleolus and that PELP1 is associated with the active ribosomal RNA transcription. Cell synchronization studies showed that PELP1 nucleolar localization varies and the greatest amount of nucleolar localization was observed during S and G2 phases. Using pharmacological compounds and CDK site mutants of PELP1, we found that CDK's activity plays an important role on PELP1 nucleolar localization. Depletion of PELP1 by siRNA decreased the expression of pre-rRNA. Reporter gene assays using ribosomal DNA (pHrD) luc-reporter revealed that PELP1WT but not PELP1MT enhanced the expression of reporter. Deletion of nucleolar domains abolished PELP1-mediated activation of the pHrD reporter. ChIP analysis revealed that PELP1 is recruited to the promoter regions of rDNA and is needed for optimal transcription of ribosomal RNA.

Conclusions/significance: Collectively, our results suggest that proto-oncogene PELP1 plays a vital role in rDNA transcription. PELP1 modulation of rRNA transcription, a key step in ribosomal biogenesis may have implications in PELP1-mediated oncogenic functions.

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Related in: MedlinePlus

PELP1 activation of ribosomal promoter depends on functional nucleolar domains.(A) Schematic representation of PELP1 nucleolar domains. (B) 293T cells were transiently transfected with PELP1 WT and Δ-Nuc-PELP1 mutant vectors. Expression of the constructs was analyzed by immunoblotting. (C) 293T cells were transfected with pHrD luciferase reporter along with PELP1 WT or Δ-Nuc-PELP1 vectors. Cells were serum starved for 24 h and then stimulated with 10% serum for 24 h and the reporter gene activity was measured. Results are the average of 3 independent experiments. Statistical analysis, paired t-test was performed and considered significant when p-value <0.05.
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pone-0021095-g003: PELP1 activation of ribosomal promoter depends on functional nucleolar domains.(A) Schematic representation of PELP1 nucleolar domains. (B) 293T cells were transiently transfected with PELP1 WT and Δ-Nuc-PELP1 mutant vectors. Expression of the constructs was analyzed by immunoblotting. (C) 293T cells were transfected with pHrD luciferase reporter along with PELP1 WT or Δ-Nuc-PELP1 vectors. Cells were serum starved for 24 h and then stimulated with 10% serum for 24 h and the reporter gene activity was measured. Results are the average of 3 independent experiments. Statistical analysis, paired t-test was performed and considered significant when p-value <0.05.

Mentions: Homology search using a bioinformatic approach revealed that PELP1 contains two nucleolar domains [19] that are commonly present in many proteins that localize in the nucleolus. These domains are localized in the N-terminal region of PELP1 comprising amino acids 79–160 (Nuc 1) and 423–489 (Nuc 2) (Figure 3A). To examine the significance of these domains in PELP1-mediated coactivation of ribosomal promoter, we deleted these two regions from full-length PELP1. Western analysis revealed expression of mutants and their migration to the expected sizes (Figure 3B). In reporter gene assays, PELP1 lacking nucleolar domains failed to activate the ribosomal promoter reporter, while PELP1WT enhanced the ribosomal promoter activity (Figure 3C). These results suggested that functional nucleolar domains in PELP1 are important for nuclear localization and ribosomal promoter activation.


Regulation of rDNA transcription by proto-oncogene PELP1.

Gonugunta VK, Nair BC, Rajhans R, Sareddy GR, Nair SS, Vadlamudi RK - PLoS ONE (2011)

PELP1 activation of ribosomal promoter depends on functional nucleolar domains.(A) Schematic representation of PELP1 nucleolar domains. (B) 293T cells were transiently transfected with PELP1 WT and Δ-Nuc-PELP1 mutant vectors. Expression of the constructs was analyzed by immunoblotting. (C) 293T cells were transfected with pHrD luciferase reporter along with PELP1 WT or Δ-Nuc-PELP1 vectors. Cells were serum starved for 24 h and then stimulated with 10% serum for 24 h and the reporter gene activity was measured. Results are the average of 3 independent experiments. Statistical analysis, paired t-test was performed and considered significant when p-value <0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113909&req=5

pone-0021095-g003: PELP1 activation of ribosomal promoter depends on functional nucleolar domains.(A) Schematic representation of PELP1 nucleolar domains. (B) 293T cells were transiently transfected with PELP1 WT and Δ-Nuc-PELP1 mutant vectors. Expression of the constructs was analyzed by immunoblotting. (C) 293T cells were transfected with pHrD luciferase reporter along with PELP1 WT or Δ-Nuc-PELP1 vectors. Cells were serum starved for 24 h and then stimulated with 10% serum for 24 h and the reporter gene activity was measured. Results are the average of 3 independent experiments. Statistical analysis, paired t-test was performed and considered significant when p-value <0.05.
Mentions: Homology search using a bioinformatic approach revealed that PELP1 contains two nucleolar domains [19] that are commonly present in many proteins that localize in the nucleolus. These domains are localized in the N-terminal region of PELP1 comprising amino acids 79–160 (Nuc 1) and 423–489 (Nuc 2) (Figure 3A). To examine the significance of these domains in PELP1-mediated coactivation of ribosomal promoter, we deleted these two regions from full-length PELP1. Western analysis revealed expression of mutants and their migration to the expected sizes (Figure 3B). In reporter gene assays, PELP1 lacking nucleolar domains failed to activate the ribosomal promoter reporter, while PELP1WT enhanced the ribosomal promoter activity (Figure 3C). These results suggested that functional nucleolar domains in PELP1 are important for nuclear localization and ribosomal promoter activation.

Bottom Line: Using pharmacological compounds and CDK site mutants of PELP1, we found that CDK's activity plays an important role on PELP1 nucleolar localization.Depletion of PELP1 by siRNA decreased the expression of pre-rRNA.Collectively, our results suggest that proto-oncogene PELP1 plays a vital role in rDNA transcription.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, and Cancer Therapy and Research Center, University of Texas Health Science Center, San Antonio, Texas, United States of America.

ABSTRACT

Background: Proline-, glutamic acid-, and leucine-rich protein (PELP1) is a novel nuclear receptor coregulator with a multitude of functions. PELP1 serves as a scaffolding protein that couples various signaling complexes with nuclear receptors and participates as a transcriptional coregulator. Recent data suggest that PELP1 expression is deregulated in hormonal cancers, and that PELP1 functions as a proto-oncogene; however, the mechanism by which PELP1 promotes oncogenesis remains elusive.

Methodology/principal findings: Using pharmacological inhibitors, confocal microscopy and biochemical assays, we demonstrated that PELP1 is localized in the nucleolus and that PELP1 is associated with the active ribosomal RNA transcription. Cell synchronization studies showed that PELP1 nucleolar localization varies and the greatest amount of nucleolar localization was observed during S and G2 phases. Using pharmacological compounds and CDK site mutants of PELP1, we found that CDK's activity plays an important role on PELP1 nucleolar localization. Depletion of PELP1 by siRNA decreased the expression of pre-rRNA. Reporter gene assays using ribosomal DNA (pHrD) luc-reporter revealed that PELP1WT but not PELP1MT enhanced the expression of reporter. Deletion of nucleolar domains abolished PELP1-mediated activation of the pHrD reporter. ChIP analysis revealed that PELP1 is recruited to the promoter regions of rDNA and is needed for optimal transcription of ribosomal RNA.

Conclusions/significance: Collectively, our results suggest that proto-oncogene PELP1 plays a vital role in rDNA transcription. PELP1 modulation of rRNA transcription, a key step in ribosomal biogenesis may have implications in PELP1-mediated oncogenic functions.

Show MeSH
Related in: MedlinePlus