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Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B.

Cojocaru M, Bouchard A, Cloutier P, Cooper JJ, Varzavand K, Price DH, Coulombe B - J. Biol. Chem. (2010)

Bottom Line: Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene.This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9.Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.

ABSTRACT
Elongation of transcription by mammalian RNA polymerase II (RNAPII) is regulated by specific factors, including transcription factor IIS (TFIIS) and positive transcription elongation factor b (P-TEFb). We show that the E3 ubiquitin ligase UBR5 associates with the CDK9 subunit of positive transcription elongation factor b to mediate its polyubiquitination in human cells. TFIIS also binds UBR5 to stimulate CDK9 polyubiquitination. Co-localization of UBR5, CDK9, and TFIIS along specific regions of the γ fibrinogen (γFBG) gene indicates that a ternary complex involving these factors participates in the transcriptional regulation of this gene. In support of this notion, overexpression of TFIIS not only modifies the ubiquitination pattern of CDK9 in vivo but also increases the association of CDK9 with various regions of the γFBG gene. Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene. This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9. Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

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UBR5 knockdown does not affect the stability of CDK9. HeLa cells were co-transfected with constructs driving the expression of RLuc fused to the N-terminal region of CDK9 and with Fluc as an internal normalization control, together with siRNAs targeting UBR5 or control nontargeting siRNAs. Cycloheximide was added to culture medium for 0, 2, and 4 h to block overall protein synthesis, and luciferase activities were measured (upper panel). Parallel control experiments using the Rluc construct alone were conducted (lower panel). Each value represents the average of four assays, including two independent experiments.
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Figure 5: UBR5 knockdown does not affect the stability of CDK9. HeLa cells were co-transfected with constructs driving the expression of RLuc fused to the N-terminal region of CDK9 and with Fluc as an internal normalization control, together with siRNAs targeting UBR5 or control nontargeting siRNAs. Cycloheximide was added to culture medium for 0, 2, and 4 h to block overall protein synthesis, and luciferase activities were measured (upper panel). Parallel control experiments using the Rluc construct alone were conducted (lower panel). Each value represents the average of four assays, including two independent experiments.

Mentions: Although endogenous CDK9 is a long-lived protein, with a half-life (t½) between 4 and 7 h depending on the cell type, the exogenously overexpressed CDK9 fusion protein was reported to degrade faster, with a t½ dependent on the level of its overexpression (31). Because we have shown that the ubiquitination of CDK9 is dependent on UBR5, we reasoned that if the role of this modification is to target CDK9 for proteosomal degradation, then the knockdown of UBR5 should have a detectable effect on the stability of this kinase. We used a luciferase-based reporter assay combined with siRNA treatment to evaluate this hypothesis. Constructs encoding N- or C-terminal CDK9-Renilla luciferase fusion proteins were co-transfected into HeLa cells with a normalizing plasmid expressing the Fluc and with UBR5-targeting or control siRNAs. After a double transfection procedure, de novo protein synthesis was blocked by incubating the cells with cycloheximide for 2–4 h, and the luciferase activity was measured with a dual luciferase assay, as described by Archambault and co-workers (32). As a control, we also performed the same set of experiments with the Renilla luciferase plasmid with no attached CDK9 moiety. The results indicate that, in our conditions, the overexpressed CDK9 has a half-life of less than 2 h (Fig. 5, upper panel), although the control protein Renilla luciferase (Rluc) was relatively stable over the duration of the experiment (Fig. 5, lower panel). In cells in which UBR5 was knocked down, the kinetics of CDK9 degradation was similar to that of cells transfected with control nontargeting siRNA or mock-treated cells (Fig. 5, upper panel). More specifically, the luciferase activity of the CDK9-Rluc fusion protein, normalized to that of Fluc in each sample, presents similar values at all time points and for all tested conditions. Similar results were obtained when the RLuc fused to the N-terminal (Fig. 5, upper panel) or C-terminal (data not shown) ends of CDK9 was analyzed. These results suggest that UBR5 is not involved in regulating CDK9 stability and that the ubiquitination event mediated by this E3 ligase does not target CDK9 for degradation by the proteasome.


Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B.

Cojocaru M, Bouchard A, Cloutier P, Cooper JJ, Varzavand K, Price DH, Coulombe B - J. Biol. Chem. (2010)

UBR5 knockdown does not affect the stability of CDK9. HeLa cells were co-transfected with constructs driving the expression of RLuc fused to the N-terminal region of CDK9 and with Fluc as an internal normalization control, together with siRNAs targeting UBR5 or control nontargeting siRNAs. Cycloheximide was added to culture medium for 0, 2, and 4 h to block overall protein synthesis, and luciferase activities were measured (upper panel). Parallel control experiments using the Rluc construct alone were conducted (lower panel). Each value represents the average of four assays, including two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037613&req=5

Figure 5: UBR5 knockdown does not affect the stability of CDK9. HeLa cells were co-transfected with constructs driving the expression of RLuc fused to the N-terminal region of CDK9 and with Fluc as an internal normalization control, together with siRNAs targeting UBR5 or control nontargeting siRNAs. Cycloheximide was added to culture medium for 0, 2, and 4 h to block overall protein synthesis, and luciferase activities were measured (upper panel). Parallel control experiments using the Rluc construct alone were conducted (lower panel). Each value represents the average of four assays, including two independent experiments.
Mentions: Although endogenous CDK9 is a long-lived protein, with a half-life (t½) between 4 and 7 h depending on the cell type, the exogenously overexpressed CDK9 fusion protein was reported to degrade faster, with a t½ dependent on the level of its overexpression (31). Because we have shown that the ubiquitination of CDK9 is dependent on UBR5, we reasoned that if the role of this modification is to target CDK9 for proteosomal degradation, then the knockdown of UBR5 should have a detectable effect on the stability of this kinase. We used a luciferase-based reporter assay combined with siRNA treatment to evaluate this hypothesis. Constructs encoding N- or C-terminal CDK9-Renilla luciferase fusion proteins were co-transfected into HeLa cells with a normalizing plasmid expressing the Fluc and with UBR5-targeting or control siRNAs. After a double transfection procedure, de novo protein synthesis was blocked by incubating the cells with cycloheximide for 2–4 h, and the luciferase activity was measured with a dual luciferase assay, as described by Archambault and co-workers (32). As a control, we also performed the same set of experiments with the Renilla luciferase plasmid with no attached CDK9 moiety. The results indicate that, in our conditions, the overexpressed CDK9 has a half-life of less than 2 h (Fig. 5, upper panel), although the control protein Renilla luciferase (Rluc) was relatively stable over the duration of the experiment (Fig. 5, lower panel). In cells in which UBR5 was knocked down, the kinetics of CDK9 degradation was similar to that of cells transfected with control nontargeting siRNA or mock-treated cells (Fig. 5, upper panel). More specifically, the luciferase activity of the CDK9-Rluc fusion protein, normalized to that of Fluc in each sample, presents similar values at all time points and for all tested conditions. Similar results were obtained when the RLuc fused to the N-terminal (Fig. 5, upper panel) or C-terminal (data not shown) ends of CDK9 was analyzed. These results suggest that UBR5 is not involved in regulating CDK9 stability and that the ubiquitination event mediated by this E3 ligase does not target CDK9 for degradation by the proteasome.

Bottom Line: Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene.This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9.Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.

ABSTRACT
Elongation of transcription by mammalian RNA polymerase II (RNAPII) is regulated by specific factors, including transcription factor IIS (TFIIS) and positive transcription elongation factor b (P-TEFb). We show that the E3 ubiquitin ligase UBR5 associates with the CDK9 subunit of positive transcription elongation factor b to mediate its polyubiquitination in human cells. TFIIS also binds UBR5 to stimulate CDK9 polyubiquitination. Co-localization of UBR5, CDK9, and TFIIS along specific regions of the γ fibrinogen (γFBG) gene indicates that a ternary complex involving these factors participates in the transcriptional regulation of this gene. In support of this notion, overexpression of TFIIS not only modifies the ubiquitination pattern of CDK9 in vivo but also increases the association of CDK9 with various regions of the γFBG gene. Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene. This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9. Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

Show MeSH