Inhibition of U6 snRNA Transcription by PTEN

Abstract

PROBLEM STATEMENT: RNA polymerase III (RNA pol III) is responsible for transcribing many of the small structural RNA molecules involved in RNA processing and protein translation, thereby regulating the growth rate of a cell. RNA pol III transcribes both gene internal (tRNA) and gene external (U6 snRNA) promoters and proper initiation by RNA polymerase III requires the transcription initiation factor TFIIIB. TFIIIB has been shown to be a target of repression by tumor suppressors such as ARF, p53, RB and the RB-related pocket proteins. Also, TFIIIB activity is stimulated by the oncogenes c-Myc and the ERK mitogen-activated protein kinase. Recently, two TFIIIB subunits, BRF1 and BRF2, have been demonstrated to behave as oncogenes, making deregulation of TFIIIB activity and thus RNA pol III transcription an important step in tumor development. PTEN is a commonly mutated tumor suppressor regulating cell growth, proliferation and survival. Thus, we sought to examine the potential role of PTEN in regulating U6 snRNA transcription. APPROACH: We examined the potential for PTEN to regulate U6 snRNA transcription using in vitro RNA pol III luciferase assays, western blotting and deletion analysis in cancer cell lines differing in their PTEN status. RESULTS: Using breast, cervical, prostate and glioblastoma cancer cells we demonstrate: (1) PTEN inhibition of gene external RNA pol III transcription is cell type specific, (2) PTEN-mediated inhibition of U6 transcription occurs via the C2 lipid-binding domain and (3) PTEN repression of U6 transcription occurs, at least in part, through the TFIIIB subunit BRF2. CONCLUSION/RECOMMENDATIONS: Our data demonstrates that regulation of the U6 snRNA gene by PTEN is mediated, in part by the TFIIIB oncogene BRF2, potentially identifying novel targets for chemotherapeutic drug design.

Fig. 1

PTEN inhibits RNA pol. III transcription in cancer cells which express functional PTEN. (A) HeLa, DU-145, MCF-7 and MCF10A cells transiently transfected with empty pGL3 vector (100 ng), pGL3-VAI (100 ng) alone or co-transfected with increasing concentrations of pCDNA3-PTEN (100, 125, 150 ng); (B) HeLa, DU-145, MCF-7 and MCF10A cells transiently transfected with empty pGL3 vector (100 ng), pGL3-U6 (100 ng) alone or cotransfected with increasing concentrations of pCDNA3-PTEN (50, 75, 100 ng). All luciferase assay results expressed as Relative Light Units (RLU): the average of the Photinus pyralis firefly activity observed divided by the average of the activity recorded from the Renilla luciferase vector. Experiments were done in triplicate, repeated three independent times and representative experiments are depicted. Statistical analysis was performed using oneway ANOVA with a Tukey post-test with a 95% confidence interval (Graphpad Prism 3.03); * = p<0.05; ** = p<0.01; *** = p<0.001

Fig. 2

PTEN inhibits RNA pol. III transcription in cancer cells which are PTEN-null. (A) PTEN-null HCC1937, PC-3 U87 cells transiently transfected with empty pGL3 vector (100 ng), pGL3-VAI (100 ng) alone or cotransfected with increasing concentrations of pCDNA3-PTEN (100, 125, 150 ng); (B) PTEN-null HCC1937, PC-3 and U87 cells transiently transfected with empty pGL3 vector (100ng), pGL3-U6 (100 ng) alone or co-transfected with increasing concentrations of pCDNA3-PTEN (50, 75,100 ng); (C) Nuclear extracts prepared from untransfected HCC1937 cells or HCC1937 cells transiently transfected with Flag-PTEN and immunoblotted with anti-actin and anti-flag antibodies. All luciferase assay results expressed as Relative Light Units (RLU): the average of the Photinus pyralis firefly activity observed divided by the average of the activity recorded from the Renilla luciferase vector. Experiments were done in triplicate, repeated three independent times and representative experiments are depicted. Statistical analysis was performed using one-way ANOVA with a Tukey post-test with a 95% confidence interval (Graphpad Prism 3.03); * = p<0.05; ** = p<0.01; *** = p<0.001

Fig. 3

U6 snRNA transcription is inhibited by PTEN deletions (A) Schematic representation of full-length PTEN and PTEN mutants. Full-length PTEN (1–403) represents the complete 403 amino acid protein. ΔP-PTEN (186–403) represents a deletion of the PTPase domain of PTEN. ΔC2/CTail-PTEN (1–185) represents a deletion of the C2 domain and the C-terminal tail. ΔCTail-PTEN (1–351) represents a deletion of the C-terminal tail. White box corresponds to the phosphatase domain which contains a black box corresponding to the PIP2-binding domain and a red box corresponding to the catalytic domain. Grey box corresponds to the C2-terminal half of PTEN containing the C2-lipid binding domain; blue boxes correspond to the PEST sequences and the yellow box corresponds to the PDZ binding domain; (B) PTEN-null HCC1937 cells transiently transfected with empty pGL3 vector (100 ng), pGL3-U6 (100 ng) alone or co-transfected with 100 ng of the following: PTEN, ΔP-PTEN, ΔC2/CTail-PTEN and ΔCTail-PTEN; (C) PTEN-null HCC1937 cells transiently transfected with empty pGL3 vector (100 ng), pGL3-VAI (100 ng) alone or co-transfected with 150 ng of the following: PTEN, ΔP-PTEN, ΔC2/CTail-PTEN and ΔCTail-PTEN. All luciferase assay results expressed as Relative Light Units (RLU): the average of the Photinus pyralis firefly activity observed divided by the average of the activity recorded from the Renilla luciferase vector. Experiments were done in triplicate, repeated three independent times and representative experiments are depicted. Statistical analysis was performed using one-way ANOVA with a Tukey post-test with a 95% confidence interval (Graphpad Prism 3.03); * = p<0.05; ** = p<0.01; *** = p<0.001

Fig. 4

PTEN phosphatase point mutations inhibit U6 snRNA transcription. Schematic of well-characterized PTEN phosphatase point mutations in the catalytic site. (A) PTEN-null HCC1937 cells transiently transfected with empty pGL3 vector (100 ng), pGL3-U6 (100 ng) alone or co-transfected with 100 ng of the following: following: pSG5L HA PTEN G129E; pSG5L Flag HA PTEN G129R; pSG5L HA PTEN C124S; (B) PTEN-null HCC1937 cells transiently transfected with empty pGL3 vector (100 ng), pGL3-VAI (100 ng) alone or co-transfected with 150 ng of the following: following: pSG5L HA PTEN G129E; pSG5L Flag HA PTEN G129R; pSG5L HA PTEN C124S. All luciferase assay results expressed as Relative Light Units (RLU): the average of the Photinus pyralis firefly activity observed divided by the average of the activity recorded from the Renilla luciferase vector. Experiments were done in triplicate, repeated three independent times and representative experiments are depicted. Statistical analysis was performed using one-way ANOVA with a Tukey post-test with a 95% confidence interval (Graphpad Prism 3.03); * = p<0.05; ** = p<0.01; *** = p<0.001

Fig. 5

The C2 domain is responsible for PTEN inhibition of U6 snRNA transcription (A) Schematic of C2-PTEN domain alone. PTEN-null HCC1937 cells transiently transfected with empty pGL3 vector (100 ng), pGL3-U6 (100 ng) alone or co-transfected with 100 ng of full-length PTEN or C2-PTEN; (B) PTEN-null HCC1937 cells transiently transfected with empty pGL3 vector (100 ng), pGL3-VAI (100 ng) alone or co-transfected with 150 ng of full-length PTEN or C2-PTEN; (C) Nuclear and cytoplasmic extracts prepared from untransfected HeLa cells or HeLa cells transiently transfected with Flag-PTEN and Flag-C2PTEN and immunoblotted with anti-actin and ntiflag antibodies; (D) PTEN-null HCC1937 cells transiently transfected with pGL3 (100 ng), pGL3-U6 (100 ng) alone, pGL3-U6 (100 ng) and full-length PTEN, or co-transfected with pGL3-U6, full-length PTEN and BRF2; (E) PTENnull HCC1937 cells transiently transfected with pGL3 (100 ng), pGL3-U6 (100ng) alone, or co-transfected with pGL3-U6 and BRF2. All luciferase assay results expressed as Relative Light Units (RLU): the average of the 10 Photinus pyralis firefly activity observed divided by the average of the activity recorded from the Renilla luciferase vector. Experiments were done in triplicate, repeated three independent times and representative experiments are depicted. Statistical analysis was performed using one-way ANOVA with a Tukey post-test with a 95% confidence interval (Graphpad Prism 3.03); * = p<0.05; ** = p<0.01; *** = p<0.001