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PRMT5 is required for cell-cycle progression and p53 tumor suppressor function - PubMed

PRMT5 is required for cell-cycle progression and p53 tumor suppressor function

A Scoumanne et al. Nucleic Acids Res. 2009 Aug.

Abstract

Protein arginine methyltransferases (PRMTs) mediate the transfer of methyl groups to arginines in proteins involved in signal transduction, transcriptional regulation and RNA processing. Tumor suppressor p53 coordinates crucial cellular processes, including cell-cycle arrest and DNA repair, in response to stress signals. Post-translational modifications and interactions with co-factors are important to regulate p53 transcriptional activity. To explore whether PRMTs modulate p53 function, we generated multiple cell lines in which PRMT1, CARM1 and PRMT5 are inducibly knocked down. Here, we showed that PRMT5, but not PRMT1 or CARM1, is essential for cell proliferation and PRMT5 deficiency triggers cell-cycle arrest in G1. In addition, PRMT5 is required for p53 expression and induction of p53 targets MDM2 and p21 upon DNA damage. Importantly, we established that PRMT5 knockdown prevents p53 protein synthesis. Furthermore, we found that PRMT5 regulates the expression of translation initiation factor eIF4E and growth suppression mediated upon PRMT5 knockdown is independent of p53 but is dependent on eIF4E. Taken together, we uncovered that arginine methyltransferase PRMT5 is a major pro-survival factor regulating eIF4E expression and p53 translation.

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Figures

Figure 1.
Figure 1.

PRMT5 is required for G1-S transition. (A) Generation of MCF7 cell lines inducibly expressing PRMT5 shRNA. Levels of PRMT5 and GAPDH were assayed in MCF7-pTR-7 and MCF7-PRMT5-KD-41/-62/-72 cells cultured in the absence (−) or presence (+) of tetracycline for 4 days. (B) PRMT5 is required for colony formation. MCF7-pTR-7 and MCF7-PRMT5-KD-41/-62/-72 cells were cultured in the absence or presence of tetracycline for 10 days. (C) PRMT5 is required for cell proliferation. Growth rate of MCF7-pTR-7 and MCF7-PRMT5-KD-41/-62/-72 cells cultured in the absence or presence of tetracycline over a 8-day period. The average ± S.D. of triplicates was plotted, and the data is representative of three independent experiments. P-values were ≤0.005 (comparison upon induction for 8 days). (D) Deficiency in PRMT5 induces G1 arrest. MCF7-PRMT5-KD-41/-62/-72 cells were uninduced or induced to knockdown PRMT5 for 5 days. The percentage of cells in each phase of the cell cycle was quantified by FACS analysis. The average ± SEM of three independent experiments is shown. P-values were ≤0.01 (comparison of cells numbers in G1).

Figure 2.
Figure 2.

CARM1 and PRMT1 are not essential for cell proliferation. (A) Generation of MCF7 cell lines inducibly expressing CARM1 or PRMT1 shRNA. Levels of CARM1, PRMT1 and Actin were assayed in MCF7-pTR-7, MCF7-CARM1-KD-12/-17 and MCF7-PRMT1-KD-6 cells cultured in the absence (−) or presence (+) of tetracycline for 4 days. (B) CARM1 or PRMT1 are not required for colony formation. MCF7-pTR-7, MCF7-CARM1-KD-12/-17 and MCF7-PRMT1-KD-6 cells were cultured in the absence or presence of tetracycline for 10 days. (C) Quantification of the number of colonies shown in (B). The percentage of colonies with a diameter <1 mm (left panel) or >1 mm (right panel) was calculated in three representative areas for each cell line. The average ± SEM was plotted as the percentage of colonies increased or decreased by tetracycline compared to parental MCF7 cells.

Figure 3.
Figure 3.

PRMT5 is required for efficient p53 stabilization and transcriptional activity in multiple cell lines. (A) Cells extracts were prepared from MCF7-pTR-7 and MCF7-PRMT5-KD-41 cells uninduced (−) or induced (+) to knockdown PRMT5 for 3 days, and then untreated or treated with 0.35 µM doxorubicin for 6, 12 or 24 h. (B) Cells extracts were prepared from RKO and HCT116 cells transiently transfected with scrambled control siRNA (−) or PRMT5 siRNA (+) for 3 days, and then untreated or treated with 0.35 µM doxorubicin for 12 or 24 h. (C) Cells extracts were prepared from SW480 and T98G cells transiently transfected with scrambled control siRNA (−) or PRMT5 siRNA (+) for 3 days. The data is representative of two independent experiments. Levels of PRMT5, p53, MDM2, p21 and GAPDH were detected by western blot analysis.

Figure 4.
Figure 4.

Knockdown of PRMT5 inhibits cell proliferation in a p53-independent manner. (A) Generation of MCF7 cell lines inducibly expressing PRMT5 shRNA and stably expressing p53 shRNA. Levels of PRMT5, p53 and GAPDH were assayed in MCF7-PRMT5-KD-41 and MCF7-PRMT5-KD/p53-KD-3/-11/-25 cells cultured in the absence (−) or presence (+) of tetracycline for 3 days. (B) PRMT5 is required for colony formation independently of p53. MCF7-PRMT5-KD-41 and MCF7-PRMT5-KD/p53-KD-3/-11/-25 cells were cultured in the absence or presence of tetracycline for 10 days. The data is representative of two separate experiments performed in triplicate wells. (C) PRMT5 is required for cell proliferation independently of p53. Growth rate of MCF7-PRMT5-KD-41 and MCF7-PRMT5-KD/p53-KD-3/-11/-25 cells cultured in the absence or presence of tetracycline over a 8-day period. The average ± S.D. of triplicates was plotted and the data is representative of two independent experiments. P-values were ≤0.01 (comparison upon induction for 8 days).

Figure 5.
Figure 5.

Knockdown of PRMT5 inhibits p53 protein synthesis. (A and B) MCF7-PRMT5-KD-41 uninduced (−) or induced (+) to knockdown PRMT5 for 3 days were (A) untreated or treated with 10 µM MG132 for 4 or 8 h, (B) followed by treatment with doxorubicin for 6 h. Cells extracts were prepared to determine levels of PRMT5, p53, and GAPDH by western blot analysis. (C) MCF7-PRMT5-KD-41 cells were cultured in the absence or presence of tetracycline for 3 days and then treated with doxorubicin for 6 h, followed by treatment with cycloheximide (CHX) for the indicated times. Levels of PRMT5, p53 and GAPDH were assayed by western blot analysis. (D) PRMT5 knockdown inhibits p53 protein synthesis. MCF7-PRMT5-KD-41 cells were uninduced (−) or induced (+) to knockdown PRMT5 for 3 days and [35S] methionine-labeled for 30 min. Whole-cell extracts and cells extracts immunoprecipitated with rabbit IgG or antibodies against p53 were resolved by SDS-PAGE and autoradiography. The data is representative of three independent immunoprecipitation experiments.

Figure 6.
Figure 6.

PRMT5 knockdown inhibits the expression of translation initiation factor 4E. (A) Knockdown of PRMT5 inhibits eIF4E expression. Cells extracts were prepared from MCF7-pTR-7 and MCF7-PRMT5-KD-41 cells uninduced (−) or induced (+) to knockdown PRMT5 for 3 days, and then untreated or treated with 0.35 µM doxorubicin for 12 or 24 h. Levels of PRMT5, eIF4E and GAPDH were detected by western blot analysis. (B) MCF7-PRMT5-KD-41 cells were transfected with pcDNA3 or pcDNA3 vector expressing Myc-tagged eIF4E for 24 h, followed by treatment with tetracycline for 3 days. Levels of PRMT5, Myc-tagged eIF4E, and GAPDH were detected by western blot analysis. (C) eIF4E expression attenuates the inhibition of cell proliferation by PRMT5 knockdown. Cells were collected and counted by Coulter counter. The average ± SEM of triplicates was plotted as percentage of cells compared to untreated cells.

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References

    1. Malkin D, Li FP, Strong LC, Fraumeni JF, Jr, Nelson CE, Kim DH, Kassel J, Gryka MA, Bischoff FZ, Tainsky MA, et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms [see comments] Science. 1990;250:1233–1238. - PubMed
    1. Harms K, Nozell S, Chen X. The common and distinct target genes of the p53 family transcription factors. Cell Mol. Life Sci. 2004;61:822–842. - PMC - PubMed
    1. Helton ES, Chen X. p53 modulation of the DNA damage response. J. Cell Biochem. 2007;100:883–896. - PubMed
    1. Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 1997;420:25–27. - PubMed
    1. Chen J, Lin J, Levine AJ. Regulation of transcription functions of the p53 tumor suppressor by the mdm-2 oncogene. Mol. Med. 1995;1:142–152. - PMC - PubMed

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