Figure 1.
PRMT5 localized with p44 in the cytoplasm but not in the nucleus in prostate cancer cells.
(A) PC3 and LNCaP cells were immunohistochemically stained with anti-PRMT5 and -p44 antibodies (panels a-g) or anti-PRMT5 plus -coilin antibodies (panel h). The fluorescent signals were observed under a confocal microscope with a red filter (to detect PRMT5) or green filter (to detect p44 or coilin). Right panels show merged images of PRMT5 and p44 or coilin staining. White and green arrowheads indicate PRMT5 and p44 or coilin signals in the nucleus, respectively. (B) Western blot of cytoplasmic and nuclear fractions of LNCaP and PC3 cells with anti-PRMT5, -p44, -HSP90, or anti-lamin B antibody. C, cytoplasm; N, nucleus.
Figure 2.
PRMT5 contains three nuclear exclusion signals.
(A) Diagrams of the PRMT5 truncations expressed as GFP-fusion proteins. The percentages of cells with GFP-PRMT5 truncations in the cytoplasm (C), nucleus (N), or cytoplasm plus nucleus (C/N) are shown on the right. (B) Subcellular localization of isolated nuclear export signals. Cells were transfected with pcDNA-f:GFP-PRMT5, -PRMT5(1–90), -PRMT5(500–560), -PRMT5(576–637), or pcDNA-GFP and observed under a confocal microscope. (C) Western blot analysis of cytoplasmic and nuclear fractions of cells transfected with pcDNA-f:GFP-PRMT5, -PRMT5(1–90), PRMT5(500–560), or PRMT5(576–637) with anti-FLAG, -HSP90, or -lamin B antibody.
Figure 3.
PRMT5 promotes p44 cytoplasmic translocation.
(A) Diagrams of the PRMT5 truncations. Cells were transfected with pcDNA-GFP-p44 and pcDNA-PRMT5 or pcDNA-PRMT5 truncations, and the percentages of cells with GFP-p44 in cytoplasm (C) or cytoplasm plus nucleus (C/N) are shown on the right. (B) Cytoplasmic translocation of GFP-p44 driven by PRMT5. Cells were transfected with pcDNA-GFP-p44 alone or together with pcDNA-f:PRMT5, -f:PRMT5(91–637), or f:PRMT5(325–637), and the GFP-p44 subcellular localization was observed under a confocal microscope. (C) Western blot analysis of cytoplasmic and nuclear fractions of Cos 7 cells described in B with anti-p44, -HSP90, or -lamin B antibody.
Figure 4.
The PRMT5-p44 interaction is essential for the PRMT5-promoted p44 cytoplasmic localization.
(A) Mutations in p44 abolished PRMT5-driven p44 cytoplasmic translocation. Cells were transfected with pcDNA-GFP-p44(WT) or pcDNA-GFP-p44(MT) alone or together with pcDNA-PRMT5. The nucleus was stained with Far-red, and the subcellular localization of GFP-p44 was observed under a confocal microscope. (B) Mutations in p44 abolished the interaction of p44 with PRMT5. Cells were transfected with pcDNA–f:p44 (WT) (lane 1) or pcDNA–f:p44 (MT) (lanes 2–5), and whole-cell lysates were prepared for immunoprecipitation with anti-FLAG antibody (M2 agarose). Western blot with anti-PRMT5 was performed to detect the precipitated PRMT5 (bottom panel). Top panel shows expression of wild-type (WT) or mutated (MT) p44 in the lysates used for the immunoprecipitation.
Figure 5.
Silencing PRMT5 or p44 expression inhibited the growth of prostate cancer cells.
(A) The shRNA-mediated silencing of PRMT5 or p44 expression in prostate cancer cells. Western blot analysis of whole-cell lysates made from LNCaP cells infected with lentivirus expressing the non-target (NT) shRNA (lanes 1, 5), PRMT5 (lanes 2–4), or p44 (lane 6) shRNAs. The shRNA-resistant PRMT5 (lane 3) or PRMT5 R368A mutant (lane 4) was expressed in the PRMT5-expressing LNCaP cells. (B) Growth curves of prostate cancer cells expressing NT shRNA, PRMT5 shRNAs, p44 shRNA, PRMT5 shRNAs plus PRMT5, p44 shRNA plus p44, or PRMT5 shRNAs plus PRMT5mt.
Figure 6.
PRMT5 and p44 co-exist in the cytoplasm.
(A) Silencing PRMT5 expression decreased p44 protein levels in the cytoplasm. LNCaP cells were infected with NT-shRNA or PRMT5 shRNA and immunostained with anti-PRMT5 or -p44 antibody. The nucleus was counterstained with Sytox green (middle panels, green). The samples were observed under a confocal microscope. (B) Western blot of cytoplasmic (C) and nuclear (N) fractions of LNCaP cells expressing NT-shRNA, PRMT5 shRNA, or p44 shRNA with anti-p44 or anti-PRMT5 antibody.
Figure 7.
The nuclear PRMT5 inhibited the growth of prostate cancer cells.
(A) The PRMT5 protein in LNCaP cells expressing PRMT5, PRMT5mt, NLS-PRMT5, or NLS-PRMT5mt was immunostained with the anti-PRMT5 antibody. The samples were observed under a confocal microscope. (B) Western blot of cytoplasmic (C) and nuclear (N) fractions of LNCaP cells expressing PRMT5, f:PRMT5mt, NLS-PRMT5, or NLS-PRMT5mt with anti-PRMT5, -p44, -lamin B, or -HSP90 antibody. (C) Growth curves of prostate cancer LNCaP cells expressing PRMT5, f:PRMT5mt, NLS-PRMT5, or NLS-PRMT5mt.
Figure 8.
PRMT5 forms a stoichiometric complex with p44 and PRMT5.
(A) SDS-PAGE of PRMT5 complexes produced by co-expression in E. coli. (B) Methylation of SmD3 and histone H4 substrates by PRMT5 and PRMT5-containing complexes. Top: autoradiography of the gel. Bottom: Coomassie blue staining of the gel.
Figure 9.
The distinct subcellular localization of PRMT5 and p44 in benign and malignant prostate tissues
. Immunohistochemical staining of p44 and PRMT5 in human benign (top panels), prostatic epithelial hyperplasia (PIN, middle panels), and malignant prostate (PCa, Gleason grade 4, bottom panels) tissues.