Journal: PLoS ONE
Article Title: PRMT5: A novel regulator of Hepatitis B virus replication and an arginine methylase of HBV core
doi: 10.1371/journal.pone.0186982
Figure Lengend Snippet: (A) Detection of monomethylation and symmetric dimethylation of HBc protein. HepG2_hNTCP cells were transfected with HBc-HA (full length, aa 1–185), HBc-ΔC-HA (C-terminal deletion, aa 1–149), or an empty vector (pcDNA). Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-HA antibodies (HBc and HBc-ΔC), and the precipitated complexes were analyzed by Western blot with monomethyl-arginine (MM-R, top panel) or symmetric-dimethyl-arginine (SDM-R, middle panel) antibodies. The quality/efficiency of anti-HA immunoprecipitation is shown by Western blot with anti-HA or anti-HBc antibodies in the bottom panels. *, non-specific WB signal. Red and green arrows show HBc protein fragments of 30 kDa and 48 kDa, respectively. (B) Cell lysates (400 μg) of HepG2.2.15 cells were precipitated with HBc antibodies and the precipitated complexes were analyzed by Western blot with monomethyl-arginine (MM-R, top panel) or symmetric-dimethyl-arginine (SDM-R, middle panel) antibodies. The expression level of HBc protein is shown in the bottom panel. *, non-specific. (C) Symmetric dimethylation of HBc is stimulated by PRMT5, PRMT7 and MEP50. HEK293T cells were co-transfected with a constant amount of HBc-HA expression plasmid in combination with expression plasmids encoding Flag-tagged PRMT1, PRMT3, PRMT5 (v1, v2), PRMT7, PRMT9, MEP50, or an empty vector. Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-HA antibodies (HBc), and the precipitated complexes were analyzed by Western blot with SDM-R antibodies. The relative levels of transfected HBc-HA and Flag-PRMTs or Flag-MEP50 in 40 μg of cell lysates were estimated by Western blots (10% input). The intensity of the symmetric dimethylation signal was quantified using the ImageQuant TL Array software and normalized to the level of HBc expression. The level of HBc methylation in mock (pcDNA) transfected cells was set to 1 (Quant. and graph below). Asterisks indicate statistically significant differences between the control (pcDNA) and PRMTs groups determined by ANOVA (*** P<0.001). Error bars represent SD of three independent experiments. (D) Down-regulation of PRMT5 and MEP50 inhibits symmetric dimethylation of HBc protein. HEK293T cells were co-transfected with a constant amount of HBc-HA expression plasmid in combination with PRMT3-, PRMT5- or MEP50-specific siRNAs, or control siRNA (ctrlsiRNA). Forty-eight hours after transfection, cell lysates (400 μg) were precipitated with anti-HA antibodies (HBc), and the precipitated complexes were analyzed by Western blot with SDM-R antibodies. The relative levels of endogenous PRMT5, MEP50, β-actin or transfected HBc-HA in 40 μg of cell lysates were estimated by Western blots (10% input). The intensity of the symmetric dimethylation signal was quantified as described in (C). The level of HBc methylation in mock (ctrlsiRNA) transfected cells was set to 1 (Quant. and graph below). Asterisks indicate statistically significant differences between the control (ctrlsiRNA) and PRMTsiRNA groups determined by ANOVA (* P<0.05; ** P<0.01). Error bars represent SD of three independent experiments. (E) Treatment with GSK591, a potent PRMT5/MEP50-specific inhibitor, reduces the symmetric arginine dimethylation of HBc protein. HepG2_hNTCP cells were transfected with equal amounts of HBc-HA expression plasmid. Twenty-four hours after transfection, the cells were treated with increasing concentrations of GSK591 (0, 0.5, 5, 50, 500 nM) for 72 hours as indicated. The cell lysates (40 μg) were analyzed by Western blotting (WB) with SDM-R, PRMT5, MEP50, HBc and β-actin antibodies. To analyze the level of symmetric-arginine dimethylation of HBc protein (bottom panel), the cell lysates (400 μg) were immunoprecipitated (IP) with anti-HA-specific antibodies followed by Western blotting with anti-SDM-R antibodies. The intensities of total cellular symmetric dimethylation and HBc protein dimethylation were quantified as described in (C) and normalized to the levels of β-actin or HBc expression, respectively. (F) Expression of HBc inhibits the PRMT5-mediated methylation of SmB/B’ and SmD3 splicing factors. HEK293T cells were co-transfected with a constant amount of HBc-HA expression plasmid in combination with expression plasmids encoding Flag-tagged PRMT5v1 and v2, or an empty vector, pcDNA, as indicated. Forty-eight hours after transfection, cell lysates (20 μg) were analyzed by Western blot with SDM-R, β-actin, Flag and HA antibodies. The levels of symmetrically dimethylated SmB/B’and SmD3 were quantified as described in (C) and normalized to the levels of β-actin expression. To confirm that the major methylated band corresponds to SmB/B’, the SmB/B’protein was immunoprecipitated from cell lysates (400 μg) with anti-SmB antibodies followed by Western blotting with anti-SDM-R antibodies (middle panels).
Article Snippet: Expression plasmids for PRMT1, PRMT3, PRMT5v1 (splice variant 1), PRMT5v2 (splice variant 2), PRMT7, PRMT9 (Q6P2P2 in the UniProt Database, previously also referred to as PRMT10) and MEP50/WDR77 tagged with myc and Flag were purchased from OriGene Technologies.
Techniques: Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Expressing, Software, Methylation, Control
