RPS10 interacts with PRMT5 both in vivo and in vitro. A and B, RPS10 interacts with PRMT5 in vivo. HEK293 cells were transfected with an expression vector encoding tagged PRMT5 and RPS10 as indicated. 24 h later, aliquots of cell lysates were saved for analysis of expressed tagged proteins. The remaining portions of the lysates were immunoprecipitated with anti-FLAG for FLAG-PRMT5, and immunocomplexes were analyzed for Myc-RPS10 (A). In reciprocal co-expression/immunoprecipitation (IP) experiments, cell lysates were subjected to immunoprecipitation with anti-Myc antibody and immunocomplexes were probed for the presence of GFP-PRMT5 (B). C, endogenous PRMT5 and RPS10 interact with each other. HEK293 cell lysates were subjected to immunoprecipitation with anti-RPS10 or IgG antibodies as indicated. The immunocomplexes were probed separately for PRMT5 and RPS10. D, PRMT5 interacts with RPS10 in vitro. GST and GST-RPS10 fusion protein for binding assays were expressed, purified, and incubated with FLAG-PRMT5 protein purified from HEK293 cell lysates with FLAG beads. GST, GST-RPS10, and FLAG-PRMT5 were detected with GST and FLAG antisera separately.

PRMT5 methylates RPS10 both in vitro and in vivo. A, alignment of the conserved RG-rich motif from human (Homo sapiens), mouse (Mus musculus), Drosophila melanogaster, Caenorhabditis elegans, and Schizosaccharomyces pombe. Highly conserved residues are highlighted in black; weakly conserved residues are highlighted in gray. B, PRMT5 methylates the C-terminal of RPS10 in vitro. Methylation of GST, GST-RPS10, or the GAR motif deletion mutant (ΔGAR) of RPS10 by immunopurified FLAG-PRMT5 was performed as described under “Experimental Procedures” and visualized by SDS-PAGE separation followed by Coomassie Brilliant Blue staining (left) and autoradiography (right). C, Arg¹⁵⁸ and Arg¹⁶⁰ in RPS10 are the residues methylated by PRMT5 in vitro. In vitro methylation assays of GST-RPS10, GST-RPS10 ΔGAR, and arginine single or double mutants of RPS10 (GST-RPS10-R158K, -R160K, or -R158K/R160K) by immunopurified FLAG-PRMT5 were performed as in B. D, PRMT5 methylates RPS10 in vivo. HEK293 cells were transfected with FLAG-tagged RPS10 or RPS10-R158K/R160K together with PRMT5. 24 h later, cell lysates were immunoprecipitated with anti-FLAG antibody and probed with SYM11 to detect symmetric dimethylarginine in RPS10. The blot was re-probed with anti-FLAG antibody for RPS10 or RPS10-R158K/R160K. E, PRMT5 is required for the methylation of RPS10 in vivo. HEK293T cells were transfected with FLAG-RPS10 together with shRNA expressing vectors targeting either luciferase or PRMT5 in a ratio of 1:5. 48 h after transfection, cell lysates were analyzed by immunoblotting with anti-PRMT5 antiserum (left panels, GAPDH used as a loading control). Immunoprecipitates (IP) obtained using the FLAG antibody from cell lysates were analyzed with anti-FLAG antibody for RPS10 and SYM11 antibody for symmetric dimethylarginine in RPS10 (right panels).

Methylation of RPS10 by PRMT5 is required for normal cell proliferation. A and B, RPS10 is required for cell proliferation. pSIREN-DNR-DsRed.siLuciferase, pSIREN-DNR-DsRed.siRPS10 A, and pSIREN-DNR-DsRed.siRPS10 B (labeled as Control, siRPS10 A, and siRPS10 B, respectively) were co-transfected into Bel7402 cells. 24 h later, cells were split into 6-well plates and allowed to grow for 5 more days. A, transfected cells are shown on days 1 (top) and 5 (bottom) under a fluorescence microscope (×10). B, the number of transfected cells was counted daily under a fluorescence microscope. The experiment was repeated three times and values represent average numbers of transfected cells and are means from duplicated cultures ± S.E. C, RPS10 shRNAs can down-regulate the expression of endogenous RPS10. Bel7402 cells were transfected as in A. 36 h later, RPS10 expression was determined by Western blotting using RPS10 antiserum. The blot was re-probed with β-tubulin as a loading control. D and E, WT RPS10 restored cell proliferation better than the methylation mutant in the RPS10 knock-down cells. Myc-tagged WT and methylation mutant siRNA-resistant RPS10 (SR-WT and SR-R158K/R160K) were generated by changing three nucleotides, as underlined, without altering the amino acid sequences (D, lower panel). Bel7402 cells were co-transfected with siRPS10 together with the control, SR-WT, or SR-R158K/R160K. 36 h later, cells were harvested, and half of the cells were split into 6-well plates for rescue experiments (E). D, expression of endogenous and exogenous RPS10 was determined as in C. The number of transfected cells was counted every 2 days for 6 days. The experiment was repeated four times and values were expressed as the means ± S.E. (day 4, p < 0.05; day 6, p < 0.01) (E).

Methylation of RPS10 plays a role in the efficient assembly of RPS10 into ribosomes and protein synthesis. A, cytoplasmic polysomes were isolated by ultracentrifugation as described under “Experimental Procedures.” The polysome fraction was extracted from HEK293 cells transfected with FLAG-tagged WT RPS10 or R158K/R160K mutants together with pCMS.EGFP. Collected fractions were analyzed by Western blotting using antibodies specific for RPS10, GFP, and GAPDH. T, S, and P represent total cell lysates, supernatant, and isolated polysomes, respectively. B, quantitative analysis of the fraction of exogenous RPS10 associated with polysomes was calculated as RPS10-FLAG in polysomes/endogenous RPS10 in polysomes and normalized with RPS10-FLAG in total cell lysates/endogenous RPS10 in total cell lysates by ImageJ software. Values are three independent experiments, mean ± S.E. (p < 0.05). C, polysome profile. 48 h after transfection, total extracts from HEK293T cells transfected with RPS10 shRNA together with either SR-RPS10-Myc or methylation mutation RPS10 were subjected to 5–45% linear sucrose density gradient sedimentation by ultracentrifugation. The linear peak represents a continuous measurement of the absorbance at 254 nm. D, RPS10 methylation mutation leads to a decreased protein synthesis rate. Cells were transfected as in C. 48 h after transfection, the medium was exchanged to a Met-free medium supplemented with [³⁵S]Met. Cells were harvested 1 h after labeling and then subjected to SDS-PAGE analysis. The SDS-PAGE gel was stained with Coomassie Blue (left, lane 1, WT SR-RPS10/RPS10 shRNA; lane 2, SR-RPS10 methylation mutant/RPS10 shRNA), dried, and exposed to x-ray film for ³⁵S-labeled proteins (right, lanes 3 and 4 correspond to lanes 1 and 2). E, quantitative analysis of total protein synthesis in D calculated as ³⁵S radioactivity/total protein (Coomassie Blue staining) using ImageJ software. Values are three independent experiments mean ± S.E. (*, p < 0.01).

The RPS10 methylation mutant is less stable than WT RPS10. A, expression of RPS10 methylation mutant is much lower than that of WT RPS10. HEK293 cells were transfected with FLAG-tagged RPS10, or different amounts of RPS10-R158K/R160K, together with 1 μg of pCMS.EGFP. 36 h later, RPS10 expression was determined by Western blotting using anti-FLAG antibody. The blot was re-probed with anti-GFP antibody as a transfection efficiency control. B and C, RPS10 methylation mutant is more unstable and can be stabilized by the proteasome inhibitor. HEK293 cells were transfected with FLAG-tagged RPS10 or RPS10-R158K/R160K. 36 h later, cells were treated with either 100 μm cycloheximide (CHX) or 10 μm MG132 and cells were collected at different time points as indicated. RPS10 expression levels were determined by Western blotting using RPS10 antiserum. The blot was re-probed with β-tubulin as a loading control. D, HEK293 cells were transfected with either WT RPS10-FLAG or RPS10-R158K/R160K-FLAG together with FLAG-PRMT5. Pulse-chase experiments were done by labeling with [³⁵S]methionine for 1 h and a subsequent chase with medium containing non-radioactive methionine for the indicated times before cells were lysed. WT and mutant RPS10 and PRMT5 proteins were immunoprecipitated with anti-FLAG beads. ³⁵S-Labeled RPS10-FLAG was analyzed with a FLA-3000 phosphorimager, and FLAG-PRMT5 was used as a control. E, the RPS10 methylation mutant is prone to degradation by the proteasomal pathway. HEK293 cells were co-transfected with a control vector (lanes 1 and 4), RPS10-FLAG (lanes 2 and 3), or RPS10-R158K/R160K (lanes 5 and 6) together with Myc-tagged ubiquitin. 36 h after transfection, the cells were treated with MG132 (lanes 4–6) as indicated. FLAG-tagged RPS10 was immunoprecipitated using anti-FLAG beads, subjected to SDS-PAGE, and immunoblot (IB) analysis to detect the ubiquitylated RPS10 or RPS10-R158K/R160K with anti-Myc antiserum. The blot was reprobed with anti-FLAG antiserum to detect RPS10 and RPS10-R158R/K160K.

Methylation of RPS10 is required for its concentration in the GC region of nucleoli. A and B, difference in the subnucleolar localization of WT RPS10 and RPS10 methylation mutant. RPS10-GFP (A) and RPS10-R158K/R160K-GFP (B) were co-transfected with B23-DsRed into U2OS cells and fixed 24 h later. RPS10 (green) and B23 (red) were observed under a confocal microscope. High magnifications of the indicated areas (squares) are shown at the bottom. Bars, 5 μm. The graphs correspond to intensities in arbitrary units of green and red labeling for each pixel of the line drawn through the axis in A and B, respectively. C and D, RPS10-GFP (C), RPS10-R158K/R160K-GFP (D), and Fibrillarin-DsRed were transfected and analyzed as in A and B. E, different distributions of WT RPS10 and methylation mutant in nucleoli. GFP-tagged RPS10 and RPS10-R158K/R160K were co-transfected either with B23-DsRed or Fibrillarin-DsRed into U2OS cells, fixed 24 h later, and observed as in A and C. RPS10 was classified as either GC region localization when co-localized with B23, or dense fibrillar component (DCF)/fibrillar component (FC) when co-localized with Fibrillarin. 100 cells were analyzed for each category. F, more RPS10-R158K/R160K tends to remain in the nucleus. HEK293 cells were transfected with FLAG-tagged WT or methylation mutant RPS10. 24 h after transfection, cytoplasmic and nuclear components were fractionated. WT and mutant forms of RPS10 in total lysate (T), cytosol (C), and nuclear (N) were analyzed by Western blot with anti-FLAG antibodies. Lamin B and GAPDH were used as the marker for nuclear and cytoplasmic fractions, respectively.

Decreased binding of RPS10 methylation mutant to B23. A and B, RPS10 interacts with B23 in vivo. HEK293 cells were transfected with FLAG or Myc-tagged RPS10 and B23 as indicated. 24 h later, cell lysates were immunoprecipitated with anti-FLAG beads and the immunocomplexes were analyzed for either Myc-tagged RPS10 or B23. C, endogenous B23 and RPS10 interact with each other. Anti-RPS10 mouse serum was used to immunoprecipitate (IP) B23 from HEK293 cell extracts with mouse IgG as a control. The cell lysates (lower panels) and immunocomplexes (upper panels) were probed separately for endogenous B23 and RPS10. D, RPS10 interacts with B23 in vitro. GST, GST-B23 fusion protein, and His-RPS10 for binding assays were expressed, purified from E. coli, and incubated with each other. GST, GST-B23, and His-RPS10 were detected with GST and His antisera separately. E, decreased binding of RPS10 methylation mutant to B23. HEK293 cells were transfected with FLAG-tagged B23 together with either Myc-tagged RPS10 or RPS10-R158R/K160K. The amount of RPS10-R158K/R160K used for transfection was doubled so that its expression level is similar to that of WT RPS10. 24 h later, cell lysates were analyzed for the expression of tagged proteins (lower panel). The remaining portions of the lysates were immunoprecipitated with anti-FLAG antibody, and the immunocomplexes were analyzed for RPS10 or RPS10-R158K/R160K with anti-Myc antibody. F, decreased binding of RPS10 methylation mutant to endogenous B23. HEK293 cells with either Myc-tagged RPS10 or RPS10-R158K/R160K. The amount of RPS10-R158K/R160K used was tripled. Cell lysates were immunoprecipitated with anti-B23 antibody, and the immunocomplexes were analyzed for RPS10 or RPS10-R158K/R160K with anti-Myc antibody and B23 with anti-B23 antibody.