Fig. 1

PRMT5 methylates Aurora-A in the Ajuba-organized protein complex. A Protein complex formation of Ajuba, PRMT5, Aurora-A and HURP. HeLa cells were applied to immune-coprecipitation assay adopting antibodies against PRMT5. Sup and ppt indicate supernatant and pellet. Western blots (WB) were followed to detect PRMT5, HURP, Aurora-A and Ajuba. B Manipulation of Ajuba expression causally determined formation of the Ajuba/PRMT5/Aurora-A/HURP complex. HeLa cells harboring HA-HURP were transfected with/or without Myc-Ajuba, or infected with viruses containing Luciferase shRNA (shLuc), Ajuba shRNA (shAjuba), and then subjected to immune-coprecipitation employing antibodies against PRMT5. Western blots were then followed to examine the formation of the protein complex. C PRMT5 methylated Aurora-A in vitro. To conduct an in vitro methylation reaction, immunoprecipitated Aurora-A or recombinant Histone 2A (H2A) was incubated with immunoprecipitated EGFP-PRMT5 in the presence of methylation reaction buffer containing H³ labelled methyl donor S-adenosylmethionine. The resulting samples were applied to autoradiography or Western blots to examine the methylation effect or immunoprecipitation results. D PRMT5 could not methylate Aurora-A R304K mutant. Immunoprecipitated FLAG-Aurora-A WT or R304K mutant was incubated with immunoprecipitated EGFP-PRMT5 for in vitro methylation reactions. Autoradiography or Western blot was performed to examine the methylation effect or immunoprecipitation results. E Knockdown or overexpression of PRMT5 reduced or elevated the level of Aurora-A m304. HeLa cells harboring shLuc, PRMT5 shRNA (shPRMT5) (left), or EGFP, EGFP-PRMT5 (right) were analyzed for the level of Aurora-A m304 and Aurora-A by Western blots. F The level of active Aurora-A was diminished on Aurora-A R304K. 293T cells transfected with pCMV2 empty vector, FLAG-Aurora-A WT or R304K were analyzed for the level of active Aurora-A, i.e. Aurora-A p288, on the immunoprecipitated FLAG-Aurora-A. G Knockdown of PRMT5 diminished the level of active Aurora-A. HeLa cells harboring shLuc or shPRMT5 were analyzed for the level of Aurora-A p288 and Aurora-A by Western blots. H Overexpression of PRMT5 elevated the level of active Aurora-A. 293 cells transfected with EGFP or EGFP-PRMT5 were analyzed for the expression of active Aurora-A and Aurora-A by Western blots. I Aurora-A R304K could not phosphorylate HURP. 293T cells were infected with shLuc or shAurora-A first, and the shAurora-A cells were further transfected with pCMV2 empty vector, FLAG-Aurora-A WT, R304K. These cells were then surveyed for the level of HURP p725, HURP and Aurora-A. Three independent experiments were performed for all the results listed above