As shown in Fig ?Fig2D,2D, nucleolar fibrillarin dispersed when cells were treated with siRNAs targeting GRWD1, suggesting that its depletion impairs nucleolar integrity and thereby induces nucleolar stress response. RPL11\mediated suppression of MDM2 ubiquitin ligase activity toward p53. These effects are mediated by the N\terminal region of GRWD1, including the acidic domain. Finally, we show that GRWD1 overexpression in combination with HPV16 E7 and activated KRAS confers anchorage\independent growth and tumorigenic capacity on normal human fibroblasts. Consistent with this, GRWD1 overexpression is associated with poor prognosis in cancer patients. Taken together, our Rubusoside results suggest that GRWD1 is a novel negative regulator of p53 and a potential oncogene. somewhat induces p53 without actinomycin D treatment (e.g., the data for time 0 in Fig ?Fig1A).1A). Also in U2OS cells, GRWD1 depletion by siRNAs induced up\regulation of p53 and accumulation of sub\G1 cells likely representing apoptotic cells (Fig ?(Fig2A2A and B). It has been suggested that GRWD1 may be required for ribosome biogenesis 18, 19, 20. Therefore, we thought it possible that GRWD1 depletion induces nucleolar stress. To address this issue, we first revisited cellular localization of GRWD1. Although GRWD1 is present in nuclei and binds chromatin 23, it tends to accumulate in nucleoli 23, 24. We examined the localization of GRWD1 by immunostaining after non\ionic detergent extraction of cells to remove nucleoplasmic proteins. This assay revealed that GRWD1 is enriched in nucleoli and co\localizes with fibrillarin, a well\known nucleolar marker (Fig ?(Fig2C).2C). Furthermore, nucleolar GRWD1, like fibrillarin, dispersed into nuclei upon nucleolar stress induced by actinomycin D (Fig ?(Fig2C).2C). We then investigated whether nucleolar integrity is affected by GRWD1 depletion. As shown in Fig ?Fig2D,2D, nucleolar fibrillarin dispersed when cells were treated with siRNAs targeting GRWD1, suggesting that its depletion impairs nucleolar integrity and thereby induces nucleolar stress response. Therefore, only with the data obtained with endogenous GRWD1\depleted cells, it would be difficult to clarify whether endogenous GRWD1 actively suppresses p53 pathway in addition to maintaining nucleolar integrity, although the hyperinduction of p53 pathway by GRWD1 depletion is in line with the idea. Open in a separate window Figure 2 GRWD1 depletion by itself impairs nucleolar integrity and induces nucleolar stress response U2OS cells were transfected with control (mixture of control DS scrambledNeg, siLuci, and siGFP) or GRWD1\targeting (mixture of siGRWD1\3 and 4 or siGRWD1\1) siRNAs for 24 h. Whole\cell extracts were then analyzed by immunoblotting with the indicated antibodies. U2OS cells treated as above were stained with propidium iodide and subjected to flow cytometry. HCT116 cells treated with actinomycin D (5 nM) or vehicle (DMSO) for 12 h were first extracted with Triton X\100 to remove nucleoplasmic proteins, double\immunostained with anti\GRWD1 (green) and anti\fibrillarin (red) antibodies as a marker for nucleoli, and counterstained with DAPI. Scale bar, 20 m. HCT116 cells were transfected with control (mixture of control siLuci and siGFP) or GRWD1\targeting (mixture of siGRWD1\3 and 4 or LAT antibody siGRWD1\1) siRNAs for 24 h and then immunostained as above. Scale bar, 20 m. ubiquitination assay to detect MDM2 autoubiquitination in H1299 cells. Lysates were prepared from H1299 cells transfected with the indicated expression vectors (His\Xpress\MDM2, 2 g; HA\Ub, 0.5 g; RPL11\FLAG, 1 g; HA\GRWD1\FLAG, 1.5 g) for Rubusoside 48 h Rubusoside and treated with proteasome inhibitors for 6 h before harvest, and then immunoprecipitated with anti\MDM2 antibody. Immunoprecipitates (IPs) and inputs were immunoblotted with the indicated antibodies. SE, short exposure. ubiquitination of p53 by immunopurified MDM2. His\Xpress\MDM2 was immunopurified from transfected 293T cells with anti\Omni probe antibody. Recombinant p53 was incubated with E1, E2 (UbcH5a), His\ubiquitin, ATP, GST\RPL11, GRWD1\His, and immunopurified.