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. 2011 Feb 4;286(5):3379-86.
doi: 10.1074/jbc.M110.188425. Epub 2010 Nov 29.

RBX1 (RING box protein 1) E3 ubiquitin ligase is required for genomic integrity by modulating DNA replication licensing proteins

Lijun Jia  1 , Jeremy S Bickel, Jiaxue Wu, Meredith A Morgan, Hua Li, Jie Yang, Xiaochun Yu, Raymond C Chan, Yi Sun
Affiliations

RBX1 (RING box protein 1) E3 ubiquitin ligase is required for genomic integrity by modulating DNA replication licensing proteins

Lijun Jia et al. J Biol Chem. .

Abstract

RBX1 (RING box protein 1), also known as ROC1 (Regulator of Cullin 1), is an essential component of SCF (Skp1/Cullins/F-box) E3 ubiquitin ligases, which target diverse proteins for proteasome-mediated degradation. Our recent study showed that RBX1 silencing triggered a DNA damage response (DDR) leading to G(2)-M arrest, senescence, and apoptosis, with the mechanism remaining elusive. Here, we show that, in human cancer cells, RBX1 silencing causes the accumulation of DNA replication licensing proteins CDT1 and ORC1, leading to DNA double-strand breaks, DDR, G(2) arrest, and, eventually, aneuploidy VSports手机版. Whereas CHK1 activation by RBX1 silencing is responsible for the G(2) arrest, enhanced DNA damage renders cancer cells more sensitive to radiation. In Caenorhabditis elegans, RBX-1 silencing causes CDT-1 accumulation, triggering DDR in intestinal cells, which is largely abrogated by simultaneous CDT-1 silencing. RBX-1 silencing also induces lethality during development of embryos and in adulthood. Thus, RBX1 E3 ligase is essential for the maintenance of mammalian genome integrity and the proper development and viability in C. elegans. .

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Figures (V体育官网)

FIGURE 1.
FIGURE 1.
RBX1 silencing induces DDR as a result of DNA DSB. H1299 cells (A) and U87 cells (B) were transfected with siRBX1 or siCONT. Cells were split 24 h later and harvested at the indicated time points for Western blot analysis (A and B) as well as for immunofluorescence staining in U87 cells using the indicated antibodies (C, at 72–120 h), with the formation of DNA damage foci clearly visualized upon RBX1 silencing (C, right panels), or comet assay in both H1299 and U87 cells (D, at 96 h).
FIGURE 2.
FIGURE 2.
RBX1 silencing triggers DNA re-replication, activates the DNA damage G2 checkpoint, and sensitizes cells to irradiation. A, RBX1 silencing induces G2 arrest and aneuploidy. U87 cells were transfected with siRBX1 or siCONT and split 24 h later. At the indicated time points post-splitting, cells were subjected to phosphohistone 3/propidium iodide double staining and FACS analysis. The percentage of cell populations at G2 and M and those with aneuploidy (4N+) is indicated. B and C, RBX1 silencing-induced G2 arrest is abrogated by CHK1 inhibition. U87 cells transfected with the indicated siRNAs for 24 h were split. At 72 h post-splitting, one portion of cells was subjected to propidium iodide staining and FACS analysis (B, upper panel), whereas the other portion was subjected to Western blot analysis (lower panel). U87 cells transfected with indicated siCONT or siRBX1 for 24 h were split. At 72 h post-splitting, cells were treated with PF-00477736 (PF) at the indicated concentrations for 24 h and subjected to propidium iodide staining and FACS analysis (C). D, RBX1 silencing sensitizes cancer cells to irradiation. U87 and H1299 cells after siRNA silencing for 48 h were subjected to standard clonogenic assay after exposure to different doses of radiation as described (24). Shown is x ± S.E. from four independent experiments: SiRBX1 versus siCONT, p < 0.05 for both H1299 and U87 cells with Student's t test. Gy, gray.
FIGURE 3.
FIGURE 3.
RBX1 silencing induces the accumulation of CDT1 and ORC1 but not oncoproteins. U87 cells transfected with siRBX1 or siCONT for 24 h were split. At 120 h post-splitting, cells were subjected to Western blot analysis for the indicated proteins with β-actin as the loading control (A). U87 (B) or H1299 (C) cells were transfected with siRBX1 or siCONT for 24 h, followed by cell splitting. At the indicated time points post-splitting, cells were harvested and subjected to Western blot analysis with β-actin as the loading control.
FIGURE 4.
FIGURE 4.
rbx-1 silencing induces DDR and lethal phenotypes in C. elegans. Wild-type C. elegans worms were fed bacteria expressing rbx-1-targeting double-stranded RNA or the blank expression vector as described under “Experimental Procedures.” A, total RNA extracted from 100 worms fed for 5 days was either mock (RT−) or reverse-transcribed (RT+) and then analyzed by PCR for the presence of the rbx-1 cDNA. Actin (act-1) served as a loading control. Isolated gonad (B and C) and intestine (D and E) from worms on the 3rd day of adulthood were examined by microscopy. Shown are micrographs of germ cells in intact gonads visualized by DAPI DNA fluorescence (B and C) and intestinal nuclei with RAD-51 immunostaining and DAPI DNA fluorescence (D and E). Scale bars = 5 μm. Bar graphs represent the percentages of offspring embryonic lethality (F) and adult lethality (G) in vector and rbx-1 RNAi worms. Offspring (eggs) were collected from RNAi-treated worms, and embryonic lethality was assessed as unhatched eggs at 48 h after egg collection (F). Adult worms were examined for lethality based on the absence of movement, pharyngeal pumping, and responsiveness to touch at Days 1 and 2 of adulthood (G). The vector and rbx-1 worms were compared by two-tailed Fisher's exact test.
FIGURE 5.
FIGURE 5.
RNAi depletion of CDT-1 suppresses the rbx-1 silencing-induced DDR but not death in C. elegans. A, RT-PCR detection of rbx-1 and actin (act-1) mRNAs in 100 worms fed for 5 days on vector control (lane 1), rbx-1 (lane 2), and cdt-1;rbx-1 (lane 3) RNAi bacterial vectors. B, Western blot of lysates from 100 worms prepared by boiling in 30 μl of SDS-PAGE loading buffer with 3.75 m urea, freeze-thawed, and reboiled in the presence of β-mercaptoethanol prior to gel electrophoresis. Each set of lysates was loaded into three lanes at 2-fold serial dilutions. The blot was probed for CDT-1 (upper panel) and RBX-1 (middle panel) proteins and α-tubulin (lower panel) as a loading control. C, micrographs of intestinal nuclei immunostained for RAD-51 and DAPI DNA fluorescence. Scale bars = 5 μm. D, the graph represents the average fraction of intestinal nuclei with RAD51 staining. Error bars represent mean ± S.D. from a total of four independent experiments (D and F). p < 0.001 for statistical comparisons for vector versus rbx-1 (*) and rbx-1 versus cdt-1;rbx-1 (**) using two-tailed Fisher's exact test. E, the graph represents the average length of the syncytial gonad from the distal tip to the end of pachytene in meiotic prophase. p < 0.001 for statistical comparisons for vector versus rbx-1 (*) and rbx-1 versus cdt-1;rbx-1 (**) using two-tailed Student's t test. Error bars represent mean ± S.E. F, the graph represents the average lethality in the RNAi-treated worms at the indicated age. Twenty worms were examined for each set of RNAi vector(s) per experiment for a total of four independent experiments.
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