Skip to main content
. Author manuscript; available in PMC: 2019 Apr 1.
Published in final edited form as: Nat Chem Biol. 2018 Aug 27;14(10):943–954. doi: 10.1038/s41589-018-0113-5

Figure 2. Nuclear RNR-α suppresses DNA replication by eliciting loss of function of ZRANB3.

Figure 2.

(a) Left: Quantification of: BrdU-positive fraction in MEFs. RNR-α+/+ (1), n=8; RNR-α+/+ (2), n=5; RNR-α+/+ (3), n=9; RNR-αD57N/D57N(1), n=8; RNR-αD57N/D57N(2), n=5; RNR-αD57N/D57N(3), n=9; where n indicates data taken from an individual frame and the bar shows mean from a specific plate. Points per bar denote fraction of cells (>20) proliferating. (Note: average of 3 wt sets proliferating is less than average 3 D57N sets proliferating, p<0.05). Two-tailed two sample t-test was applied. No adjustments were made. Precise p-values between each combination are: between RNR-α+/+ (1) and RNR-αD57N/D57N(1): 7e-5; between RNR-α+/+ (1) and RNR-αD57N/D57N(2): 1e-5; between RNR-α+/+ (1) and RNR-αD57N/D57N(3): 0.02; between RNR-α+/+ (2) and RNR-αD57N/D57N(1): 0.0002; between RNR-α+/+ (2) and RNR-αD57N/D57N(2): 0.0001; between RNR-α+/+ (2) and RNR-αD57N/D57N(3): 0.02; between RNR-α+/+ (3) and RNR-αD57N/D57N(1): 8e-6; between RNR-α+/+ (3) and RNR-αD57N/D57N(2): 9e-6; between RNR-α+/+ (3) and RNR-αD57N/D57N(3): 0.001. Right: nucleus:cytosol fraction of RNR-α in primary MEFs derived from different embryos Each bar derived from a separate embryo with points per bar denoting separate cells: [RNR-α+/+ (1), n=205; RNR-α+/+ (2), n=46; RNR-α+/+ (3), n=137; RNR-αD57N/D57N(1), n=100; RNR-αD57N/D57N(2), n=73; RNR-αD57N/D57N(3), n=276; where n indicates individual cells]. Average of 3 wt sets is greater than average 3 D57N sets, p<0.001. Two-tailed two sample t-test was applied. No adjustments were made. Precise p-values between each combination are: between RNR-α+/+ (1) and RNR-αD57N/D57N(1): 1e-18; between RNR-α+/+ (1) and RNR-αD57N/D57N(2): 6e-15; between RNR-α+/+ (1) and RNR-αD57N/D57N(3):5e-43; between RNR-α+/+ (2) and RNR-αD57N/D57N(1): 5e-31; between RNR-α+/+ (2) and RNR-αD57N/D57N(2): 2e-22; between RNR-α+/+ (2) and RNR-αD57N/D57N(3): 1e-56; between RNR-α+/+ (3) and RNR-αD57N/D57N(1): 1e-25; between RNR-α+/+ (3) and RNR-αD57N/D57N(2): 6e-20; between RNR-α+/+ (3) and RNR-αD57N/D57N(3): 4e-56. For box plots, center lines indicate medians, box limits are the first and third quartiles and whisker ends represent 10-90%. Data not included between the whiskers are plotted as an outlier with a dot. See also Supplementary Fig. 9. (b) Schematics for dual-pulse labeling, single-DNA-fiber-staining, and single-DNA-fiber-combing. For single-DNA-fiber combing, 3× wash cycles were performed after IdU treatment. See Methods. (c) Overexpression of wt-ZRANB3 partially rescues nuclear-RNR-α-induced DNA-synthesis suppression. The normalized DNA-synthesis efficiency (reported by “normalized BrdU-incorporation efficiency”) was determined by sequential dual-pulse labeling using EdU/BrdU in tetracycline-inducible isogenic cells expressing wt-RNR-α-NLS compared to the nuclear-translocation-defective hypomorph RNR-α(D57N). These cells were also transfected with either empty vector or a vector encoding wt-ZRANB3. Data for cells transfected with either empty- or ZRANB3-plasmid are respectively divided by the mean of the RNR-α(D57N) cells transfected with either empty- or ZRANB3-plasmid. Data show mean +/− s.e.m. [RNR-α(D57N), n=558; RNR-α-NLS, n=376; RNR-α(D57N), ZRANB3, n=368; RNR-α-NLS, ZRANB3, n=397]. Also see Fig. 2f and Supplementary Fig. 15b (presented using a ‘non-normalized’ Y-axis). (d) Single-DNA-fiber analysis shows ZRANB3-knockdown fails to reduce DNA-synthesis further in tetracycline-induced RNR-α-NLS-overexpressing-isogenic Flp-In T-REx™ 293 cells. Data show mean+/−s.e.m. (Tet−, siCont-1 n=291; Tet−, siZRANB3–1 n=312; Tet+, siCont-1 n=450; Tet+, siZRANB3-1 n=289) The p-value between siCont-1, Tet+ and siZRANB3-1, Tet+ is 0.49. See also Supplementary Fig. 8, 10–17. (e) HEK293T cells were transfected with indicated siRNA/plasmid combination and were analyzed after 2 days by dual pulse imaging. Data show mean +/− s.e.m. (Left panel: siCont-1, n=430; siZRANB3-1, n=399; siZRANB3-1+wt-ZRANB3, n=215; siZRANB3+2Xwt-ZRANB3, n=443; right panel: siCont-1 Empty vector, n=327; siZRANB3-1 Empty vector, n=634; siZRANB3 wt-ZRANB3 n=441; siZRANB3 Δ-PIP n=408; siZRANB3 Δ-APIM n=363; siZRANB3 Q519A n=313; siZRANB3 H1021A n=312; siCont-1 wt-ZRANB3 n=334). In the right panel, the p-values for n’s are: between empty vector, siZRANB3 and ΔPIP, siZRANB3: 0.10; between empty vector, siZRANB3 and ΔAPIM, siZRANB3: 0.16; between empty vector, siZRANB3 and Q519A, siZRANB3: 0.86. (f) Single-DNA-fiber analysis from cells with/without Tet-induced-RNR-α-expression shows only the overexpression of wt-RNR-α, but not the nuclear-translocation-defective hypomorph RNR-α(D57N), reduces the DNA-synthesis rate. [Data show mean+/−s.e.m. RNR-α(D57N), Tet+ n=151; RNR-α, Tet+ n=151, RNR-α(D57N), Tet− n=208; RNR-α, Tet− n=59]. The p-value between D57N, Tet- and wt, Tet- is 0.20. For full-view blots relevant to Fig. 2 and supplementary figures referred to above, see Supplementary Fig. 33–40, and 42. For Fig. 2(c)–(f), two-tailed t-test was applied. n indicates number of fibers or cells measured.