Fig 2: USP48 interacts, deubiquitinates, and extends Aurora B protein half-life. (A) The endogenous interactions between the USP48 and Aurora B proteins were examined by immunoprecipitation (IP) experiments in HeLa cells. Cell lysates from HeLa cells were immunoprecipitated and immunoblotted with specific USP48 or Aurora B antibodies as indicated. (B) Exogenous IP experiments to demonstrate the interaction between Myc-Aurora B and Flag-USP48 were performed in HEK293 cells. Samples were immunoprecipitated and immunoblotted using either anti-Flag or anti-Myc antibodies as indicated. GAPDH was used as a loading control. (C) Myc-Aurora B and HA-ubiquitin were co-transfected along with either Flag-USP48 or Flag-USP48CS in HEK293 cells. The ubiquitination level of Aurora B protein was confirmed by performing IP with an anti-Myc antibody and immunoblotting with the anti-HA (ubiquitin) and anti-Myc antibody as indicated. (D) The cycloheximide assay was performed to demonstrate the half-life of endogenous Aurora B protein in cells transfected with mock control, sgRNA1 targeting USP48, and upon the reconstitution with Flag-USP48 in sgRNA1 targeted USP48 HeLa cells. Experiments were performed in triplicates and band intensities were estimated using ImageJ software with reference to the GAPDH control band and graphically represented. Two-way ANOVA followed by Tukey’s post hoc test was used and the p values are represented.

Fig 3: USP48 regulated Aurora B protein stability. The efficiencies of sgRNA1 and sgRNA2, targeting the USP48 gene, in regulating the (A) endogenous or (B) exogenous Aurora B protein stabilization was determined by transfecting in HeLa and HEK293 cells, respectively, along with Cas9. (C) HeLa cells were transfected with an increasing amount of Flag-USP48 (0,1,2, and 3 µg), and the endogenous expression of Aurora B protein was analyzed by Western blot. (D) HEK293 cells were transfected with an increasing amount of Flag-USP48 (0,1,2, and 3 µg), along with a constant amount of Myc-Aurora B (0.5 µg), and the expression of Myc-Aurora B protein was analyzed by Western blot. (E) HeLa cells were transfected with increasing amounts of catalytically inactive Flag-USP48CS (0,1,2, and 3 µg), and the endogenous expression of Aurora B protein was analyzed by Western blot. (F) HEK293 cells were transfected with increasing amounts of Flag-USP48CS (0,1,2, and 3 µg), along with a constant amount of Myc-Aurora B (0.5 µg). The expression of Myc-Aurora B protein was analyzed by Western blot. Reconstitution experiments were performed to validate the specificity of USP48 for the stabilization of (G) endogenous or (H) ectopically expressed Aurora B in HeLa and HEK293 cells, respectively. All the experiments were performed in triplicates and band intensities were estimated using ImageJ software with reference to the GAPDH control band and graphically represented. One-way ANOVA followed by Tukey’s post hoc test was used and the p values are represented on the figures (ns = non-significant).

Fig 4: USP48 regulates Aurora B function during mitosis. (A) The spindle assembly factors in mock (left panel) and USP48KO (right panel) HeLa cells were stained with TPX2 antibodies. DAPI was used to stain the nuclei. Scale bar = 10 µM. Mitotic defects were observed upon depletion of USP48. The graph represents the percentage of cells exhibiting (B) normal mitotic phases in mock (74 ± 2), and USP48KO (48 ± 7) and (C) abnormal mitotic phases in mock (26 ± 2) and USP48KO (51 ± 7.55) (n = 3, p values are represented on each graph). (D) USP48KO HeLa cells (lane 2) were transfected with either Flag-USP48 (lane 3) or Flag-USP48CS (lane 4) and compared with wild-type HeLa cells (lane 1). Transfected cells were synchronized with 100 ng/mL Nocodazole for 18 h prior and analyzed by Western blot against the indicated antibodies.

Fig 5: CRISPR-based genome-scale screening of USP sub-family proteins exhibiting a reduction in Aurora B protein levels. (A) Screening for DUBs regulating Myc-Aurora B was performed using a CRISPR-Cas9-based DUBKO sgRNA kit. HEK293 cells were transfected with Myc-Aurora B and the indicated sgRNAs along with Cas9. Equal concentrations of proteins from DUBKO HEK293 cell lysates were subjected to Western blot analyses. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control band for each individual sgRNA (Myc-Aurora B/GAPDH). The loss of USPs leading to the downregulation of the Aurora B protein level is marked in red. (B) The putative DUBs USP7, USP13, USP21, USP35, USP48, and USP50, which potentially regulate Aurora B protein levels, were transfected in HeLa cells along with Cas9. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control band for each individual sgRNA (Aurora B/GAPDH). The protein band intensity of HeLa cells transfected with sgRNA targeting USP48 showed the highest reduction in the endogenous Aurora B is represented in red. (C) Schematic of RNA-guided engineered nuclease targeting the sequences in exon 2 and exon 3 of the human USP48 gene using sgRNA1 and sgRNA2, respectively. PAM sequences are represented in blue, while the sgRNA target sequences are represented in red. (D) T7E1 assays were performed in HEK293 cells to determine the cleavage efficiency of sgRNA1 (T1) and sgRNA2 (T2). The cleaved band intensity (indicated by arrow) obtained from the T7E1 assay was estimated using ImageJ software and represented as indel percentage (indel %). Scrambled sgRNA-transfected cells were used as control cells (C). A marker is shown for size reference.