The TDRD3-USP9X complex and MIB1 regulate TOP3B homeostasis and prevent deleterious TOP3B cleavage complexes

Abstract

TOP3B is stabilized by TDRD3. Hypothesizing that TDRD3 recruits a deubiquitinase, we find that TOP3B interacts with USP9X via TDRD3. Inactivation of USP9X destabilizes TOP3B, and depletion of both TDRD3 and USP9X does not promote further TOP3B ubiquitylation. Additionally, we observe that MIB1 mediates the ubiquitylation and proteasomal degradation of TOP3B by directly interacting with TOP3B independently of TDRD3. Combined depletion of USP9X, TDRD3 and MIB1 causes no additional increase in TOP3B levels compared to MIB1 knockdown alone indicating that the TDRD3-USP9X complex works downstream of MIB1. To comprehend why cells degrade TOP3B in the absence of TDRD3, we measured TOP3Bccs. Lack of TDRD3 increases TOP3Bccs in DNA and RNA, and induced R-loops, γH2AX and growth defect. Biochemical experiments confirm that TDRD3 increases the turnover of TOP3B. Our work provides molecular insights into the mechanisms by which TDRD3 protect cells from deleterious TOP3Bccs which are otherwise removed by TRIM41.

Fig. 1. TDRD3 maintains TOP3B protein levels in HEK293 and HCT116 cells.

a, b TDRD3 stabilizes cellular TOP3B protein in HEK293 cells. Cell lysates from TDRD3 siRNA transfected cells were immunoblotted with TOP3B and TDRD3 antibodies (GAPDH as loading control). Panel a is a representative blot and panel b the quantitation of TOP3B and TDRD3 protein levels from three independent experiments. Data are plotted as means ± standard deviations (SD). c TDRD3 depletion by siRNA destabilizes TOP3B protein in HCT116 cells. d TOP3B expression levels in HCT116 wild-type and TDRD3KO cells. e, f Depletion of TDRD3 decreases the half-life of TOP3B. HEK293 cells were transfected with TDRD3 siRNA and treated with CHX (10 μg/mL) for the indicated time. Panel e is a representative western blot and panel f the quantitation of three independent experiments. Data are plotted as means ± standard deviations (SD). g, h TDRD3 increases half-life of TOP3B protein in HCT116 cells. HCT116 wild-type and TDRD3KO cells were treated with CHX (10 μg/mL) for the indicated time. Panel g is a representative western blot and panel h displays quantitation of TOP3B protein levels from three independent experiments. Data are plotted as means ± standard deviations (SD). i, j Ectopic expression of TDRD3 stabilizes TOP3B in HEK293 cells. Cells were transfected with TDRD3-FLAG constructs. Panel i is a representative western blot and panel j the quantitation of three independent experiments. Data are plotted as means ± standard deviations (SD). Two-tailed paired t-test. **P value = 0.0003 (TOP3B in no transfected vs TDRD3 transfected), **P value = 0.0002 (TDRD3 in no transfected vs TDRD3 transfected). k, l Similar experiment in TDRD3 knockout HCT116 cells. Ectopic expression of TDRD3 stabilizes TOP3B in TDRD3KO HCT116 cells. TDRD3KO HCT116 cells were transfected with TDRD3-FLAG constructs. Panel k is a representative western blot and panel l displays quantitation of TOP3B protein level from three independent experiments. Data are plotted as means ± standard deviations (SD). Two tailed unpaired t-test. **P value = <0.0001. Source data are provided as a Source Data file.

Fig. 2. Enhanced ubiquitylation of TOP3B in the absence of TDRD3 and recruitment of USP9X by TDRD3.

a Enhanced TOP3B ubiquitylation in the absence of TDRD3 in HEK293 cells. Cells were transfected with HA-Ubiquitin construct and TDRD3 siRNA (as indicated) for 48 h and subjected to TOP3B pulldown. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TOP3B and TDRD3 antibodies. The figure is representative of three independent experiments. b Enhanced ubiquitylation of TOP3B in TDRD3KO HCT116 cells. HCT116 wild-type and TDRD3KO cells were subjected to TOP3B pulldown. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TOP3B and TDRD3 antibodies. The figure is representative of three independent experiments. c Ectopic expression of TDRD3 decreases TOP3B ubiquitylation in TDRD3KO HCT116 cells. TDRD3KO HCT116 cells were transfected with TDRD3-FLAG constructs for 48 h and subjected to TOP3B pull down. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TOP3B and TDRD3 antibodies. The figure is representative of three independent experiments. d TOP3B pulldown-LC-MS/MS showing that endogenous TOP3B interacts with USP9X and TDRD3 in HEK293 cells. Shown are the number of identified peptide spectral matches (PSM). e HA-tag pulldown-LC-MS/MS showing TOP3B interaction with USP9X in HEK293 cells. After transfection of HA-TOP3B, HEK293 cells were subjected to HA-tag pulldown followed by LC-MS. f, g USP9X pulldown-Western blot experiments showing that depletion of TDRD3 suppresses TOP3B-USP9X interaction in HEK293 cells. Panel f is a representative Western blot and panel g is the quantitation of three independent experiments. Data are plotted as means ± standard deviations (SD). Two tailed unpaired t-test. *P value = 0.002. h USP9X pulldown-Western blot experiments showing the absence of TOP3B-USP9X interaction in TDRD3KO HCT116 cells. Source data are provided as a Source Data file.

Fig. 3. USP9X deubiquitylates and protects TOP3B from proteasomal degradation.

a Loss of USP9X destabilizes both TOP3B and TDRD3 proteins in HEK293 cells. b, c Depletion of USP9X decreases the half-lives of both TDRD3 and TOP3B in HEK293 cells. Cells were transfected with USP9X siRNA and treated with CHX (10 μg/ mL) for the indicated time. Panel b is a representative Western blot and panel c the quantitation of TOP3B and TDRD3 protein levels from three independent experiments. Data are plotted as means ± standard deviations (SD). d Inhibition of USP9X with the DUB inhibitor Degrasyn (WP1130) decreases both TOP3B and TDRD3 protein levels in HEK293 cells. Cells were treated with Degrasyn (WP1130; 2 μM, 24 h), pan-DUB inhibitor PR619 (5 μM, 24 h) or transiently transfected with TDRD3 siRNA. e USP9X can deubiquitylate TOP3B and TDRD3-USP9X axis is important for the deubiquitylation process. HEK293 cells were transfected with HA-Ubiquitin construct and siTDRD3, siUSP9X or both (as indicated) for 48 h and subjected to TOP3B pulldown. Pulled down and input samples were resolved on SDS-PAGE and probed with ubiquitin, TOP3B, TDRD3, and USP9X antibodies (GAPDH as loading control for input samples). The figure is representative of three independent experiments. f Treatment with the proteasome inhibitor MG132 rescues the TOP3B protein levels in USP9X-depleted cells. HEK293 cells were transfected with USP9X siRNA for 48 h. Before harvest, cells were treated with MG132 (1 µM, 24 h) as indicated and lysates were subjected to western blotting with USP9X, TOP3B, and TDRD3 antibodies (GAPDH loading control). g USP9X downregulation does not affect TOP3B protein level in TDRD3KO HCT116 cells. After transfecting the cells with USP9X siRNA for 48 h, lysates were subjected to Western blotting with USP9X and TOP3B antibodies (GAPDH as loading control). h Ectopic expression of USP9X increases both TOP3B and TDRD3 protein levels. After transfection of HEK293 cells with USP9X-FLAG construct for 48 h, cell lysates were subjected to western blotting with TOP3B, TDRD3, and USP9X antibodies (GAPDH as loading control). Source data are provided as a Source Data file.

Fig. 4. MIB1 ubiquitylates and drives the proteasomal degradation of TOP3B independently of TDRD3 and TDRD3-USP9X complex works downstream of MIB1 to limit TOP3B degradation.

a TOP3B pulldown-Western blot experiments showing TOP3B-MIB1 interaction in HEK293 cells. b MIB1 pulldown-Western blot experiments confirming TOP3B-MIB1 interaction in HEK293 cells. c TDRD3 pulldown-Western blot experiments showing TDRD3-MIB1 interaction in HEK293 cells. d TOP3B pulldown-Western blot experiments showing that TOP3B interacts with MIB1 both in wild type and siTDRD3-transfected HEK293 cells. e MIB1 depletion increases endogenous TOP3B protein level without affecting TDRD3 levels. f MIB1 ubiquitylates TOP3B independently of TDRD3. HEK293 cells were transfected with HA-Ubiquitin construct and siTDRD3, siMIB1 or both. Before harvest, cells were treated with MG132 (1 µM, 24 h) and subjected to TOP3B pulldown. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TOP3B, TDRD3, and MIB1 antibodies (GAPDH as loading control). The figure is representative of three independent experiments. g Ectopic expression of MIB1 increases TOP3B ubiquitylation. HEK293 cells were transfected with HA-Ubiquitin construct and MIB1-FLAG construct. Before harvest, cells were treated with MG132 (1 µM, 24 h) as indicated and subjected to TOP3B pulldown. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TOP3B, TDRD3, and MIB1 antibodies (GAPDH as loading control). The figure is representative of three independent experiments. h MIB1 depletion does not affect TDRD3 ubiquitylation. Cells were transfected with HA-Ubiquitin construct and siMIB1 as indicated for 48 h. Before harvest, cells were treated with MG132 (1 µM, 24 h) as indicated and subjected to TDRD3 pulldown. Pulled down and input samples were resolved on SDS-PAGE and probed with Ubiquitin, TDRD3 and MIB1 antibodies (GAPDH as loading control for input samples). The figure is representative of three independent experiments. i USP9X antagonizes MIB1 to stabilize TOP3B protein. HEK293 cells were transfected with the indicated siRNAs for 48 h. j Schematic representation of the homeostatic regulation of TOP3B protein levels by TDRD3-USP9X and MIB1. The TOP3B interacting E3 ligase MIB1 ubiquitylates and targets TOP3B for proteasomal degradation while the TDRD3-USP9X complex deubiquitylates and protects TOP3B from proteasomal degradation. Source data are provided as a Source Data file.

Fig. 5. Cellular depletion of TDRD3 causes accumulation of aberrant TOP3Bccs and genomic instability.

a Representative immunoblots showing expression of HA-TOP3B and FLAG-TDRD3 in TDRD3KO HCT116 cells transfected for 48 h. b, c Increased TOP3Bccs in the absence of TDRD3. RADAR assay samples were prepared from TDRD3KO HCT116 cells transfected with HA-TOP3B alone or together with TDRD3-FLAG constructs (as indicated). Samples were immunoprecipitated with anti-HA antibody, eluted from the beads, ethanol precipitated and resuspended. Samples were slot blotted and TOP3Bccs were detected with anti-HA antibody. Panel b displays a representative slot blot. Panel c is the quantitation of TOP3Bcc formation from three independent experiments. Data are means ± SD. A.U. is arbitrary units. d, e TDRD3 depletion enhances both DNA and RNA TOP3Bccs. Protein-nucleic acid adducts were isolated by RADAR assay from TDRD3KO HCT116 cells transfected with HA-TOP3B alone or together with TDRD3-FLAG constructs (as indicated). TOP3Bccs were enriched by IP using HA antibody. Samples were digested either with excess RNase A (200 μg/mL) and RNase T1 (200 units/ml) mix, or with DNase 1 (10 units), ethanol-precipitated, resuspended and slot-blotted. TOP3Bccs were detected with anti-HA antibody. Representative slot blot of DNA and RNA TOP3Bccs is displayed in panel d. Panel e is the quantitation of TOP3Bccs from independent experiments. Data are means ± standard deviations (SD; n = 3). A.U. is arbitrary units. f, g R-loop accumulation in TDRD3-depleted cells. transfected with TOP3B Genomic DNA isolated from TDRD3KO HCT116 cells transfected as indicated was slot blotted and probed with S9.6 antibody. Panel G is the quantitation from three independent experiments. Data are means ± SD. A.U. is arbitrary units. h γH2AX induction in TDRD3-depleted cells transfected with TOP3B. Immunoblots showing γH2AX levels in TDRD3KO HCT116 cells transfected with HA-TOP3B alone or together with TDRD3-FLAG constructs (as indicated). i, j Representative images of colony formation assay of TDRD3KO HCT116 cells transfected with HA-TOP3B alone or together with TDRD3-FLAG constructs (panel i). Quantitative representation of colony formation assays as shown in left panel (panel j). Data are provided as means ±  SD (n = 3). Two-tailed unpaired t-test with Welch’s correction. **P value = 0.0004. Source data are provided as a Source Data file.

Fig. 6. TDRD3 prevents the accumulation of TOP3B DNA and RNA cleavage complexes.

a Scheme of the in vitro TOP3Bcc formation assay in steady-state condition. DNA or RNA oligo substrate labeled at the 3′-end with a fluorophore is incubated with recombinant TOP3B in the presence or absence of recombinant TDRD3. TOP3Bcc formation results in a slower migrating band and can be detected via the fluorophore at the 3′-ends of oligo construct. b Reduced steady-state levels of DNA and RNA TOP3Bccs with increasing TDRD3 (stoichiometric ratio indicated below the gel). c Scheme of the TOP3Bcc reversal kinetics assay. TOP3Bccs formed (as indicated above) in the absence and presence of TDRD3 (TOP3B: TDRD3 = 1: 4) were diluted 3-fold and allowed to reverse for 0–30 min. d Time-course showing the stimulation of DNA and RNA TOP3Bcc reversal by TDRD3. Source data are provided as a Source Data file.

Fig. 7. Homeostatic regulation of TOP3B by TDRD3, USP9X, and MIB1.

(1) Free TOP3B is ubiquitylated by MIB1. (2) Ubiquitin (Ub)-modified TOP3B is degraded by the proteasome. (3) The binding of TOP3B to TDRD3 recruits the deubiquitinase USP9X (4), which stabilizes TOP3B by preventing its proteasomal degradation. (5) It also increases the turnover of TOP3B. (6) Excessive TOP3Bccs formed in the absence of TDRD3 are ubiquitylated by TRIM41 prior to their proteasomal processing and excision by TDP2 (not shown).