Fig 2: Identification and validation of TULP3 protein–protein interactions.A, silver stained gel showing proteins coimmunoprecipitated alongside mock-FLAG or FLAG-TULP3 from 293T cells. The band corresponding to FLAG-TULP3 is indicated by a red arrow. B, bar graph ranking the top 35 high-confidence hits in (A) as identified by mass spectrometry. C, STRING diagram outlining the interconnectedness of TULP3 interactors identified in (B). Western blots showing coimmunoprecipitation of FLAG-TULP3 with (D) SIRT1, (E) RAD18, and (F) PP6R3 in 293T cells.

Fig 3: Effect of CYLD on RAD18, DTL, POLH and PCNA monoubiquitination. (A) MCF-7 cells were transfected with CYLD-targeted siRNA (siCYLD) or siControl. At 65 h post-transfection, the cells were irradiated at 20 J/m2 UV and harvested 1 or 6 h post-irradiation. (B) MCF-7 cells were transfected with siRNA against CYLD, RAD18 and DTL or their double or triple combinations. At 65 h post-transfection, the cells were UV-irradiated at 20 J/m2 and harvested 6 h post-irradiation. Whole-cell extracts were fractionated by SDS-PAGE and analyzed by Western blot with indicated antibodies. Protein levels were normalized to GAPDH and presented each relative to its level in unirradiated or UV-irradiated siControl-treated cells.

Fig 4: Effect of TENT4A and PAXIP1 on the stability of TLS-related mRNAs and level of TLS-related proteins. (A,B) TENT4A and/or PAXIP1 expression was knocked down in MCF-7 cells for 48 h, after which the cells were treated with 5 μg/mL Actinomycin D for up to 8 h. The expression levels of POLH and RAD18 were each determined by qPCR relative to the expression level at time 0. Half-life was calculated using a non-linear fit one-phase decay curve equation. The significance of the differences was calculated using Student’s t-test (one-tailed); Data are presented as mean ± SEM from three independent experiments. ns, non-significant; p > 0.05. (C) MCF-7 cells were transfected with TENT4A and/or PAXIP1 or non-targeting control siRNA for a final combined concentration of 100 nM. At 65 h post-transfection, the cells were UV-irradiated at 20 J/m2 and harvested 1 or 6 h post-irradiation. Whole-cell extracts were fractionated by SDS-PAGE followed by Western blot with indicated antibodies. Amounts are presented relative to those in extracts of unirradiated cells treated with siControl and the values are shown in the corresponding position of the blots.

Fig 5: Effect of TENT4A on binding and stability of TLS-related mRNAs. (A) FLAG-tagged TENT4A expressed in HEK293T cells was immunoprecipitated with an anti-FLAG antibody and RNA bound to it was analyzed by qPCR. The results are shown as fold-change relative to empty vector. The error bars indicate SEM (two independent experiments). (B–D) Representative gel images of RAD18, CYLD and POLH poly(A) tail are shown, each with the poly(A) tail densitometric trace underneath, using Image J. The position of TVN is indicated by an asterisk that predominantly recognizes A12, serving as an internal control. (E–H), TENT4A and/or TENT4B expression was knocked down in MCF-7 cells for 48 h, after which the cells were treated with 5 µg/mL Actinomycin D for up to 8 h. The expression levels of RAD18, CYLD, POLH and POLD3 genes were each determined by qPCR relative to the expression level at time 0. Half-life was calculated by using non-linear fit one-phase decay curve equation. The significance of the differences was calculated using Student’s t-test (one-tailed); Data are presented as mean ± SEM from three independent experiments. ns, non-significant; p > 0.05.