Fig 2: RBM15 associates with DBP5 in vivo and in vitro. The expression plasmids encoding epitope-tagged proteins (1–5 µg each) were transiently transfected in human 293 cells as indicated, and coprecipitation assays were performed from RNase A-treated extracts (30). (A–C) The extracts were adjusted to 400 mM NaCl before adding the antibodies, and the precipitates were washed in the presence of 400 mM NaCl and 2 M urea prior to elution. The epitopes detected on western blots and the positions of detected proteins are indicated. Input, 1:100 of the extract before IP; Control, omission of bait protein and n/t, non-transfected cells. (D) Binding of E.coli-produced, immobilized GST-tagged DBP5 or isolated GST moiety (left panel, GST proteins) to metabolically radiolabeled RBM15 and luciferase (Luc) proteins which were synthesized in reticulocyte lysates (input) (26). Prior to binding, RNase A was added to reticulocyte samples to exclude the RNA-mediated interactions. Right panel (GST pulldowns), SDS–PAGE/radiofluorography analysis of the bound fractions. Asterisk shows a truncated RBM15 protein.

Fig 3: Essential paralogs underlying SL.(A) QBF of high-confidence SL hits for PTEN KO cells. WT_Average means the mean QBF of all four repeated WT screens and is presented as means ± SD. (B) Pearson correlation analysis of gene dependency for ARID1A-mutant features in all available cell lines in the DepMap portal. (C) Relative mRNA levels of ARID1B in 293A WT and all TSG KO cells. Data are presented as means ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA. ****P < 0.0001. (D) EV- or PTEN-overexpressed MDA-MB-468 cells were seeded at 4 × 105 cells per well in a six-well plate and maintained for 48 hours, and then cells were harvested for immunoblotting with the indicated antibodies or real-time quantitative PCR to measure the mRNA levels of ARID1B. Data are presented as means + SD of three biological replicates with statistical analysis by unpaired t test. ***P < 0.001. (E) Same conditions described in (D), except that the cell line was PC-3. (F) Negative correlation between DDX19B expression and DDX19A essentiality. TPM, transcripts per kilobase million. (G) 293A WT, NF2 KO, and ARID1A KD cells were infected with lentiviruses containing LentiCRISPRv2 sgControl or two independent sgRNAs targeting DDX19A; 24 hours later, cells were selected with puromycin (2 μg/ml) for 3 days and harvested for immunoblotting with the indicated antibodies. (H) Same conditions described in (G), except that the cells after puromycin selection were seeded at 800 cells per well in a 96-well plate with four replicates, and luminescence detection was performed every 2 days according to the standard protocol.

Fig 4: RBM15 stimulates crosslinking of DBP5 to mRNA. (A) Exponentially growing human 293 cells were UV-irradiated with a dose of 400 mJ when indicated (UV+) and fractionated into the C, N1 and N2 extracts. Poly(A)+RNA was isolated under denaturing conditions, and the endogenous RBM15, DBP5 and, as negative control, ACTB proteins were analyzed in the poly(A)+ fractions on Western blots, after RNase digestion (polyA+); and in 1:1000 aliquots of crude extracts (input). (B and C) Human 293 cells were transiently transfected with 1 µg of HA-NXF1, 1 µg of HA-DBP5, and the indicated amounts of HA-RBM15 plasmids (B) or with 1 µg of each of the indicated plasmid (C). At day 1 posttransfection, cells were UV-irradiated (B, lanes 1–4; C, lanes 1–6) as in (A). Poly(A)+ RNA was isolated from soluble cell fractions (C + N1), and the crosslinked proteins were analyzed as in (A). Input, a 1:200 aliquot of crude extract. The positions of proteins and the antibodies used are indicated. In panel B, staining with the RBM15 antibody (pAb 10587-1-AP, Proteintech) detected both the exogenous and endogenous proteins with the expected mobility difference [endogenous, isoform 2-L, 100 kDa; exogenous, HA-tagged isoform 1 − S + AE, 108 kDa (30)]. The identity of the crosslinked DBP5 bands was verified by staining with protein-specific antibodies (data not shown). Similar data were obtained in three independent transfection experiments.