Fig 2: Biological responses of eRNAs in clinical samples and cell growth(A) Pearson’s correlation between PRRX2 mRNA and eRNA levels in nine breast cancer tumors was determined (two-tailed p value, *p < 0.05).(B) Stimulatory effect of targeted PRRX2 eRNA on cell proliferation. Cell proliferation assay of dCas9-expressing MCF-7 cells with various PRRX2 sgRNA constructs. See (C) for a description of the statistics. Each point represents the mean ± standard error of the mean (n = 3 biological/technical replicates). The asterisks indicate significant differences from the corresponding empty control (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, ****p < 0.0001).(C) Depletion of BCAS2 abolishes the stimulatory effect of targeted PRRX2 eRNA on cell proliferation. Cell proliferation assay of dCas9-expressing MCF-7 cells with PRRX2 sgRNA constructs upon control or BCAS2 knockdown. Each point represents the mean ± standard error of the mean (n = 3 biological/technical replicates). The asterisks indicate significant differences from the corresponding control (siControl, sgRNA at the specific time point) (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, ****p < 0.0001).(D) Metagene representations from ERa ChIP-seq upon BCAS2 depletion between ERa binding sites (ERBS) containing eRNAs with or without FERM. The metagene plots represent the average read counts normalized to their respective input controls around the ERBS (±1.5 kb) in each group—n(without FERM) = 940, n(with FERM) = 339. The genomic assays were performed as two biological and two technical replicates.(E) Boxplots of depleted ERa peaks upon BCAS2 depletion at ERa binding sites (ERBS) containing eRNAs with or without FERM (±1.5 kb). A cut-off of 2-fold decrease between siBCAS2/siCtrl at 45 min and at 0 min was used to define depleted ERa peaks. The boxes represent the 25th–75th percentile (band at median), with whiskers at 1.5 × interquartile range. Bars marked with different letters are significantly different—n(without FERM) = 207, n(with FERM) = 70 (two-tailed Mann-Whitney test, p = 0.0164). The genomic assays were performed as two biological and two technical replicates.(F) Depletion of BCAS2 alters E2-regulated genes in MCF-7 cells. Heatmap of RNA-seq data representing changes in the expression of E2-regulated genes from MCF-7 cells treated with control or BCAS2 knockdowns for 48 h before E2 stimulation for 6 h. In total, 1,075 genes were differentially expressed between control and BCAS2 knockdown upon 6 h E2 treatment—n(upregulated) = 403, n(downregulated) = 672. The genomic assays were performed as two biological and two technical replicates.(G and H) GO analysis of (G) upregulated and (H) downregulated genes upon BCAS2 knockdown in MCF-7 cells treated with E2 for 6 h.(I) A model for estrogen-regulated eRNAs in enhancer assembly and gene expression. Details are provided in the text.See also Figure S7.

Fig 3: Depletion of the Prp19 complex disturbs spindle formation in Xenopus egg extracts.(A): Control Xenopus egg extracts or extracts after depletion of the Prp19 complex (deBCAS2) were released from metaphase (times after release are indicated) and the degradation and re-accumulation of cyclin B was monitored by immunoblot. Tubulin served as a loading control. (B): Extracts were depleted of the Prp19 complex by antibodies against BCAS2 (deBCAS2) or Prp19 (dePrp19) and complemented with 100 nM purified human Prp19 complex (see C, Coomassie staining). Extract samples, and the immunoglobulin beads after depletion, were analyzed by Immunoblot with Xenopus BCAS2 and Prp19 antibodies. Tubulin served as a loading control for the extract samples. (D): Spindle assembly was monitored in control, BCAS2 (deBCAS2), or Prp19 (dePrp19) depleted egg extracts, or in depleted extracts after re-addition of 100 nM Prp19 complex (see panel C) after a complete cell cycle in the presence of sperm nuclei (DAPI, DNA, blue in merge) and Cy3-tubulin (red in merge). Scalebars: 20 µm. (E): Quantification of normal (grey) and weak (red) spindles in control, depleted (dePrp19) and reconstituted extracts (rescue) spindles in 20 µl extract. The graph (left) shows values from three independent experiments +/- s.e.m. after depletion using BCAS2 antibodies, and from one representative experiment after depletion using Prp19 antibodies (right). Significance values (left) were calculated using an unpaired two-tailed T-test. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

Fig 4: Knockdown of Prp19 complex components in human cells compromises mitosis.(A): HeLa cell lysates analyzed by immunoblotting with Prp19 complex antibodies, after knockdown of BCAS2 by siRNA (1 and 2). Numbers indicate protein levels relative to Tubulin. (B-E) BCAS2 (oligo 1) knockdown (siRNA BCAS2, kd BCAS2) and control (siRNA ctrl). YFP-Xenopus laevis (X.l.) BCAS2 was expressed to complement knockdown of the human (h.s.) BCAS2 (B): Immunoblot to show knockdown of endogenous human BCAS2 and re-expression of the Xenopus ortholog. (C) Quantification of mitotic indices determined from three independent experiments (N=500); graph shows mean values +/- s.e.m. Significance values were calculated using an unpaired two-tailed T-test. ***: P < 0.001. (D) BCAS2 expression and localization were analyzed by indirect immunofluorescence with BCAS2 (green in merge) and α-Tubulin (red in merge) antibodies. DAPI (blue in merge) was used to stain the DNA. (E and F) Cells after knockdown (kd) of BCAS2 (oligo 1), or control cells were immunostained with antibodies against the centromere protein CENPA (green) and the spindle protein TPX2 (red). Cells expressing X.l. YFP-BCAS2 were analyzed for the rescue situation. (E) Representative images. (F) the distance between white arrowheads (E) was quantified as interkinetochore distance; orange arrowheads (E) exemplify microtubule to kinetochore end-on attachments. Nuf2 knock-down cells served as a positive control. Quantifications were determined from three independent experiments (N=60); graph shows mean values +/- s.e.m. Significance values were calculated using an unpaired two-tailed T-test. ***: P < 0.001. Scalebars: 10 µm; 1 µm in magnifications in (E).