Doxo globally rewires ELAVL1 binding to RNA within cells, with a shift toward exonic and cytosolic RNA. (A) MCF-7 cell growth following transfection with siRNAs targeting ELAVL1 (siELAVL1) or a negative-control siRNA (siCTRL) and subsequent treatment with Doxo or vehicle for the indicated time point. Cells proliferate more in the siCTRL-Doxo condition than in the other ones. (B) Western blot analysis of ELAVL1 protein levels in nuclear and cytosolic fractions of MCF-7 cells transfected with the indicated siRNAs and treated with Doxo or vehicle for 6 h. GAPDH and lamins were used as cytosolic and nuclear markers, respectively. (C) Experimental design of CCS-iCLIP analyses of ELAVL1-RNA interactions in different fractions of MCF-7 cells treated with Doxo or vehicle for 6 h. (Tot) Total cells (whole cells), (Nuc) nucleus, (Cyto) cytosol. (D) Number of ELAVL1 binding sites (peaks found in two biological replicates) identified in each fraction of vehicle-treated cells. (E) Density of nuclear and cytosolic ELAVL1 binding sites from vehicle-treated cells in exons, introns, 3′ UTRs overlapping introns, and 3′ UTRs overlapping the last exon of genes. (F) Proportion of ELAVL1 binding sites from each fraction that are either up- or down-regulated by Doxo treatment. (G) Density of Doxo up- and down-regulated ELAVL1 binding sites from total cells in exons, introns, 3′ UTRs overlapping introns, and 3′ UTRs overlapping the last exon of genes.
