Fig. 4: MIR31HG interacts with YBX1 and YBX1 knock-down phenocopies MIR31HG depletion.

a Schematic representation of the pulldown procedure. b Cellular extracts from BJ ER:BRAF (Control or siMIR31HG) treated with 1 μM 4-OHT for 72 h were incubated with antisense oligos, enriched proteins extracted and subjected to LC-MS analysis (see ‘Methods'). The volcano plot highlights proteins enriched in the MIR31HG pulldown analysis compared to a luciferase control pull down. Marked in red are protein with p value <0.05. c Heat map showing the relative expression of significant differentially expressed SASP genes in BJ ER:BRAF cells (control or siMIR31HG) treated with ethanol (Control 1-3) or 1 μM 4-OHT for 48 h (Sen 1-3, Sen siMIR31HG 1-3, Sen YBX1-KD1-3). d Relative RNA expression of selected components of the SASP normalized to housekeeping genes (HPRT1 and RPLP0) in BJ ER:BRAF cells transfected with the indicated siRNAs (Control or siYBX1, 1–2), treated with ethanol (Control) or 1 μM 4-OHT for 48 h. The graph shows results compared to control ethanol-treated set to 1 (n = 4). e Secreted IL6 and CXCL1 (pg/ml) measured by ELISA in the CM of BJ ER:BRAF (Control or siYBX1, 1–2) treated for 72 h with ethanol (Control) or 1 μM 4-OHT (n = 3). f Western blot for IL1A in BJ ER:BRAF cells (Control or siYBX1, 1–2) were treated with ethanol (Control) or 1 μM 4-OHT for 72 h. Molecular weight marker is shown in kDa (n = 3). g Quantification of the IL1A western blot band intensities relative to Vinculin (n = 3). h Relative RNA binding using GFP-YBX1 in 4-OHT-treated BJ ER:BRAF, represented as the percentage of the input bound relative to an empty GFP cell line. YBX1 was used as a positive control (blue), MALAT1, ND1, and ND4 binding as negative controls (green) and cytokines binding (pink). The results are shown as the percentage of input relative to an empty GFP cell line treated in the same conditions (n = 3). i Top, schematic representation of MIR31HG with the putative YBX1-binding sites (BS) found in published iCLIP data. BS1-3 were found in Goodarzi et al. iCLIP data and BS5-4 were retrieved from Wu et al. dataset. Bottom, representation of MIR31HG truncations (Mut1 to Mut5). j EMSA showing the in vitro binding of 2 nM of the corresponding radiolabelled transcript and the indicated amounts of recombinant YBX1. BSA at the highest concentration (400 ng) was used as a negative control (n = 3). k EMSA showing the in vitro binding of 400 ng of YBX1 to 2 nM of the radiolabelled MIR31HG wild type in competition with the indicated amounts of unlabelled Mut5 or Mut3 (n = 3). All statistical significances were calculated using two-tailed Student’s t-tests, *p < 0.05; ns non-significant. All error bars represent means ± s.d. Source data are provided as a Source Data file.