Fig. 6: MIR31HG promotes YBX1 phosphorylation by facilitating YBX1 interaction with its kinase RSK.
From: The long non-coding RNA MIR31HG regulates the senescence associated secretory phenotype
a Western blot analysis for p-YBX1, total YBX1, GAPDH and H3 in total and cytoplasmic fractions of BJ ER:BRAF cells (Control or siMIR31HG1-2) treated with ethanol (Control) or 1 μM 4-OHT for 72 h (n = 3). b Quantification of the pYBX1 band intensities relative to total YBX1 and Vinculin from three independent experiments. c PLA representative images showing the interaction (red dots) between YBX1 and RSK in BJ ER:BRAF cells (Control or siMIR31HG1) were treated with ethanol (Control) or 1 μM 4-OHT for 72 h. DAPI staining (blue) shows the nucleus. d Quantification of the number of interactions (dots) per cell in the conditions indicated in a representative experiment from four independent replicates (n = 4). Scale bar: 20 μm. e Left, in vitro kinase experiment incubating recombinant YBX1 in the presence of 32P-ATP and recombinant RSK1 in the presence of the indicated in vitro-transcribed RNA, run in 4–12% NuPAGE Bis–Tris gel and exposed to an Amersham Hyperfilm ECL film. Right, quantification of the band intensities represented as p-YBX1 related to p-RSK (n = 4). f Western blot for p-YBX1, total YBX1 and Vinculin in BJ ER:BRAF cells expressing a doxycycline-induced empty, MIR31HG WT, Mut3 or Mut5 treated for 36 h with ethanol (Control) or 1 μM 4-OHT. g Quantification of the p-YBX1 band intensities relative to total YBX1 and Vinculin (n = 3). h Scheme of the working model: (1) During OIS MIR31HG is induced and locates in the cytoplasm. (2) At the same time, transcription of IL1A mRNA is induced. (3) IL1A mRNA is exported to the cytoplasm where it interacts with YBX1. MIR31HG facilitates YBX1 interaction with the kinase RSK promoting its phosphorylation which in turns promotes IL1A translation. (4) IL1A signalling induces the transcription of other SASP mRNAs through CEBPB and NF-κB. (5) These RNAs will be translated in the cytoplasm and the SASP components will be secreted (6). Statistical significance was calculated using two-tailed Student’s t-tests, *p < 0.05; **p < 0.01; ***p < 0.001. Error bars represent means ± s.d. Source data are provided as a Source Data file.
