Fig. 3: Polyamines bind acidic phosphorylatable motifs of splicing factors and prevent their phosphorylation.
From: Polyamine-dependent metabolic shielding regulates alternative splicing
a, Bar plot of significantly altered phosphorylation sites (P < 0.05, FC ± 0.2) in U2 snRNP SF3 subcomplex members after inducible AMD1 silencing (2 days, DU145 cells). b, Violin plot of isoelectric points of clusters that are altered upon polyamine deprivation by AMD1 silencing in DU145 cells. White circles show medians, box limits indicate 25th and 75th percentiles, whiskers extend to 1.5 times the interquartile range from the 25th and 75th percentiles, and polygons represent density estimates of data and extend to extreme values. c, Heat map of amino acid abundance surrounding phosphorylation sites from cluster 1 after AMD1 silencing (2 days, Dox versus no Dox). d, Representative binding modes for Spd (top) and Spm (bottom) on SF3A3. Spd and Spm are shown as green sticks, phosphorylatable residues (Ser365, Ser367, Ser369) are in yellow sticks and glutamic acid residues are shown as red sticks. e, Selected 1H-1H-total correlation spectroscopy (TOCSY) NMR spectra of SF3A3 fragment in without (black) or with (blue to red) increasing Spd (top) or Spm (bottom). Two spectra were omitted for clarity. f, Averaged chemical shift perturbation (CSP) from spectra in e, fitted to a 1:1 binding model with dissociation constant (Kd (± s.e.m.)). ppm, parts per million. g, Autoradiography showing 32P incorporation into SF3A3 and CK1 after external addition of SF3A3 and CK1 with or without increasing Spd (left) or Spm (right). Kinetic titration: n = 1 independent biological replicate; reference concentration: n = 2 independent biological replicates. h, z-scored PSI heat map of alternative splicing exonic events altered by AMD1 silencing (2 days) with or without the CK2 inhibitor CX4945 (10 µM, 24 h) in DU145 cells (Abs(ΔPSI) > 10%, P < 0.1). i, l/s ratios for differential events in DU145 cells treated as in h determined by gel densitometry. n = 6 independent biological replicates. Two-tailed Mann–Whitney U-test (b); one-tailed one-sample t-test with 1 as hypothetical value (i). # indicates P values from one-tailed paired t-test for CX4945 versus control under polyamine deprivation. Data are mean ± s.e.m.
