Fig. 7: p21 is a component of the MC and antagonizes DHX9 and NPM1 effect on miRNA biogenesis.

a KIMAT1 KD induces MYC downregulation and p21 upregulation. Representative images of 2 biological replicates. b Expression levels of primary, precursors and mature forms of the indicated miRNAs were analyzed by qPCR after silencing or overexpression of p21 in H1299 cells. Primary and precursor miRNAs were normalized to β-actin and mature miRNAs were normalized to RNU48. sip21 vs siCtrl pre-miRNA and mature miRNA p value= 0.013 (pre-miR-7), 0.003 (miR-7-5p), 0.001 (pre-miR-27a), 0.018 (miR-27a-3p), 0.0496 (pre-miR-27b), 0.008 (miR-27b-5p), 0.00015 (pre-miR-139), 0.008 (miR-139-5p), 0.004 (pre-let-7b), 0.0011 (let-7b-3p), 0.011 (pre-miR-17), 0.0024 (miR-17), 0.0004 (pre-miR-18a), 0.001 (miR-18a), 3.448E-05 (pre-miR-375), 0.0015 (miR-375), 0.001 (pre-miR-10b), 0.0014 (miR-10b-5p); p21 OE vs Ev pre-miRNA and mature miRNA p value = 0.002 (pre-miR-7), 0.001 (miR-7-5p), 1.06E-06 (pre-miR-27a), 0.0002 (miR-27a-3p), 0.0014 (pre-miR-27b), 0.007 (miR-27b-5p), 7.9E-05 (pre-miR-139), 0.002 (miR-139-5p), 0.002 (pre-let-7b), 0.0001 (let-7b-3p), 0.0019 (pre-miR-17), 0.0001 (miR-17), 0.0009 (pre-miR-18a), 0.003 (miR-18a), 0.03 (pre-miR-375), 0.002 (miR-375), 0.005 (pre-miR-10b), 0.0007 (miR-10b-5p). c p21 interacts with Drosha in p53 null and p53 wild-type cells. Representative images of 2 biological replicates. d CLIP analysis of the association between the indicated primary miRNAs and DHX9 or NPM1 upon p21 OE. Cells were transfected with p21 and immunoprecipitated with anti-DHX9 or anti-NPM1 antibody. Pri-miR-17-5p/miR-18a/miR-375/miR-10b expression was analyzed by qRT-PCR. UBC was used as a negative control. Error bars represent mean ± S.D (n = 3), **p value < 0.001, *p value < 0.05 by a two-tailed Student’s t test. (Left) From left to right p values = 0.0013436, 0.001265, 0.0004582, 0.002409, 0.000873, 0.014035, 7.022E−05, 0.001565; (Right) From left to right p values = 0.0013656, 0.013152, 0.0321765, 0.029651, 0.0043712, 6.92E−05, 0.0088363, 0.000348. e p21 OE abrogates the interaction between DHX9 and DDX5 and between NPM1 and DDX5. Representative images of 2 biological replicates. f p21 enforced expression reduces DDX5 and NPM1 protein level. Representative images of 2 biological replicates. g p21 OE in p53 null or wild-type cells induces prominent cell death.