Fig. 5.

RMRP inhibits p53 activity through SNRPA1. (A) Identification of RMRP-interacting proteins. RMRP and the antisense of RMRP were synthesized and biotinylated in vitro followed by the RNA pull-down assay. The silver staining reveals the specific bands (red arrows) that were subjected to MS analysis. (B and C) RMRP interacts with SNRPA1. SNRPA1 is pulled down with RMRP, but not with the antisense of RMRP, by the RNA pull-down assay (B). RMRP, but not another lncRNA MALAT1, is coimmunoprecipitated with SNRPA1 by the RIP assay using an anti-SNRPA1 antibody (C). (D and E) p53 expression is negatively correlated with SNRPA1. Overexpression of RMRP reduces the p53 level, whereas increases the SNRPA1 level in HCT116 ^p53+/+ cells (D). Knockout of RMRP elevates p53 expression but decreases SNRPA1 expression in HCT116 ^p53+/+ cells (E). (F and G) Exogenous SNRPA1 interacts with exogenous p53 by reciprocal IP assays. (H and I) Endogenous interactions between SNRPA1 and p53 in HCT116 ^p53+/+ cells. (J) Overexpression of SNRPA1 promotes p53 protein degradation in the presence of MDM2 in HCT116 ^p53+/+ cell. (K) Knockdown of SNRPA1 increases p53 protein level in HCT116 ^p53+/+ cells. (L) SNRPA1 promotes MDM2-induced ubiquitination of p53. (M) Overexpression of SNRPA1 accelerates proliferation of HCT116 ^p53+/+ cells by the cell viability assay. (N) Knockdown of SNRPA1 inhibits proliferation of HCT116 ^p53+/+ cells by the cell viability assay. (O) Overexpression of SNRPA1 has no effect on HCT116 ^p53−/− cell proliferation by the cell viability assay. (P) Knockdown of RMRP has no effect on HCT116 ^p53−/− cell proliferation by the cell viability assay. (Q) Knockdown of SNRPA1 abolishes RMRP inhibition of p53. (R) Knockdown of SNRPA1 abrogates RMRP-induced cancer cell proliferation. (S) Overexpression of SNRPA1 restores cell proliferation impaired by RMRP depletion. **P < 0.01 by two-tailed Student’s t test. n.s. indicates no significance.