Inactivation of the tumor suppressor p53 by long noncoding RNA RMRP

Abstract

p53 inactivation is highly associated with tumorigenesis and drug resistance. Here, we identify a long noncoding RNA, the RNA component of mitochondrial RNA-processing endoribonuclease (RMRP), as an inhibitor of p53. RMRP is overexpressed and associated with an unfavorable prognosis in colorectal cancer. Ectopic RMRP suppresses p53 activity by promoting MDM2-induced p53 ubiquitination and degradation, while depletion of RMRP activates the p53 pathway. RMRP also promotes colorectal cancer growth and proliferation in a p53-dependent fashion in vitro and in vivo. This anti-p53 action of RMRP is executed through an identified partner protein, SNRPA1. RMRP can interact with SNRPA1 and sequester it in the nucleus, consequently blocking its lysosomal proteolysis via chaperone-mediated autophagy. The nuclear SNRPA1 then interacts with p53 and enhances MDM2-induced proteasomal degradation of p53. Remarkably, ablation of SNRPA1 completely abrogates RMRP regulation of p53 and tumor cell growth, indicating that SNRPA1 is indispensable for the anti-p53 function of RMRP. Interestingly and significantly, poly (ADP-ribose) polymerase (PARP) inhibitors induce RMRP expression through the transcription factor C/EBPβ, and RMRP confers tumor resistance to PARP inhibition by preventing p53 activation. Altogether, our study demonstrates that RMRP plays an oncogenic role by inactivating p53 via SNRPA1 in colorectal cancer.

Keywords: PARP inhibition; RMRP; SNRPA1; long noncoding RNA; p53.

Fig. 1.

LncRNA RMRP is overexpressed in colorectal cancer and associated with unfavorable prognosis. (A) RMRP expression is higher in colon cancer (n = 79) compared with normal tissues (n = 14). (B) RMRP expression is higher in colon cancer tissues (n = 14) compared with the paired adjacent tissues (n = 14). Values are expressed as the median with interquartile range in A and B. (C) A higher level of RMRP predicts poorer prognosis in 79 colon cancer patients. (D) RMRP expression is higher in cancerous tissues compared with the adjacent normal tissues through a colorectal cancer tissue array determined by RNA in situ hybridization. (E) Statistical analysis of RMRP staining in the colorectal cancer tissues (n = 93) and the adjacent tissues (n = 87). Statistical significance was assessed using Fisher’ s exact test. (F) Higher level of RMRP is associated with worse overall survival in 93 colorectal cancer patients. (G) Higher expression of RMRP is associated with worse overall survival in the TCGA rectum adenocarcinoma cohort.

Fig. 2.

RMRP represses p53 activity by enhancing MDM2-induced p53 proteasomal degradation. (A and B) Overexpression of RMRP reduces p53 target gene expression in HCT116 ^p53+/+ and H460 cells. (C and D) Knockdown of RMRP increases p53 target gene expression in HCT116 ^p53+/+ and H460 cells. (E) CRISPR-Cas9–mediated ablation of RMRP induces p53 target gene expression in HCT116 ^p53+/+ cells. (F) Overexpression of RMRP decreases the protein level of p53 in HCT116 ^p53+/+ and H460 cells. (G and H) Knockdown of RMRP elevates the protein levels of p53 and p21 in HCT116 ^p53+/+ and H460 cells. (I) Knockout of RMRP induces p53 and p21 protein levels in HCT116 ^p53+/+ cell. (J and K) Overexpression of RMRP impairs 5-FU–induced p53 activation determined by IB (J) and RT-qPCR (K). (L) The proteasome inhibitor MG132 blocks RMRP-mediated p53 degradation in HCT116 ^p53+/+ cells. Cells were treated with MG132 (20 μM) for 6 h before harvested for IB. (M) The p53’s half-life is extended upon RMRP depletion. The ctrl-Cas9 and RMRP-sg-1 cell lines were treated with 100 μg/mL of cycloheximide (CHX) and harvested at the indicated time points for IB (Left). (Right) The ratios of p53/GAPDH. (N) RMRP promotes MDM2-dependent ubiquitination of p53. HCT116 ^p53−/− cells were transfected with combinations of plasmids encoding p53, RMRP, HA-MDM2, and His-Ub as indicated and treated with MG132 (20 μM) for 6 h before harvested for in vivo ubiquitination assay. *P < 0.05, **P < 0.01 by two-tailed Student’s t test.

Fig. 3.

RMRP promotes colorectal cancer cell growth and proliferation through inactivation of p53. (A) Overexpression of RMRP accelerates proliferation of HCT116 ^p53+/+ cells by the cell viability assay. (B) Overexpression of RMRP has no effect on HCT116 ^p53−/− cell proliferation. (C) Overexpression of RMRP enhances the colony-forming ability of HCT116 ^p53+/+ cells but not HCT116 ^p53−/− cells. The quantification of colonies is shown in the right panel. (D) Knockdown of RMRP inhibits proliferation of HCT116 ^p53+/+ cells. (E) Knockdown of RMRP has no effect on proliferation of HCT116 ^p53−/− cells. (F) Knockdown of RMRP impedes the colony-forming ability of HCT116 ^p53+/+ cells but not HCT116 ^p53−/− cells. (Right) The quantification of colonies. (G) Knockout of RMRP by CRISPR/Cas9 dramatically prompts proliferation of HCT116 ^p53+/+ cells. (H) RMRP knockout had a marginal inhibitory effect on HCT116 ^p53−/− cell proliferation. (I) RMRP knockout drastically inhibits the colony-forming ability of HCT116 ^p53+/+ cells but has a marginal effect on colony formation of HCT116 ^p53−/− cells. (Right) The quantification of colonies. (J) Knockdown of RMRP leads to reduction of S-phase population in HCT116 ^p53+/+ cells. The quantification of S-phase is shown in the right panel. (K) Knockdown of RMRP has no effect on HCT116 ^p53−/− cell cycle progression. The quantification of S-phase is shown in the right panel. *P < 0.05, **P < 0.01 by two-tailed Student’s t test.

Fig. 4.

RMRP endorses tumor growth in vivo by inactivating p53. (A) Lentivirus-based overexpression of RMRP in HCT116 ^p53+/+ cells significantly elevates tumor volume in average compared with the control group. (B and C) The dissected tumors show that RMRP overexpression increases the weight and mass of tumors derived from HCT116 ^p53+/+ cells. Data are represented as mean ± SD, n = 6. (D) Overexpression of RMRP inhibits p53 and p21 protein expression in vivo. (E) Overexpression of RMRP inhibits the mRNA expression of p21 and PUMA examined in three pairs of xenograft tumors (mean ± SD). (F–H) Overexpression of RMRP has a marginal effect on the growth of tumors derived from HCT116 ^p53−/− cells. Data are represented as mean ± SD, n = 6. (I and J) Overexpression of RMRP does not affect p53 target gene expression in three pairs of xenograft tumors derived from HCT116 ^p53−/− cells (mean ± SD). (K) CRISPR/ Cas9–mediated depletion of RMRP in HCT116 ^p53+/+ cells significantly suppresses tumor volume in average compared with the control group. (L and M) The dissected tumors show that knockout of RMRP diminishes the weight and mass of tumors derived from HCT116 ^p53+/+ cell. Data are represented as mean ± SD, n = 8. (N) RMRP knockout bolsters p53 and p21 protein expression in vivo. (O) RMRP knockout activates the mRNA expression of p21 and PUMA examined in three pairs of xenograft tumors (mean ± SD). (P–R) Knockout of RMRP has a marginal effect on the growth of tumors derived from HCT116 ^p53−/− cells. Data are represented as mean ± SD, n = 8. (S and T) Knockout of RMRP does not affect p53 target gene expression in three pairs of xenograft tumors derived from HCT116 ^p53−/− cells (mean ± SD). *P < 0.05, **P < 0.01 by two-tailed Student’s t test. n.s. indicates no significance.

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.

Fig. 6.

RMRP prevents lysosomal proteolysis of SNRPA1 by perturbing CMA. (A and B) SNRPA1 protein is stabilized by the autophagy inhibitor CQ but not by the proteasome inhibitor MG132. HCT116 ^p53+/+ (A) and H460 (B) cells were treated with MG132 (20 μM, 6 h) and CQ (50 μM, 8 h) before harvested for IB. (C) The presence of a KFERQ-like motif, LKERQ, in SNRPA1 and mutation of this motif to LKEAA. (D) Exogenous SNRPA1 interacts with exogenous HSPA8 via the LKERQ motif. HCT116 ^p53−/− cells were transfected with combinations of plasmids encoding Myc-HSPA8, Flag-SNRPA1, and Flag-SNRPA1-Mut followed by co-IP–IB assays. (E) Exogenous SNRPA1 interacts with exogenous LAMP2A. HCT116 ^p53−/− cells were transfected with plasmids encoding Flag-SNRPA1 and Myc-LAMP2A followed by co-IP–IB assays. (F) Overexpression of LAMP2A reduces the protein level of SNRPA1 in HCT116 ^p53+/+ cells. (G) Knockdown of LAMP2A increases the expression of SNRPA1 in HCT116 ^p53+/+. (H) The cellular distribution of SNRPA1 protein in HCT116 ^p53+/+ cells. GAPDH and Lamin B indicate the cytosolic and nuclear fractions, respectively. (I) CRISPR-Cas9–mediated ablation of RMRP reduces the SNRPA1 level in the nucleus, while increases the cytosolic accumulation of SNRPA1, in HCT116 ^p53+/+ cells. (J) Knockout of RMRP enhances the endogenous interaction of SNRPA1 and HSPA8.

Fig. 7.

The C/EBPβ-RMRP axis confers colorectal cancer resistance to PARP inhibition. (A) Evaluation of the RMRP promoter activity by the luciferase reporter assay. (B) RMRP expression was determined by individually knocking down 11 TFs in HCT116 ^p53+/+ cells. (C) Schematic of the ∼500-bp region of the RMRP promoter. Two potential consensus binding sites of C/EBPs are indicated. (D) Overexpression of C/EBPβ up-regulates the RMRP level in HCT116 ^p53+/+ cells. (E) Overexpression of C/EBPβ triggers RMRP promoter (∼500 bp) activity as determined by the luciferase reporter assay. (F and G) C/EBPα and β associate with the RMRP promoter by the ChIP assay. The bound DNA elements were analyzed by PCR. (H) The expression of RMRP was induced by PARP inhibitors, including Olaparib, Niraparib, and Talazoparib. HCT116 ^p53+/+ cells were treated with the PARP inhibitors for 16 h before harvested for RT-qPCR. (I) Knockdown of PARP-1 up-regulates the RMRP level in HCT116 ^p53+/+ determined by RT-qPCR. (J and K) Knockout of RMRP sensitizes cancer cells to Olaparib-induced p53 activation. (L) A low dose of Olaparib significantly inhibits proliferation of RMRP-knockout, but not RMRP-proficient, colorectal cancer cells. (M) Knockout of RMRP sensitizes colorectal cancer cells to Olaparib as determined by the IC50. (N and O) Knockout of RMRP sensitizes colorectal cancer cells to the combination use of Olaparib with the genotoxic agents, Cisplatin (N) and 5-FU (O), as determined by the IC50. *P < 0.05, **P < 0.01 by two-tailed Student’s t test. (P) A schematic for RMRP-induced colorectal cancer resistance to PARP inhibitors by preventing p53 activation. Treatment of cancer cells with PARP inhibitors induces RMRP expression through the TF C/EBPβ. RMRP interacts with and sequesters SNRPA1 in the nucleus, thus blocking CMA-mediated lysosomal proteolysis of SNRPA1. The nuclear SNRPA1 then binds to p53 and promotes MDM2-induced p53 ubiquitination and proteasomal degradation (Left). Knockout of RMRP prompts cytosolic enrichment and lysosomal degradation of SNRPA1, leading to reactivation of p53 and tumor cell sensitization to PARP inhibitors (Right).