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An N6-methyladenosine at the transited codon 273 of p53 pre-mRNA T
promotes the expression of R273H mutant protein and drug resistance of
Mohammad B. Uddin a,1, Kartik R. Roya, Salman B. Hosaina, Sachin K. Khistea, Ronald A. Hilla, Seetharama D. Joisa, Yunfeng Zhaob, Alan J. Tackettc, Yong-Yu Liua, a School of Basic Pharmaceutical and Toxicological Sciences, University of Louisiana at Monroe, Monroe, LA 71201, USA b Department of Pharmacology, Toxicology & Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA c Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
Tumor suppressor p53
N6-methyladenosine Drug resistance
Mutant p53 proteins that promote cancer cell invasive growth, metastasis and drug resistance emerge as ther-apeutic targets. Previously, we reported that suppression of ceramide glycosylation restored wild-type p53 protein and tumor suppressing function in cancer cells heterozygously carrying p53 R273H, a hot-spot missense mutation; however, the mechanisms underlying the control of mutant protein expression remain elusive. Herein, we report that an N6-methyladenosine (m6A) at the point-mutated codon 273 (G > A) of p53 pre-mRNA de-termines the mutant protein expression. Methylation of the transited adenosine was catalyzed by methyl-transferase like 3 (METTL3), and this m6A-RNA promoted a preferential pre-mRNA splicing; consequently, the produced p53 R273H mutant protein resulted in acquired multidrug resistance in colon cancer cells. Furthermore, glycosphingolipids (particularly globotriaosylceramide) generated from serial ceramide glycosy-lation were seen to activate cSrc and β-catenin signaling so as to upregulate METTL3 expression, in turn pro-moting expression of p53 R273H mutant protein, with consequent drug resistance. Conversely, either silencing METTL3 expression by using small interfering RNA (siRNA) or inhibiting RNA methylation with neplanocin A suppressed m6A formation in p53 pre-mRNA, and substantially increased the level of phosphorylated p53 protein (Ser15) and its function in cells heterozygously carrying the R273H mutation, thereby re-sensitizing these cells to anticancer drugs. Concordantly, suppression of ceramide glycosylation repressed METTL3 ex-pression and m6A formation in p53 pre-mRNA, thus sensitizing cells carrying R273H to anticancer drugs. This study uncovers a novel function of pre-mRNA m6A as a determinant of mutant protein expression in cancer cells heterozygously carrying the TP53 R273H mutation. Suppressing both RNA methylation and ceramide glycosy-lation might constitute an efficacious and specific approach for targeting TP53 missense mutations coding for a G > A transition, thereby improving cancer treatments.