GSE173530
A single N6-methyladenosine site in lncRNA HOTAIR regulates its function in breast cancer cells
N6-methyladenosine (m6A) modification of RNA plays important roles in normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we investigate whether m6A regulates the function of the human HOTAIR lncRNA, which contributes to multiple pro-tumor phenotypes in triple-negative breast cancer (TNBC) cells. We identify 14 individual m6A sites within HOTAIR, with a single site (A783) consistently methylated. Mutation of A783 impairs cellular proliferation and invasion in HOTAIR-overexpressing TNBC cells. m6A at A783 regulates HOTAIR's ability to localize to chromatin and induce gene expression changes. A783 mutant HOTAIR reverses wild-type effects beyond baseline, demonstrating a potential antimorphic behavior. HOTAIR interacts with nuclear m6A reader YTHDC1 and YTHDC1-HOTAIR interactions are required for chromatin localization and gene repression. Our work demonstrates how modification of one base in a lncRNA can elicit a distinct gene regulation mechanism and drive disease-associated phenotypic changes. Overall design: MeCLIP: M6A identification in 2 cell lines (MDA-MB-231 pB-HOTAIRA783U (3 replicates) and MDA-MB-231 pB-AntiLuciferase (1 replicate)) using modified eCLIP-based library preparation and custom analysis pipeline. RNAseq: PolyA-selected high-throughput RNA sequencing for 3 cell lines, 3 replicates each (MDA-MB-231 with pB-HOTAIRWT, pB-HOTAIRA783U, and pB-AntiLuciferase).