N6-methyladenosine (m6A) is one of the most abundant RNA modifications with important roles in normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. In this study, we investigate whether m6A plays a role in regulating the function of the 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) being consistently methylated.Mutation of A783 impairs cellular proliferation and colony formation in TNBC cells. We find that HOTAIR interacts with the nuclear m6A reader YTHDC1 at methylated A783 and additional sites. Interestingly, we determine that modifications at different sites in HOTAIR have differential effects on HOTAIR regulation. Specifically, m6A at A783 regulates HOTAIR localization to chromatin, whereas other m6A sites mediate high HOTAIR levels. We further find that YTHDC1-HOTAIR interactions are required for gene repression, independent of expression level and chromatin recruitment. Altogether, our work suggests a mechanism whereby m6A regulates the function of HOTAIR via mediating the interaction of YTHDC1 with specific m6A sites, promoting chromatin-mediated repression and breast cancer cell aggressiveness.

N6-methyladenosine (m6A) modification of RNA regulates normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we reveal that m6A regulates the breast cancer-associated human lncRNA HOTAIR. Mapping m6A in breast cancer cell lines, we identify multiple m6A sites on HOTAIR, with 1 single consistently methylated site (A783) that is critical for HOTAIR-driven proliferation and invasion of triple-negative breast cancer (TNBC) cells. Methylated A783 interacts with the m6A “reader” YTHDC1, promoting chromatin association of HOTAIR, proliferation and invasion of TNBC cells, and gene repression. A783U mutant HOTAIR induces a unique antitumor gene expression profile and displays loss-of-function and antimorph behaviors by impairing and, in some cases, causing opposite gene expression changes induced by wild-type (WT) HOTAIR. Our work demonstrates how modification of 1 base in an lncRNA can elicit a distinct gene regulation mechanism and drive cancer-associated phenotypes.