A single N6-methyladenosine site in lncRNA HOTAIR regulates its function in breast cancer cells

Abstract

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.

The m6A landscape of polyadenylated nuclear (PAN) RNA and its related methylome in the context of KSHV replication

“…This is also a crucial parameter for investigating the biological significance of PAN m 6 A. For example, it has been shown that only one out of fourteen m 6 A detected on HOX antisense intergenic lncRNA (HOTAIR), which displayed the most consistent modification, plays a critical role in HOTAIR-mediated cellular proliferation, colony formation, and HOTAIR nuclear retention (22). Also, one out of four m 6 A detected in MALAT1, which shows over 80% modification frequency in two cell lines (101), is attributed to the m 6 A-induced structural change associated with m 6 A reader binding (100).…”

“…EpiNano software was utilized to determine the position of m 6 A (21). The SquiggleKit was used to view current intensities (22) The membranes were washed with 1X PBS with 0.1% Tween 20 (PBST) and blocked with 4% (w/v) non-fat milk in 1X PBST for 1 h at room temperature. The membranes were incubated with a specific primary antibody (Table 2) for 1 h at room temperature, washed thrice with 1X PBST, and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (Table 2) for 45 min at room temperature.…”

Long Non-Coding RNA Epigenetics

“…It is likely that the development of new sequencing techniques, such as nanopore sequencing, will facilitate the search for new modified lncRNAs. Modified lncRNAs are involved in oncogenesis [ 176 , 187 ] and they can therefore be excellent biomarkers of neoplastic processes [ 188 ]. The underlying mechanisms by which lncRNA modifications contribute to gene regulation and whether and how the RNA epigenetics of mRNAs differs from that of lncRNAs, as well as which proteins are involved in both RNAs remain to be elucidated.…”

“…HOX transcript antisense RNA (HOTAIR) lncRNA is a transcript of the antisense strand of the hoxC gene, which is spliced and polyadenylated [175] Within HOTAIR, 14 individual m 6 A sites were identified, with a single site (A783) being consistently methylated. HOTAIR interacts with the nuclear m 6 A reader YTHDC1 at the methylated A783 and at additional sites [176]. Localization in chromatin strongly depends on the m6A modification at site A783 of HOTAIR, while the modification of other m 6 A sites mediates high HOTAIR levels.…”

The non-coding epitranscriptome in cancer

“…Similarly, many m 6 A sites were identified on HOTAIR transcripts [ 3 ]. Importantly, one of the m 6 A sites was shown to regulate HOTAIR recruitment to chromatin and promote breast cancer cells proliferation [ 115 ].…”