N6-Methyladenosine Modification in a Long Noncoding RNA Hairpin Predisposes Its Conformation to Protein Binding

Abstract

N6-Methyladenosine (m6A) is a reversible and abundant internal modification of messenger RNA (mRNA) and long noncoding RNA (lncRNA) with roles in RNA processing, transport, and stability. Although m6A does not preclude Watson–Crick base pairing, the N6-methyl group alters the stability of RNA secondary structure. Since changes in RNA structure can affect diverse cellular processes, the influence of m6A on mRNA and lncRNA structure has the potential to be an important mechanism for m6A function in the cell. Indeed, an m6A site in the lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was recently shown to induce a local change in structure that increases the accessibility of a U5-tract for recognition and binding by heterogeneous nuclear ribonucleoprotein C (HNRNPC). This m6A-dependent regulation of protein binding through a change in RNA structure, termed “m6A-switch”, affects transcriptome-wide mRNA abundance and alternative splicing. To further characterize this first example of an m6A-switch in a cellular RNA, we used nuclear magnetic resonance and Förster resonance energy transfer to demonstrate the effect of m6A on a 32-nucleotide RNA hairpin derived from the m6A-switch in MALAT1. The observed imino proton nuclear magnetic resonance resonances and Förster resonance energy transfer efficiencies suggest that m6A selectively destabilizes the portion of the hairpin stem where the U5-tract is located, increasing the solvent accessibility of the neighboring bases while maintaining the overall hairpin structure. The m6A-modified hairpin has a predisposed conformation that resembles the hairpin conformation in the RNA–HNRNPC complex more closely than the unmodified hairpin. The m6A-induced structural changes in the MALAT1 hairpin can serve as a model for a large family of m6A-switches that mediate the influence of m6A on cellular processes.