Thermodynamic insights into N6-methyladenosine-modified ribonucleic acids and their interactions with the RNA recognition motif of heterogeneous nuclear ribonucleoprotein C.

N6-methyladenosine (m6A) is a prevalent RNA modification that regulates key functions such as splicing, transport, translation, and stability across various RNA types, including mRNA, tRNA, rRNA, and lncRNA. Transcriptome-wide studies reveal that approximately one-third of mammalian mRNAs carry 3-5 m6A modifications, enriched in the consensus motif RRA*CH. While some studies suggest m6A induces structural changes in RNA to facilitate protein binding through an "m6A switch" mechanism, others propose it primarily primes RNA for enhanced protein interactions, emphasizing the need for further exploration of m6A's role. Here, we investigated how m6A influences the binding of heterogeneous nuclear ribonucleoprotein C (hnRNPC), which recognizes poly (U) tracts via its RNA recognition motif (RRM). Using naturally occurring m6A-modified RNAs, including lncRNA MALAT1, we examined the effects of m6A on RNA folding and protein binding. Biophysical experiments (UV melting, circular dichroism, and molecular dynamics simulations) revealed that m6A subtly alters RNA stability and folding. Binding studies using EMSA, Microscale Thermophoresis (MST), and Isothermal Titration Calorimetry (ITC) showed m6A primes RNA for hnRNPC recognition rather than inducing structural switches. These findings refine our understanding of m6A's role in RNA-protein interactions, highlighting its regulatory importance in RNA metabolism.