N ⁶-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions

Abstract

RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBM) ^{1- 3} . However, RBMs can be buried within their local RNA structures ^{4- 7} , thus inhibiting RNA-protein interactions. N ⁶-methyladenosine (m ⁶A), the most abundant and dynamic internal modification in eukaryotic messenger RNA ^{8- 19} , can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs ¹⁵ , but how m ⁶A achieves wide-ranging physiological significance needs further exploration. Here we show that m ⁶A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism “m ⁶A-switch”. We found that m ⁶A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (hnRNP C), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing ^{20- 24} . Combining PAR-CLIP and m ⁶A/MeRIP approaches enabled us to identify 39,060 m ⁶A-switches among hnRNP C binding sites; and global m ⁶A reduction decreased hnRNP C binding at 2,798 high confidence m ⁶A-switches. We determined that these m ⁶A-switch-regulated hnRNP C binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m ⁶A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m ⁶A-dependent RNA structural remodeling, and provide a new direction for investigating RNA-modification-coded cellular biology.