N6-methyladenosine-dependent RNA structural switches regulate RNA–protein interactions

Abstract

The binding motifs for many RNA-binding proteins are normally buried within structured regions; now, the N6-methyladenosine modification is shown to act as a switch to remodel these regions, expose the motif, and thereby facilitate binding of RNA-binding proteins. N6-methyladenosine (m6A) is a common internal modification found in many eukaryotic mRNAs and long non-coding RNAs. It is involved in the control of various cellular functions including circadian rhythm, meiosis and stem cell development. This study uncovers a previously unknown mechanism by which m6A regulates RNA–protein interactions. Tao Pan and colleagues show that the binding motifs for many RNA binding proteins (RBPs) are normally buried within structured regions. The m6A modification acts as a switch to reorganize these regions, inducing structural changes that expose the motif and thereby facilitate RBP binding. This finding implies that m6A functions as an RNA structure remodeller, for example, to affect mRNA maturation through interference with post-transcriptional regulator binding activities. RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs)1,2,3. However, RBMs can be buried within their local RNA structures4,5,6,7, thus inhibiting RNA–protein interactions. N6-methyladenosine (m6A), the most abundant and dynamic internal modification in eukaryotic messenger RNA8,9,10,11,12,13,14,15,16,17,18,19, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs15, but how m6A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m6A controls the RNA-structure-dependent accessibility of RBMs to affect RNA–protein interactions for biological regulation; we term this mechanism ‘the m6A-switch’. We found that m6A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing20,21,22,23,24. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m6A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m6A-switches among HNRNPC-binding sites; and global m6A reduction decreased HNRNPC binding at 2,798 high-confidence m6A-switches. We determined that these m6A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m6A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m6A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology.