N6-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 structures4-7, thus inhibiting RNA-protein interactions. N6-methyladenosine (m6A), the most abundant and dynamic internal modification in eukaryotic messenger RNA8-19, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs15, but how m6A achieves wide-ranging physiological significance needs further exploration. Here we show that m6A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism “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 (hnRNP C), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing20-24. Combining PAR-CLIP and m6A/MeRIP approaches enabled us to identify 39,060 m6A-switches among hnRNP C binding sites; and global m6A reduction decreased hnRNP C binding at 2,798 high confidence m6A-switches. We determined that these m6A-switch-regulated hnRNP C 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 remodeling, and provide a new direction for investigating RNA-modification-coded cellular biology.

Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

“…The identity of any of the other proteins that act together with METTL3 remained unknown until it was shown in Arabidopsis that FIP37 (FKBP12 INTERACTING PROTEIN 37) was a partner protein of MTA (the homologue of METTL3) (Zhong et al ., 2008; Shen et al ., 2016). Following this initial discovery, the homologues of FIP37, S. cerevisiae MUM2 (Muddled Meiosis 2), and mammalian WTAP (Wilms tumour 1 associated protein), were shown to interact with METTL3 (MTA) orthologues and to be required for mRNA methylation in their respective model organisms (Agarwala et al ., 2012; Ping et al ., 2014; Wang et al ., 2014b; Liu et al ., 2015). More recently, another human methylase, METTL14, phylogenetically related to METTL3 (Bujnicki et al ., 2002), was shown to form a complex with METTL3 and WTAP, and to be required also for m 6 A formation (Ping et al ., 2014; Wang et al ., 2014b; Liu et al ., 2015).…”

Specific RNP capture with antisense LNA/DNA mixmers

“…HNRNPC may thus interact with the pre-rRNA before cleavage of the internal transcribed spacer 1 (ITS1) that separates 18S and 28S rRNA. A recent study revealed that RNA binding of HNRNPC is enhanced upon addition of m 6 A modifications (Liu et al 2015), and it is possible that HNRNPC interacts with rRNA in an m 6 A-dependent manner. Furthermore, HNRNPD was identified in the same study as being involved in large ribosomal subunit maturation (Henras et al 2015).…”

Nuclear TARBP2 drives oncogenic dysregulation of RNA splicing and decay

“…Based on this model, it is plausible that pre-mRNA m 6 A marks may modulate local binding of regulators of the splicing machinery. To address this possibility, we compiled a list of RNA-binding proteins that differentially bind methylated RNA (Alarcón et al, 2015b;Edupuganti et al, 2017;Liu et al, 2015). We then systematically analyzed the distribution of binding sites of each RBP (derived from CLIP-seq data and known RBP binding motifs, 33 RBPs were included in this analysis) to determine if each candidate RBP binds to introns (and their flanking exons) that are also bound by TARBP2 and/or contain m6A marks.…”

“…The prevalent internal RNA modification mark N(6)-methyladenosine (m 6 A) has been reported to play a role in regulating most facets of the RNA life cycle, including regulation of pre-mRNA splicing, mRNA stability, and mRNA translation (Lin et al, 2016;Liu et al, 2015;Wang et al, 2015Wang et al, , 2014Xiao et al, 2016;Zhao et al, 2014). Recently, work by us (Alarcón et al, 2015) and others (Ke et al, 2017;Knuckles et al, 2017) has established that m 6 A marks are deposited in the nucleus and are proposed to function in many nuclear regulatory processes, including microRNA and messenger RNA processing.…”