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A Potentially Abundant Junctional RNA Motif Stabilized by M6a and Mg2+

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A Potentially Abundant Junctional RNA Motif Stabilized by M6a and Mg2+ ARTICLE DOI: 10.1038/s41467-018-05243-z OPEN A potentially abundant junctional RNA motif stabilized by m6A and Mg2+ Bei Liu1, Dawn K. Merriman2, Seung H. Choi1, Maria A. Schumacher1, Raphael Plangger3, Christoph Kreutz3, Stacy M. Horner 4,5, Kate D. Meyer1 & Hashim M. Al-Hashimi 1,2 N6-Methyladenosine (m6A) is an abundant post-transcriptional RNA modification that influences multiple aspects of gene expression. In addition to recruiting proteins, m6A can 1234567890():,; modulate RNA function by destabilizing base pairing. Here, we show that when neighbored by a 5ʹ bulge, m6A stabilizes m6A–U base pairs, and global RNA structure by ~1 kcal mol−1. The bulge most likely provides the flexibility needed to allow optimal stacking between the methyl group and 3ʹ neighbor through a conformation that is stabilized by Mg2+. A bias toward this motif can help explain the global impact of methylation on RNA structure in transcriptome-wide studies. While m6A embedded in duplex RNA is poorly recognized by the YTH domain reader protein and m6A antibodies, both readily recognize m6A in this newly identified motif. The results uncover potentially abundant and functional m6A motifs that can modulate the epitranscriptomic structure landscape with important implications for the interpretation of transcriptome-wide data. 1 Department of Biochemistry, Duke University School of Medicine, Durham, NC 27710, USA. 2 Department of Chemistry, Duke University, Durham, NC 27710, USA. 3 Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, 6020 Innsbruck, Austria. 4 Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA. 5 Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA. Correspondence and requests for materials should be addressed to H.M.A.-H. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:2761 | DOI: 10.1038/s41467-018-05243-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05243-z 6-methylated adenosine (m6A) is the most abundant transcriptome-wide RNA structure mapping data indicate that internal RNA modification in eukaryotic mRNAs1–3 and m6A destabilizes RNA structure in vitro and in vivo28,35, the N 4,5 6 long non-coding RNAs (lncRNA) and is also found in destabilization is not observed at the m A nucleotide per se, but viral RNAs6–8. The modification is dynamically regulated by rather, at the immediate 5ʹ neighbor which favors single-stranded methyl transferase9–11 and demethylase enzymes12,13 that are conformations in methylated RNA28. The mechanism by which linked to human disease14,15. It frequently occurs at the 3ʹ and 5ʹ m6A destabilizes its 5ʹ-neighbor is currently unknown. untranslated regions of mRNA where it can influence RNA Fewer studies have examined the potential for m6A to stabilize splicing16,17, mRNA nuclear exportation13,18, RNA decay19, and RNA even though there is precedent for this in the literature. translation initiation20,21. The modification is implicated in a Prior studies have shown that when placed at dangling ends growing number of processes including stem cell fate determi- of duplexes28 or in apical loops29,m6A stabilizes RNA by nation, stress response, DNA damage repair, microRNA bio- 0.1–1.2 kcal mol−1 most likely due to favorable stacking interac- genesis, lncRNA-mediated transcription repression, viral tions between the methyl group and adjacent bases. Here, by infection, and in the mechanisms of cancer22–25. attempting to harness the destabilizing effects of m6A in the m6A is thought to exert its biological effects through two design of an RNA secondary structural switch, we discovered a general mechanisms. First, it can help recruit proteins that reg- bulge motif that is stabilized by m6AinaMg2+-dependent ulate mRNA fate by influencing splicing16, export26, decay19,27, manner. This motif is recognized by the YTH domain and m6A and translation initiation efficiency20,27. Many of these m6A antibodies and can potentially help explain the global impact of reader proteins contain YTH domains, which specifically recog- m6A on RNA structure observed in transcriptome-wide studies. nize the methyl group5. Second, m6A can exert biological effects by modulating RNA structure. In particular, studies have shown Results 6 – that m A destabilizes A U base pairing and RNA duplexes by m6A stabilizes a junctional A–U base pair – −1 28,29 . Our initial motiva- 0.5 1.7 kcal mol . This destabilization has been proposed tion was to examine whether the destabilizing effects of m6Aon to occur due to steric contacts between the N6-methyl group and – 28 6 A U base pairing could be harnessed to induce a change in RNA the base (Fig. 1a). Studies have shown that m A can promote secondary structure. We designed an RNA hairpin (B5ʹ, Fig. 1b) RNA melting and thereby enhance binding of single-stranded 4,5 6 6 30–32 fi containing the most common (GGm ACU) m A consensus RNA binding proteins . The modi cation can also disrupt sequence (DRACH, where D denotes A, G, or U; R is A or G; and non-canonical A–G base pairs (bps) required for protein bind- 36–38 39 33 6 H is A, C or U) . This sequence is predicted to fold into two ing . Presence of m A in mRNA also impedes tRNA accom- iso-energetic secondary structures in which the m6A forms an modation within the ribosome and translation-elongation most – – 34 A U bp either at the junction next to a bulged guanine or deeper likely by destabilizing A U bps . Interestingly, while within the upper helix (Fig. 1b). The sequence is also predicted to a b Steric clash ΔG = 0 kcal/mol ΔG = 0 kcal/mol H C anti B5′ D 3 UC UC UC 10 13 10 13 10 13 N N H O U G U G U G 7 CG C G C G 8 8 15 8 15 8 15 56 45 U A U A U A 9 C G C G C G N 6 17 6 17 6 17 4 A 1N H N3 6 U U U U 5 A A 18 A R 3 2 2 1 G G C G C N N GC18 UC 4G 4G C19 10 13 2C G20 U G 2C G20 2C G21 H syn O R G C C G G C G C 5′GC 8U A15 5′G C 5′G C C G N N CH3 O 6 7 A 17 8 G U 56 45 4G C18 N9 4 A 1 N H N3 U 6 2C G20 R 3 2 1 G C N 2 N 5'G C O R ΔG = 0.6 kcal/mol c G5/14 G5 D –m6A e D B5′ G20 6 2+ U8 +m A Mg – + – + G16G2 G4 U17 G13 1 U10 ) –1 U11 0.5 G19/4* d U8 G16 0 G14 B5' G2 G13 (Kcal mol U17 U10 –0.5 U11 37°c –1 G 15 14 13 12 11 10 9 ΔΔ –1.5 1H (p.p.m.) Fig. 1 m6A stabilizes junctional A–U base pairs with 5ʹ bulge. a The methyl group in m6A destabilizes A–U pairing through steric contacts. b Design of an m6A-dependent RNA structural switch. Secondary structures and free energies computed using the MC-Fold web server39. The modified adenine (A6) is highlighted in red. c, d Comparison of 1H NMR spectra of unmodified and m6A6 substituted (c) D and (d)B5ʹ recorded in 15 mM sodium phosphate, 25 mM NaCl, 0.1 mM EDTA and 3 mM Mg2+ at pH 6.4 and 10 °C. e Impact of methylation on the thermal stability of the hairpins with and without 3 mM Mg2+. Shown are the differences in the free energy of melting between methylated and unmodified RNA, i.e., ΔΔG = ΔG(methylated) − ΔG(unmodified). The uncertainty reflects the standard deviation from at least three measurements (see Supplementary Table 1) 2 NATURE COMMUNICATIONS | (2018) 9:2761 | DOI: 10.1038/s41467-018-05243-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05243-z ARTICLE form an alternative conformation in which the m6A is bulged out B5ʹ (Supplementary Fig. 2a), indicate that in the absence of Mg2+, and in which the 5ʹ neighboring guanine forms a non-canonical unmodified B5ʹ folds into many distinct and slowly (on the NMR G–U wobble (Fig. 1b). This alternative conformation is predicted chemical shift timescale) exchanging conformations. The U17 to be destabilized by 0.6 kcal mol−1, which is within the range of imino resonance in methylated B5ʹ is significantly broadened, m6A destabilization of A–U bps in duplex RNA28,29. Thus, we consistent with local melting of the m6A6-U17 bp in the absence reasoned that m6A could destabilize the junctional A–U bp and of Mg2+ (Supplementary Fig. 2a and Fig. 3d). Here, the NOE data promote formation of the alternative conformation containing indicate that the methyl group adopts an anti conformation the G–U mismatch, which can be readily detected by 1D 1H (Supplementary Fig. 1) and that G5 forms a G5-U17 mismatch NMR40. As a control, we also carried out experiments on a (Supplementary Fig. 2a). Thus, the addition of Mg2+ induces a corresponding RNA duplex (denoted D), in which a cytosine conformational switch relative to the structure without Mg2+ by nucleotide was included to pair with the bulged guanine (Fig. 1b). stabilizing the junctional m6A6-U17 bp with a 5ʹ neighboring G5 Experiments were carried out in the presence of 3 mM Mg2+. bulge. The m6A substitution resulted in small perturbations in NMR spectra of the control duplex D (Fig.
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