N6-Methyladenosine Modification in a Long Noncoding RNA Hairpin Predisposes Its Conformation to Protein Binding

Abstract

N6-methyladenosine (m6A) is a reversible and abundant internal modification of messenger RNA (mRNA) and long noncoding RNA (lncRNA) with roles in RNA processing, transport, and stability. Although m6A does not preclude Watson-Crick base pairing, the N6-methyl group alters the stability of RNA secondary structure. Since changes in RNA structure can affect diverse cellular processes, the influence of m6A on mRNA and lncRNA structure has the potential to be an important mechanism for m6A function in the cell. Indeed, an m6A site in the lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was recently shown to induce a local change in structure that increases the accessibility of a U5-tract for recognition and binding by heterogeneous nuclear ribonucleoprotein C (HNRNPC). This m6A-dependent regulation of protein binding through a change in RNA structure, termed ‘m6A-switch,’ affects transcriptome-wide mRNA abundance and alternative splicing. To further characterize this first example of an m6A-switch in a cellular RNA, we used nuclear magnetic resonance (NMR) and Förster resonance energy transfer (FRET) to demonstrate the effect of m6A on a 32-nucleotide RNA hairpin derived from the m6A-switch in MALAT1. The observed imino proton NMR resonances and FRET efficiencies suggest that m6A selectively destabilizes the portion of the hairpin-stem where the U5-tract is located, increasing the solvent accessibility of the neighboring bases while maintaining the overall hairpin structure. The m6A-modified hairpin has a predisposed conformation that resembles the hairpin conformation in the RNA–HNRNPC complex more closely than the unmodified hairpin. The m6A-induced structural changes in the MALAT1 hairpin can serve as a model for a large family of m6A-switches that mediate the influence of m6A on cellular processes.

TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code

“…In the context of tRNA and rRNA, studies have mostly focused on the impact of Ψ on RNA structure, where it is thought to contribute to structural stability through the potential formation of an extra hydrogen bond Davis 1997, 1999;Kierzek et al 2014). Such a structural role, if present on mRNA, could modulate the ability of RNA binding proteins to bind to the mRNA, perhaps in an analogous manner to the role recently reported for N 6 -methyladenosine Zhou et al 2015) and, in this manner, impact its localization, stability, or translation. In yeast, we previously observed that steady state levels of mRNAs pseudouridylated via Pus7 were ∼25% decreased following knockout of PUS7, suggestive of a potential role for pseudouridine in stabilizing messages (Schwartz et al 2014).…”

Non-coding RNAs: long non-coding RNAs and microRNAs in endocrine-related cancers

“…Additional protein binding partners of SRA1 were recently reviewed (Liu, et al 2016). SRA1 interacts with DICER (Redfern, et al 2013).…”

“…SRA1 interacts with DICER (Redfern, et al 2013). By its interaction with SLIRP (SRA stem-loop interacting RNA binding protein), SRA1 has a repressive function by recruiting corepressors (reviewed in (Liu et al 2016)). SLIRP represses NR transactivation in an SRA1-dependent manner (Hatchell, et al 2006).…”

Structural Basis for Cooperative Function of Mettl3 and Mettl14 Methyltransferases

“…For most mRNAs, m 6 A modification is most frequently observed in long exons, near stop codons, and in 3′ UTRs (Dominissini et al, 2012; Meyer et al, 2012). Addition of m 6 A can alter mRNA stability, induce RNA conformational changes, modulate protein-RNA interactions, and even modify microRNA processing (Alarcón et al, 2015; Liu et al, 2015b; Wang et al, 2014a; Wang et al, 2015; Zhou et al, 2016). Several studies investigating the sequence specificity of m 6 A modification discovered RRACH (R represents A or G, and H represents A, C or U) as the consensus sequence, with the central A as the methyl acceptor (Csepany et al, 1990; Dominissini et al, 2012; Harper et al, 1990; Meyer et al, 2012; Rottman et al, 1994; Schibler et al, 1977).…”