N6-methyl-adenosine modification in messenger and long non-coding RNA

Abstract

N6-methyl-adenosine (m(6)A) is the most abundant modification in mammalian mRNA and long non-coding RNA. First discovered in the 1970s, m(6)A modification has been proposed to function in mRNA splicing, export, stability, and immune tolerance. Interest and excitement in m(6)A modification has recently been revived based on the discovery of a mammalian enzyme that removes m(6)A and the application of deep sequencing to localize modification sites. The m(6)A demethylase fat mass and obesity associated protein (FTO) controls cellular energy homeostasis and is the first enzyme discovered that reverses an RNA modification. m(6)A Sequencing demonstrates cell-type- and cell-state-dependent m(6)A patterns, indicating that m(6)A modifications are highly regulated. This review describes the current knowledge of mammalian m(6)A modifications and future perspectives on how to push the field forward.

Fig. 1. mRNA and lncRNA modifications

The functional groups introduced through modifications are shown in red. (A) The three abundant modifications are m⁶A, 5′ cap and m⁵C. The 5′ cap contains both modifications at the 5′ triphosphate and 2′O-methylation of the first and sometimes the second nucleotide. The estimated number of modifications per type, per RNA molecule, is shown in parentheses ^{, ,} . Some known human enzymes that catalyze the forward and reverse reactions are also shown ^{, , ,} . MTA70: METTL3 methyltransferase like 3; FTO: fat mass and obesity associated; AlkBH5: alkB alkylation repair homolog 5; NSUN2: NOP2/Sun RNA methyltransferase family, member 2. (B) Pseudouridine modification (Ψ) may also be present, although the extent of Ψ is currently unknown. (C) RNA editing commonly refers to deamination of A to inosine and C to uridine at specific sites in mRNA.

Fig. 2. m⁶A sequencing method and results ^,

Starting from total polyadenylated RNA, step 1 is to chemically fragment RNA to ~50–100 nucleotides. This sample is split in two; one is used as input control, and the other immunoprecipitated with anti-m⁶A antibody to isolate RNA fragments containing m⁶A modification. Both samples are deep sequenced (step 2), and the sequencing results compared to identify m⁶A-containing RNA segments (step 3). The m⁶A modification is markedly enriched around the stop codon of mRNA, although the reason for this enrichment is unclear.

Fig. 3. Functional effects of m⁶A modification

(A) m⁶A modification could alter RNA structure either through weakening of base pairing or through the loss of one of the two hydrogen bond donors at the N⁶-position of A. This H-bond donor could be involved in RNA tertiary structure. (B) m⁶A modification could generate a new binding site for proteins that recognize m⁶A modified RNA. The m⁶A binding proteins can recruit additional complexes involved in cellular processes such as mRNA splicing, export, stability and immune tolerance.