A new paper in Cell provides a transcriptome-wide survey of the methylation of adenosine residues in RNAs. Meyer et al find that this epitranscriptomic post-transcriptional modification is widespread and dynamically regulated, and likely to play important roles in cellular regulation.
Methylation of the N6 position of adenosine residues (m6A) has been known to be a post-transcriptional modification of RNAs for many years. Research in the 1960’s and 70’s demonstrated that m6A is present in tRNAs, rRNAs and viral RNAs, and made up between 0.1% and 0.4% or total adenosines in cellular RNA. However as m6A was not easily detectable by commonly available methods, research on this modified base foundered. A recent spur to experimentation on m6A has come from the analysis of a gene linked to obesity. FTO (fat mass and obesity associated) is a major regulator of metabolism and energy utilisation. It appears that the major catalytic function of FTO is the demethylation of N6-methyladensosine (m6A), suggesting that m6A has important physiological roles in humans and other mammals.
As m6A is not detectable by sequencing or hybridisation based techniques, nor susceptible to chemical modification, Meyer et al. based their experiments on the use of an anti-m6A antibody (ά-m6A). They first showed that m6A was present in RNA from a wide selection of different mouse tissues and cell lines. It was especially enriched in liver, kidney, and brain, and showed a dramatic increase in adult neural tissue as opposed to embryonic. m6A was found to be present in RNAs of all sizes, and was enriched in the polyadenylated fraction (ie. mRNAs), but not present in the poly(A) tails themselves.
To look in more detail at the distribution of m6A throughout the transcriptome, Meyer et al. developed a high throughput technique called MeRIP-Seq. Cellular RNA is fragmented into ~100nt fragments, and then m6A containing fragments are immunoprecipitated using ά-m6A. The RNA fragments are then deep sequenced. m6A residues should be detected on multiple RNA fragment sequence reads, allowing the detection of m6A peaks, that can be assigned to their approximate position on RNA molecules. Using adult mouse brain RNA in multiple MeRIP-Seq experiments, Meyer et al. identified 41, 072 distinct peaks in the RNAs of 8,843 genes. However they used a smaller, highly reproducible, subset of 13, 471 peaks in 4, 654 genes for their further analyses.
94.5% of the m6A peaks occurred in mRNAs, but more than 3% were found within long non-coding RNAs, showing that ncRNAs are also targets for adenosine methylation. mRNAs from a wide variety of genes were found to contain methylated adenosines, including many involved in cellular regulation, and genes linked to neurodevelopmental and neurological disorders.
The largest proportion of m6A containing mRNAs exhibited a single m6A peak (46%) (equating to either a single m6A residue or a cluster of adjacent m6As), whilst 48.5% contained two or three peaks. However, mRNAs can contain more than 15 peaks along their lengths. Although MeRIP-Seq doesn’t allow one to say exactly which adenosines are methylated, it does give one a good idea of their positions on RNAs. m6A levels are low in the 5’ ends of mRNAs. They increase steadily throughout the coding sequence, peak in the vicinity of the stop codon, remain high in the first portion of the 3’ UTR and then rapidly decline. This linkage between the region of the translational stop codon and m6A is the most important finding of the paper.
Meyer et al. went on to show that regions of m6A occurrence are more likely to be conserved in vertebrates. They also found a correlation between m6A in 3’UTRs and the presence of microRNA binding sites.
Adenosine methylation has therefore been shown to be a widespread and dynamically regulated post-transcriptional modification of mRNAs and lncRNAs in mammals. Its functional significance however, is still difficult to gauge. So far, the pathways responsible for adenosine methylation of RNAs are not characterised. It is also unclear as to whether FTO is the primary enzyme responsible for adenosine demethylation. FTO knockout mice survive, but display postnatal growth retardation and decreased locomotor activity. The linkages between m6A, stop codons and miRNA binding await mechanistic study, but are suggestive of important regulatory roles for RNA methylation. With MerIP-Seq, Meyer et al. have invented a useful technique for the analysis of this important modification.
Meyer, K., Saletore, Y., Zumbo, P., Elemento, O., Mason, C., & Jaffrey, S. (2012). Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and near Stop Codons Cell DOI: 10.1016/j.cell.2012.05.003
A follow-up to this post on 5-methylcytosine in RNAs: Patterns of RNA methylation 2