N6-methyladenosine (m6A) in mRNA

• Dynamic chemical modifications of DNA and proteins influence their function.
• Cytosine methylation in DNA to form 5-methylcytosine (5mC) affects gene expression
• Protein phosphorylation, initially thought to be limited, is involved in most cellular physiology.
• mRNA was recently found to undergo chemical modifications affecting its function.

N6-methyladenosine (m6A) in mRNA

• In 2012, it was shown that a large fraction of cellular mRNA contains adenosine residues methylated to form N6-methyladenosine (m6A).
• Adenosine methyl-transferases and m6A demethylating enzymes regulate adenosine methylation in mRNA
• mRNA is susceptible to dynamic, reversible chemical modification.
• Adenosine methylation in mRNA represents an additional layer of regulation influencing mRNA function and gene expression.

Physiological Processes Linked to N6-methyladenosine

• N6-methyladenosine is linked to several physiological processes, including obesity, synaptic signaling, cancer, sperm development, stem cell differentiation, circadian periods, yeast meiosis, and plant development.

Obesity

• Mutations in intron 1 of the human fat mass and obesity-associated protein (FTO) are associated with increased body weight.
• Carriers of one or two copies of the mutant allele are, on average, 1.7 kg or 3.5 kg heavier than individuals with normal alleles.
• Fto–/– mice have lower body mass; FTO overexpression increases food intake and obesity.
• Intronic mutations in humans might affect FTO gene expression, but the direct connection between FTO and body mass is unclear.

Synaptic Signaling

• Fto-knockout mice show impaired dopamine release, reduced dopaminergic receptor responses, and altered locomotor responses to cocaine
• Select mRNAs in dopaminergic signaling pathways are hypermethylated in these mice, potentially affecting neurobiological and behavioral phenotypes.
• mRNA hypermethylation effects on transcript stability and protein production are complex.

Cancer

• Intron 1 FTO mutations are risk factors for estrogen receptor-negative breast cancer,
• Mutations in intron 8 of FTO increase melanoma risk, independent of body mass index.
• FTO single nucleotide polymorphisms predispose to cancer.

Sperm Development

• ALKBH5 (α-ketoglutarate-dependent dioxygenase alkB homologue 5) is highly expressed in testes.
• Alkbh5-knockout mice have reduced testicular size, abnormal morphology, impaired spermatogenesis, and increased apoptosis.
• The phenotype in Alkbh5-knockout mice is probably due to altered expression levels of spermatogenesis and p53 apoptotic pathway genes.

Stem Cell Differentiation

• N6-methyladenosine (m6A) is required for the self-renewal capacity of human embryonic stem cells.
• Dynamic changes in m6A levels are seen during stem cell differentiation.

Circadian Periods

• The global methylation inhibitor 3-deazaadenosine (3-DZA) extends the normal circadian period in cultured cells and mice.
• m6A decreases in several clock genes with 3-DZA treatment, correlating with prolonged nuclear retention in clock transcripts Per2 and Arntl.
• Similar results were observed after methyltransferase-like 3 (Mettl3) knockdown.
• Adenosine methylation may influence the cyclic expression of mRNAs that encode clock genes.

Yeast Meiosis

• mRNA methylation in Saccharomyces cerevisiae occurs selectively during meiosis.
• Methylation is mediated by the core RNA methyltransferase complex (MIS), which includes Ime4, Mum2, and Slz1.
• The MIS complex localizes to nucleoli during meiosis and is required for the proper time course of meiosis.

Plant Development

• m6A contributes to plant embryonic development.
• m6A is required for normal growth patterns, apical dominance, and plant development.

Drosophila melanogaster oogenesis

• m6A is required for Notch signaling during oogenesis.
• Phenotypes in D. melanogaster cells deficient in the adenosine methyltransferase Ime4 support this.

Techniques for Detecting N6-methyladenosine

• N6-methyladenosine immunoblotting
• m6A-sensitive ligase reaction
• m6A-sensitive reverse transcription
• SCARLET (site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography)
• PacBio sequencing
• Nanopore sequencing

MeRIP-Seq Profiling of N6-methyladenosine

• Methylated RNA immunoprecipitation followed by sequencing enables transcriptome-wide profiling of N6-methyladenosine (m6A).
• RNA is fragmented into ~100 nucleotide fragments
• m6A-containing RNAs are immunoprecipitated with m6A-specific antibodies for selective enrichment.
• After sequencing, reads are mapped to the genome, and m6A peaks are identified
• A pre-immunoprecipitation RNA-Seq data set is incorporated during peak calling.
• MeRIP-Seq experiments carried out with two different m6A-specific antibodies ensures 'true' m6A peaks

Enzymes That Mediate m6A Methylation

• Enzymes catalyzing adenosine methylation are important for delineating mRNA methylation regulation.

The METTL3-METTL14 Complex

• A large (>1 MDa) multiprotein complex mediates adenosine methylation.
• Nuclear preparations methylate RNA in a SAM-dependent manner in vitro.
• mRNAs containing GAC were more efficiently methylated than mRNAs containing AAC.
• The complex did not induce formation of N,N-dimethyladenosine.
• The catalytic component, methyltransferase-like 3 (METTL3), crosslinks to [3H]–SAM and exhibits specificity towards GAC and AAC sequences.
• METTL3 has a classic SAM-binding methyltransferase domain.
• Homologues exist in plants (MTA), Saccharomyces cerevisiae (Ime4), and Drosophila melanogaster (Ime4).
• METTL14 is highly similar to METTL3 being a predicted methyltransferase and it selectively methylates GAC sequences in vitro.
• Methyltransferase activities of METTL3 and METTL14 are synergistically increased.
• Knockdown of either Mettl3 or Mettl14 reduces m6A peaks in most, confirming that METTL3 and METTL14 physiologically target mRNAs for methylation.

Wilms Tumour 1-Associated Protein (WTAP)

• WTAP associates with METTL3–METTL14 and is required for efficient mRNA methylation.
• WTAP binds to Wilms tumour protein (WT1), but WT1 is not involved in mRNA methylation.
• WTAP interacts with numerous cellular proteins.
• WTAP, lacking methyltransferase activity, binds to the METTL3–METTL14 complex and induces its localization to nuclear speckles to facilitate mRNA methylation.

N6-Methyladenosine Methylation and Demethylation Pathways

• Methyltransferase-like 3 (METTL3) and METTL14 synergize to methylate adenosines in RNA.
• Wilms tumour 1-associated protein is an additional component of this complex which lacks methyltransferase activity but influences m6A formation.
• FTO and ALKBH5 are the two mammalian m6A mRNA demethylases that have been identified.

Location

• Owing to the readily detectable levels of METTL3, METTL14 and WTAP in nuclear speckles, this is likely to be an important site for methylation.
• WTAP knockdown prevents METTL3 localization to nuclear speckles and reduces cellular m6A levels.
• The localization of METTL3 to nuclear speckles suggests that this protein may methylate speckle-associated RNAs, such as small nuclear RNAs (snRNAs) and pre-mRNA, and functions during splicing reactions.
• Additional support for nuclear mRNA methylation comes from PAR-CLIP (photoactivatable- ribonucleoside-enhanced crosslinking and IP) analysis of METTL3-binding sites in the transcriptome, which revealed an exceptionally high number of binding sites in intronic mRNA sequences.

Where Methylation Occurs

• Early studies also detected methyltransferase activity in cytosolic extracts, which supports the idea that at least some methylation could occur in the cytoplasm. Cytoplasmic methylation would imply a role for m6A in the control of mRNA fates that do not affect splicing or nuclear export.
• Furthermore, the localization of METTL3 that was observed using different antibodies showed a granular cytoplasmic or perinuclear labelling
• Ribosome function is predominantly cytosolic.
• Metagene analysis of methylated RNA immunoprecipitation data shows that N6-methyladenosine (m6A) is enriched in discrete regions of mRNA transcripts.
• m6A residues can be found at the first encoded residue, often as a N6,2'-O-dimethyladenosine (m6Am) and are also found at each of the other indicated positions in mRNAs and is particularly enriched near the stop codon in mRNAs. UTR, untranslatedregions.
• Localization in the transcriptome shows that m6A residues are highly enriched around stop codons.
• Knockdown of either Mettl3 or Mettl14 results in reduced m6A peaks in mouse embryonic stem cells.