We report here that FTO (fat mass and obesity-associated protein) exhibits efficient oxidative demethylation activity of abundant N6-methyladenosine (m6A) residues in RNA in vitro. FTO knockdown with siRNA led to an increased level of m6A in mRNA, whereas overexpression of FTO resulted in a decreased level of m6A in human cells. We further show that FTO partially colocalizes with nuclear speckles, supporting m6A in nuclear RNA as a physiological substrate of FTO.

SUMMARY N6-methyladenosine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m6A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m6A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m6A modification has fundamental and broad functions in mammalian cells.

RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBM)1-3. However, RBMs can be buried within their local RNA structures4-7, thus inhibiting RNA-protein interactions. N6-methyladenosine (m6A), the most abundant and dynamic internal modification in eukaryotic messenger RNA8-19, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs15, but how m6A achieves wide-ranging physiological significance needs further exploration. Here we show that m6A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism “m6A-switch”. We found that m6A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (hnRNP C), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing20-24. Combining PAR-CLIP and m6A/MeRIP approaches enabled us to identify 39,060 m6A-switches among hnRNP C binding sites; and global m6A reduction decreased hnRNP C binding at 2,798 high confidence m6A-switches. We determined that these m6A-switch-regulated hnRNP C binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m6A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m6A-dependent RNA structural remodeling, and provide a new direction for investigating RNA-modification-coded cellular biology.

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N6-methyladenosine (m6A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N1-methyladenosine (m1A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m1A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m1A in promoting translation of methylated mRNA.

Despite its biological importance, transfer RNA (tRNA) could not be adequately sequenced by standard methods due to abundant post-transcriptional modifications and stable structure, which interfere with cDNA synthesis. We achieve efficient and quantitative tRNA sequencing using engineered demethylases to remove base methylations and a highly processive thermostable group II intron reverse transcriptase to overcome these obstacles (DM-TGIRT-seq). Our method should be applicable to investigations of tRNA in all organisms.