Abstract
N6-methyladenosine (m6A) is the most abundant base modification found in messenger RNAs (mRNAs). The discovery of FTO as the first m6A mRNA demethylase established the concept of reversible RNA modification. Here, we present a comprehensive transcriptome-wide analysis of RNA demethylation and uncover FTO as a potent regulator of nuclear mRNA processing events such as alternative splicing and 3΄ end mRNA processing. We show that FTO binds preferentially to pre-mRNAs in intronic regions, in the proximity of alternatively spliced (AS) exons and poly(A) sites. FTO knockout (KO) results in substantial changes in pre-mRNA splicing with prevalence of exon skipping events. The alternative splicing effects of FTO KO anti-correlate with METTL3 knockdown suggesting the involvement of m6A. Besides, deletion of intronic region that contains m6A-linked DRACH motifs partially rescues the FTO KO phenotype in a reporter system. All together, we demonstrate that the splicing effects of FTO are dependent on the catalytic activity in vivo and are mediated by m6A. Our results reveal for the first time the dynamic connection between FTO RNA binding and demethylation activity that influences several mRNA processing events.
Highlights
Reversible N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) [1,2,3,4,5,6,7,8]
The distribution of CLIP reads in RNA classes was reproducible and most of the reads were mapped to mRNAs (42%), followed by ribosomal RNAs (26%), and small nuclear RNAs (4%) (Figure 1A, Supplementary Figure S1C)
Despite the increasing number of m6A studies, it is still unclear why adenosines are preferentially modified at specific locations within transcripts, or what are the functional consequences of such patterns of methylation
Summary
Reversible N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic mRNA [1,2,3,4,5,6,7,8]. The most striking feature that emerged from these studies was the widespread presence of m6A in more than half of all mammalian mRNAs and few hundred non-coding RNAs (ncRNAs) [1,2,17]. The RNA modification by m6A affects multiple aspects of mRNA metabolism, such as alternative splicing (AS) The mRNA m6A writer is represented by a multi-subunit methyltransferase complex consisting of the core MMW complex that is formed by the methyltransferase-like 3 and 14 (METTL3 and METTL14), Wilms’ tumor 1-
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