METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing

Abstract

N 6-methylation of 2′-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2′-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4’s in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as 3′ splice-site weakness and an increase in exon inclusion. These findings suggest that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.

Spliceosomal snRNA Epitranscriptomics

“…Additionally, the level of base methylation could also be severely reduced when changing the 5' and 3' nucleotides, pointing toward sequence recognition by the METTL4. In an independent study, Goh et al (2020) confirmed METTL4 as the enzyme responsible for the m 6 Am30 modification (with Am being the true substrate). The authors also confirmed the base modification identity with HPLC-MS/MS ( Goh et al, 2020 ).…”

“…In an independent study, Goh et al (2020) confirmed METTL4 as the enzyme responsible for the m 6 Am30 modification (with Am being the true substrate). The authors also confirmed the base modification identity with HPLC-MS/MS ( Goh et al, 2020 ). Using transcriptome-wide sequencing, they further showed that this modified nucleotide contributed to splicing regulation.…”

“…Since m 6 Am30 is located almost immediately upstream of the branch site recognition sequence, it has recently drawn some attention. Chen et al (2020) and Goh et al (2020) have independently identified METTL4 as the methyltransferase responsible for the formation of m 6 Am of mammalian U2 snRNA at position 30. In their study, Chen et al generated knocked-out METTL4 human cells and observed an effect on splicing in those cells when compared to wild-type cells.…”

Methyladenosine Modification in RNAs: Classification and Roles in Gastrointestinal Cancers

“…The m 6 Am MTase was previously purified with a molecular weight of~65 KD in 1978, and phosphorylated CTD-interacting factor 1 (PCIF1) was recognized as the first m 6 Am MTase in 2019. It was named by its ability to directly bind to the phosphorylated carboxylterminal domain (CTD) of RNA polymerase II (RNAP II) by its WW domain, also called cap-specific adenosine methyltransferase (CAPAM) (106,112,113). Unlike the m 6 A core readers that work in the form of a methyltransferase complex, PCIF1 is a "stand-alone" RNA MTase and functions in an m 7 G cap-dependent manner.…”

Regulatory Role of N6-methyladenosine (m6A) Modification in Osteosarcoma

“…N6methyladenosine (m 6 A) modification refers to the addition or deletion of the methyl group to/from the nitrogen on the 6th carbon of the adenine nucleotide, which is one of the most abundant epigenetic modifications found in RNA molecules (13). First discovered by Desrosiers in the 1970s, m 6 A modification has been found to be involved in almost all steps of RNA processing and metabolism, including pre-mRNA splicing, export, translation, stabilization, and degradation (14)(15)(16)(17)(18)(19). With the burgeoning advances in molecular biology and sequencing, m 6 A modification has been reported to be implicated in virtually all cellular functions and multiple diseases (including osteosarcoma) (20)(21)(22)(23).…”