CG14906 (mettl4) mediates m6A methylation of U2 snRNA in Drosophila

Abstract

While the eukaryotic candidate m 6 A methyltransferases belong to multiple distinct methylase lineages, the most widespread group belongs to the MT-A70 family exemplified by the yeast messenger RNA (mRNA) adenine methylase complex Ime4/Kar4. At the structural level, all of these enzymes are characterized by a 7-β-strand methyltransferase domain at their C terminus, fused to a predicted α-helical domain at their N terminus and require S -adenosyl- l -methionine (SAM) as a methyl donor. The catalytic motif, [DSH]PP[YFW], present in many members of this family, has shown to be critical for METTL3/METTL4-mediated mRNA m 6 A methylation 1 . The high degree of amino acid sequence conservation among the predicted N6-methyladenosine methyltransferases motivates further explorations into their potential functional conservation. METTL4 is a member of the MT-A70-like protein family, which is conserved during evolution (Fig. 1a ) 2 . Previous studies suggested that METTL4 regulates DNA 6mA in vivo and therefore is a candidate DNA 6mA methyltransferase 3 , 4 , 5 . However, the enzymatic activity of METTL4 in vitro has not been demonstrated. Fig. 1: CG14906 (mettl4) methylates U2 snRNA in Drosophila melanogaster . The alternative text for this image may have been generated using AI. Full size image a Cladogram of mettl4 in model organisms based on their sequence similarity, the pink rectangle indicates the MT-A70 domain. b Enrichment analysis of eCLIP-seq data for different RNA types. tRNA and snRNA are enriched among all RNA types in general and snRNA is the top enriched RNA molecules targeted by mettl4 in vivo. c Enrichment analysis of eCLIP-seq data for subgroups of snRNA. U2, U4, and U6atac are the top enriched subgroups. d In vitro enzymatic activity is measured by LS-MS/MS using substrates, including U2, U4, U6atac, and U2, with different point mutations and deletions, and tRNA and DNA with U2 sequences. Results show that U2 is the best substrate for fly mettl4 and that adenosine at position 29 in U2 is methylated by mettl4. e Michaelis–Menten kinetics of recombinant mettl4 was determined using U2 probes as substrate by LC-MS/MS analysis. f U2 m 6 A analysis in WT, KO, and rescued (wt: wild-type mettl4 ; mut: catalytic dead mutant mettl4 , DPPW→NPPW) cells by LC-MS/MS. g In vivo U2 m 6 A analysis by LC-MS/MS of WT and KO flies. Error bars indicate mean ± SD ( n = 3). h Genes with differential alternative splicing were used for the GO analysis. The top 20 enriched biological processes are shown in the bar plot. i Growth curves of WT and mettl4 KO cells in a course of 5 days. Error bars indicate mean ± SD ( n = 7). Permutation test was used to determine the significant level of the difference between two groups of growth curves. Statistical significance is determined as: n.s., P > 0.05; * P < 0.05; *** P < 0.001; **** P < 0.0001.

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