M6A‐mediated alternative splicing coupled with nonsense‐mediated mRNA decay regulates SAM synthetase homeostasis. (21st June 2021) - Watabe, Eichi
Abstract: Alternative splicing of pre‐mRNAs can regulate gene expression levels by coupling with nonsense‐mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS‐NMD) in an organism, we performed long‐read RNA sequencing of poly(A) + RNAs from an NMD‐deficient mutant strain of Caenorhabditis elegans, and obtained full‐length sequences for mRNA isoforms from 259 high‐confidence AS‐NMD genes. Among them are the S‐adenosyl‐L‐methionine (SAM) synthetase ( sams ) genes sams‐3 and sams‐4 . SAM synthetase activity autoregulates sams gene expression through AS‐NMD in a negative feedback loop. We furthermore find that METT‐10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation in␣vivo, and specifically methylates the invariant AG dinucleotide at the distal 3′ splice site (3′SS) in␣vitro . Direct RNA sequencing coupled with machine learning confirms m 6 A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m 6 A modification at the 3′SS of the sams genes. SYNOPSIS: Alternative splicing (AS) of pre‐mRNAs can regulate gene expression levels by coupling with nonsense‐mediated mRNA decay (NMD). Here, assessment of the organismal substrate repertoire of this process reveals SAM synthetase ( sams ) gene expression regulation via m 6 A modification in C. elegans . Long‐read sequencing of poly(A)+ RNAs from an NMD‐deficient mutant strain reveals 259 high‐confidence AS‐NMD genes, including the SAM synthetase ( sams ) genes. SAMS activity negatively autoregulates AS‐NMD of the sams genes in␣vivo . Direct RNA sequencing coupled with machine learning reveals m 6 A modification of endogenous sams mRNAs at the distal 3′ splice sites. Methyltransferase METT‐10 catalyzes m 6 A modification specifically at the distal 3′ splice site of sams pre‐mRNA in␣vitro . METT‐10 regulates alternative splicing of the sams genes in␣vivo . Abstract : Full‐length sequences of natural NMD isoforms in C. elegans are elucidated by direct RNA sequencing. SAM synthetase homeostasis is regulated via pre‐mRNA splicing of the sams genes through m 6 A modification at the invariant AG of the distal 3′SS.