RESUMEN
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 inâ£vivo, and specifically methylates the invariant AG dinucleotide at the distal 3' splice site (3'SS) inâ£vitro. Direct RNA sequencing coupled with machine learning confirms m6 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 m6 A modification at the 3'SS of the sams genes.
Asunto(s)
Empalme Alternativo/genética , Homeostasis/genética , Ligasas/genética , Metionina Adenosiltransferasa/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , ARN Mensajero/genética , S-Adenosilmetionina/metabolismo , Animales , Caenorhabditis elegans/genética , Metiltransferasas/genética , Precursores del ARN/genéticaRESUMEN
RBM20 is a major regulator of heart-specific alternative pre-mRNA splicing of TTN encoding a giant sarcomeric protein titin. Mutation in RBM20 is linked to autosomal-dominant familial dilated cardiomyopathy (DCM), yet most of the RBM20 missense mutations in familial and sporadic cases were mapped to an RSRSP stretch in an arginine/serine-rich region of which function remains unknown. In the present study, we identified an R634W missense mutation within the stretch and a G1031X nonsense mutation in cohorts of DCM patients. We demonstrate that the two serine residues in the RSRSP stretch are constitutively phosphorylated and mutations in the stretch disturb nuclear localization of RBM20. Rbm20 S637A knock-in mouse mimicking an S635A mutation reported in a familial case showed a remarkable effect on titin isoform expression like in a patient carrying the mutation. These results revealed the function of the RSRSP stretch as a critical part of a nuclear localization signal and offer the Rbm20 S637A mouse as a good model for in vivo study.
