5-methylcytosine promotes mRNA export — NSUN2 as the methyltransferase and ALYREF as an m(5)C reader

5-methylcytosine (m(5)C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m(5)C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m(5)C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m(5)C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m(5)C profiles of mammalian transcriptomes and suggests an essential role for m(5)C modification in mRNA export and post-transcriptional regulation.