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To obtain a transcriptome-wide landscape of m 5C profiles, we performed RNA-BisSeq analysis on RNA samples purified from human HeLa cells. Our data provide a comprehensive description of m 5C methylomes in mammalian transcriptomes and further illustrate an essential role of m 5C in regulating mRNA export. We further identified that NSUN2 is the main enzyme catalyzing m 5C formation, while Aly/REF export factor (ALYREF, an mRNA transport adaptor, also named THOC4) functions as a specific mRNA m 5C-binding protein regulating mRNA export. The distributive analysis showed that m 5C sites were mainly enriched in the CG context and also in regions immediately downstream of translation initiation sites of mRNAs, and displayed conserved, tissue-specific and dynamic features in mammalian transcriptomes. In this study, we mapped transcriptome-wide m 5C profiles in human HeLa cells and multiple mouse tissues using RNA-BisSeq. Therefore, it is crucial to clarify how m 5C modifications are distributed on mRNAs in different cell types, tissues, and organisms, and to identify and characterize the roles of the protein factors responsible for adding/removing/reading these modifications in order to gain better understanding of the potential significance of m 5C modification for RNA metabolism. However, this feature was not detected in Arabidopsis 28.

One recent study observed that m 5C sites preferentially occur in the vicinity of the translational start codon in mouse embryonic stem (ES) cells and brain 27. However, the knowledge about the prevalence and transcriptome-wide distribution of m 5C in different species and tissues is still very limited. Based on available published data, a clear view of m 5C modification in non-coding RNAs (ncRNAs) has been established, and many m 5C sites have also been identified in mRNAs 23, 25, 26, 27, 28. These include RNA bisulfite sequencing (RNA-BisSeq) 22, 23, m 5C-RNA immunoprecipitation (RIP) 24, 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP) 25 and methylation-individual-nucleotide-resolution crosslinking and immunoprecipitation (miCLIP) 26. Subsequently, many known or novel m 5C sites have been validated by using advanced high-throughput techniques combined with next-generation sequencing (NGS). All these lines of emerging evidence point to the logical speculation that reversible RNA modifications may serve as novel epigenetic markers with profound biological significance in RNA metabolism.Īnother important RNA modification, 5-methylcytosine (m 5C), was first identified in stable and highly abundant tRNAs and rRNAs 20, 21, 22. More recently, another two RNA modifications, pseudouridine (ψ) 15, 16, 17 and N1-methyladenosine (m 1A) 18, 19, have also been shown to play a vital role in posttranscriptional gene regulation. Since then, accumulating lines of evidence have pointed to a broad effect of m 6A on mRNA metabolism 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. The identification of fat mass and obesity-associated (FTO) as the first discovered RNA m 6A demethylase 1 established the reversible nature of m 6A modification. Our study provides comprehensive m 5C profiles of mammalian transcriptomes and suggests an essential role for m 5C modification in mRNA export and post-transcriptional regulation. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Moreover, m 5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m 5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. Here, we reveal that m 5C 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. However, its regulatory role in mRNA metabolism is still largely unknown.

5-methylcytosine (m 5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs.

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