5-methylcytosine is a post-transcriptional modification common in tRNA and rRNA, playing an important role in tRNA function and stability as well as ribosome biogenesis and assembly. The 5-methylcytosine modifications is mediated by the NOP2/Sun (NSUN) family of RNA methyltransferases, which consists of seven members, each targeting a distinct subset of sites. Of this RNA methyltransferase family, NSUN2 is by far the best studied, although targets for other methyltransferases have recently started to emerge. The main target substrates for NSUN2 are tRNAs and tRNA methylation plays an important role in decoding, tRNA stability and stress response. However, NSUN2 has also been shown to target other RNA biotypes such as snoRNAs, lncRNAs and mRNAs, but the role of 5-methylcytosine in these remains unclear.

Several aspects of mRNA biology could be affected by 5-methylcytosine such as splicing or alternative exon usage, stability, RNA degradation or miRNA targeting as well as translation. Putative roles in transcript stability and miRNA targeting have previously been suggested due to enrichment of the mark in the UTRs and near Argonaute binding sites along with depletion at miRNA seed regions. Recently several studies suggested a role of 5-methylcytosine in translation and stability. 5-methylcytosine was shown to increase mRNA stability and/or enhance translation of distinct transcripts involved in senescence. However, this was only shown for a handful of transcripts and the precise methylated sites remain elusive.

To gain a global understanding of the role of 5-methylcytosine in mRNA biology we analysed the effect of loss of 5-methylcytosine, by means of NSUN2 knock-down, on transcript stability and also endeavoured to gain initial insight into alternative exon usage. Further we analysed the translation efficiency of mRNA molecules marked with 5-methylcytosine compared to their unmodified counterparts by Bis-RNAseq of RNA isolated from lowly to highly translated regions of a polysome gradient. Here, we present our findings, unravelling the role of 5-methylcytosine with regards to transcript stability and translation at a transcritome-wide level as well as highlighting a few distinct examples.