Here we present a transcriptome-wide analysis of the role of 5-methylcytosine in RNA biology, opening a gateway into fully unraveling the role of this mark in cellular processes.
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 are primarily mediated by the NOP2/Sun (NSUN) family of RNA methyltransferases, which consists of seven members. NSUN2, the best understood member, targets tRNAs for methylation, which plays an important role in decoding, tRNA stability and stress response. 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 poorly characterised.
Focusing specifically on mRNAs, 5-methylcytosine could affect processes such as splicing, stability, RNA degradation or miRNA targeting as well as translation. Recently roles for 5-methylcytosine in mRNA translation and stability was proposed for specific mRNAs. To gain an understanding of mRNA regulation by 5-methylcytosine at a transcriptome-wide level, we analyzed the effect of loss of 5-methylcytosine, by means of NSUN2 knock-down, on mRNA steady-state level. A large proportion of mRNAs affected by loss of NSUN2, showing either up- or down-regulation, are predicted to also harbour 5-methylcytosine. Further, we investigated the extent of ribosome association of modified mRNAs compared to their unmodified counterparts, by employing bsRNA-seq of RNA isolated from lowly to highly translated regions of a polysome gradient. Utilizing clustering analyses we reveal that a subset of mRNAs show either positive or negative correlation of translation state with 5-methylcytosine modification at specific sites.
We discuss our overall findings and highlight the regulation of specific examples in detail.