Abstract
RNA:5-methylcytosine (m5C) methyltransferases are currently the focus of intense research following a series of high-profile reports documenting their physiological links to several diseases. However, no methods exist which permit the specific analysis of RNA:m5C methyltransferases in cells. Herein, we described how a combination of biophysical studies led us to identify distinct duplex-remodelling effects of m5C on RNA and DNA duplexes. Specifically, m5C induces a C3′-endo to C2′-endo sugar-pucker switch in CpG RNA duplex but triggers a B-to-Z transformation in CpG DNA duplex. Inspired by these different ‘structural signatures’, we developed a m5C-sensitive probe which fluoresces spontaneously in response to m5C-induced sugar-pucker switch, hence useful for sensing RNA:m5C methyltransferase activity. Through the use of this probe, we achieved real-time imaging and flow cytometry analysis of NOP2/Sun RNA methyltransferase 2 (NSUN2) activity in HeLa cells. We further applied the probe to the cell-based screening of NSUN2 inhibitors. The developed strategy could also be adapted for the detection of DNA:m5C methyltransferases. This was demonstrated by the development of DNA m5C-probe which permits the screening of DNA methyltransferase 3A inhibitors. To our knowledge, this study represents not only the first examples of m5C-responsive probes, but also a new strategy for discriminating RNA and DNA m5C methyltransferase activity in cells.
Highlights
The DNA and RNA of all living organisms, as well as that of viruses, mitochondria and chloroplasts, undergo a wide range of modifications [1,2]
There has not been a systematic study directly comparing the structural impact of m5C methylation on CpG RNA duplexes and CpG DNA duplexes
We examined whether m5C-probe 9a could function as fluorogenic substrate for RNA:m5C MTase by testing it against NOP2/Sun RNA methyltransferase 2 (NSUN2), which is of particular interest in light of emerging evidence linking NSUN2 with m5C methylation of human mRNA [14] and its association with human cancers [25,26,27,28,29]
Summary
The DNA and RNA of all living organisms, as well as that of viruses, mitochondria and chloroplasts, undergo a wide range of modifications [1,2] These modifications expand the structural diversity of nucleic acids, and provide an epigenetic mechanism to fine-tune their biological functions [3,4]. The significance of m5C modification in mRNA was not fully appreciated until recently, following the landmark discovery of widespread m5C sites in the transcriptomes of diverse organisms [9,10,11,12,13,14,15,16], suggesting that the m5C modification is far more pervasive in human mRNA than previously realised This has reignited intense interest in the study of this epitranscriptomic mark. C-5 cytosine methylation of DNA is catalysed by at least three DNA:m5C MTases (i.e. DNMT1, 3A and 3B), whereas m5C methylation of RNA is catalysed by the NOL1/NOP2/Sun domain (NSUN) RNA methyltransferase family, which includes NSUN1-7, as well as the DNA
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