Transcriptome-wide identification of 5-methylcytosine by deaminase and reader protein-assisted sequencing
5-Methylcytosine (m5C) is one of the major post-transcriptional modifications in mRNA and is highly involved in the pathogenesis of various diseases. However, the capacity of existing assays for accurately and comprehensively transcriptome-wide m5C mapping still needs improvement. Here, we developed a deaminase and reader protein assisted RNA methylation analysis, termed DRAM, in which deaminases (APOBEC1 and TadA-8e) are fused with m5C reader proteins (ALYREF and YBX1) to identify the m5C sites through deamination events neighboring the methylation sites. This antibody-free and bisulfite-free approach provided transcriptome-wide editing regions which were highly overlapped with the publicly available BS-seq datasets. Notably, DRAM-seq even discovered a new m5C methylation locus in KAT7, a previously demonstrated key aging regulator. In addition, DRAM system even supports ultra-low input RNA (10ng) and monitor the dynamic accumulation of cellular m5C. We anticipated that the DRAM system could pave the way for uncovering further biological functions of m5C modifications. Overall design: To comprehensively detect the m5C locus, the readers of m5C (ALYREF and YBX1) were separately fused to the C-terminus of the deaminases (APOBEC1 and TadA-8e), namely DRAM system.DRAM-ABE (mixture of TadA-8e-ALYREF and TadA-8e-YBX1) and DRAM-CBE (mixture of APOBEC1-ALYREF and APOBEC1-YBX1) were then transfected into the human HEK293T cells, respectively. To verify that the detection of DRAM occurs in the presence of m5C, we individually depleted NSUN2 and NSUN6 in HEK293T cells and performed DRAM transfection.Eventually, we performed RNA-seq after DRAM transfection to enable a transcriptome-wide detection of m5C.