5-Formylcytosine (f5C) modification is present in human mitochondrial methionine tRNA (mt-tRNAMet) and cytosolic leucine tRNA (ct-tRNALeu), with their formation mediated by NSUN3 and ALKBH1. f5C has also been detected in mRNA of yeast and human cells, but its transcriptome-wide distribution in mammals has not been studied. Here we report f5C-seq, a quantitative sequencing method to map f5C transcriptome-wide in HeLa and mouse embryonic stem cells (mESCs). We show that f5C in RNA can be reduced to dihydrouracil (DHU) by pic-borane, and DHU can be exclusively read as T during reverse transcription (RT) reaction, allowing the detection and quantification of f5C sites by a unique C-to-T mutation signature. We validated f5C-seq by identifying and quantifying the two known f5C sites in tRNA, in which the f5C modification fractions dropped significantly in ALKBH1-depleted cells. By applying f5C-seq to chromatin-associated RNA (caRNA), we identified several highly modified f5C sites in HeLa and mouse embryonic stem cells (mESC).
Keywords: 5-Formylcytosine; mutation rate; quantitative sequencing; read-through rate; transcriptome-wide.