Dynamic transcriptomic m5C and its regulatory role in RNA processing

Transcriptome‐wide sequencing methods for m⁵C. (a) m⁵C‐RIP‐seq. RNA fragments with m⁵Cs were specifically pulled down by anti‐m⁵C antibody and then constructed for sequencing. The m⁵C peaks were then identified by comparing with the regular RNA‐seq as background. (b) miCLIP‐seq. The overexpressed mutant NSUN2 (C271A) targeted the potential m⁵C residues and then RNA‐protein complex with strong covalent bond, which might induce stop position during RT‐PCR (red dash line), was obtained by UV crosslinking. The RNA‐protein complex was then pulled down by anti‐NSUN2 antibody and the RNA was washed for library construction. Finally, m⁵C loci was identified as significant truncation site along transcriptome. (c) AZA‐IP‐seq. The cytidine analog 5‐azaC was incubated with cells overexpressing NSUN2, in which NSUN2 recognized the potential m⁵C residues but could not be released from RNA. The pulled down RNAs were then constructed for sequencing and the precise m⁵C sites were identified as C‐to‐G transversion (red dash box). (d) RNA‐BisSeq. RNA fragments were first treated with bisulfite and the unmodified C could be converted to U. m⁵Cs were then identified as those unchanged C (red dash box) and methylation levels were estimated at ratio of unconverted reads to total reads. (e) WO‐seq and TAWO‐seq. For WO‐seq, hm⁵C can be converted to ^thT (trihydroxylated‐thymine) by peroxotungstate and then identified as mutation of C‐to‐T by sequencing. Combined with WO‐seq, TAWO‐seq was designed to identify the original m⁵C that could be converted to hm⁵C by TET demethylase and further to ^thT, whereas the original hm⁵C was protected from conversion to ^thT by labeling with β‐glucosyltransferase (βGT). Then original m⁵C, but not original hm⁵C, could be detected by mutation of C‐to‐T from TAWO‐seq. TGIRT: thermostable group II intron reverse transcriptase. (f) Nanopore sequencing. The nanopore sequencing was based on a protein nanopore which was inserted into membrane. The current flow was recorded when the RNA molecules were driven through the nanopore, so this platform could determine the status of each nucleic acid in a molecule directly. Combined with analysis model, the modification signals were then identified along RNA fragments

Diverse molecular functions of m⁵C in mRNA post‐transcriptional modulation. In eukaryotic cells, RNA m⁵C level is dynamically regulated by “writers” and “eraser”, and recognized by “readers”. The diversity of cellular processes m⁵C involved in is mainly contributed by different “readers”. The nuclear m⁵Cs involve in mRNA export via ALYREF, while the cytoplasmic m⁵C enhances mRNA stability by recruiting YBX1. And, m⁵C has been found it involve in lots of biogenesis and tumorigenesis in various species, such as urothelial carcinoma of bladder, embryonic development in zebrafish, ovarian germ line stem cell development in Drosophila, thermotolerance in rice, long‐distant transport in Arabidopsis thaliana, and viral replication