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Dynamic transcriptomic m5C and its regulatory role in RNA processing

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Abstract RNA 5‐methylcytosine (m5C) is a prevalent RNA modification in multiple RNA species, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and noncoding RNAs (ncRNAs), and broadly distributed from archaea, prokaryotes to eukaryotes. The multiple detecting techniques of m5C have been developed, such as m5C‐RIP‐seq, miCLIP‐seq, AZA‐IP‐seq, RNA‐BisSeq, TAWO‐seq, and Nanopore sequencing. These high‐throughput techniques, combined with corresponding analysis pipeline, provide a precise m5C landscape contributing to the deciphering of its biological functions. The m5C modification is distributed along with mRNA and enriched around 5′UTR and 3′UTR, and conserved in tRNAs and rRNAs. It is dynamically regulated by its related enzymes, including methyltransferases (NSUN, DNMT, and TRDMT family members), demethylases (TET families and ALKBH1), and binding proteins (ALYREF and YBX1). So far, accumulative studies have revealed that m5C participates in a variety of RNA metabolism, including mRNA export, RNA stability, and translation. Depletion of m5C modification in the organism could cause dysfunction of mitochondria, drawback of stress response, frustration of gametogenesis and embryogenesis, abnormality of neuro and brain development, and has been implicated in cell migration and tumorigenesis. In this review, we provide a comprehensive summary of dynamic regulatory elements of RNA m5C, including methyltransferases (writers), demethylases (erasers), and binding proteins (readers). We also summarized the related detecting technologies and biological functions of the RNA 5‐methylcytosine, and provided future perspectives in m5C research. This article is categorized under: RNA Processing > RNA Editing and Modification
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
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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
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