Advances in mRNA 5-methylcytosine modifications: Detection, effectors, biological functions, and clinical relevance

Abstract

5-methylcytosine (m⁵C) post-transcriptional modifications affect the maturation, stability, and translation of the mRNA molecule. These modifications play an important role in many physiological and pathological processes, including stress response, tumorigenesis, tumor cell migration, embryogenesis, and viral replication. Recently, there has been a better understanding of the biological implications of m⁵C modification owing to the rapid development and optimization of detection technologies, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and RNA-BisSeq. Further, predictive models (such as PEA-m⁵C, m⁵C-PseDNC, and DeepMRMP) for the identification of potential m⁵C modification sites have also emerged. In this review, we summarize the current experimental detection methods and predictive models for mRNA m⁵C modifications, focusing on their advantages and limitations. We systematically surveyed the latest research on the effectors related to mRNA m⁵C modifications and their biological functions in multiple species. Finally, we discuss the physiological effects and pathological significance of m⁵C modifications in multiple diseases, as well as their therapeutic potential, thereby providing new perspectives for disease treatment and prognosis.

Keywords: 5-methylcytosine; RNA epigenetics; disease; mRNA; post-transcriptional modification; prediction.

Graphical abstract

Figure 1

5-methylcytosine (m⁵C) detection methods based on chromatography and mass spectrometry (MS) Nuclease is used to digest the RNA molecule into its constituting nucleotides, which are dephosphorylated. These nucleosides are prepared as input for capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), and liquid chromatography tandem MS (LC-MS/MS). CE: in high-voltage electric field drive, nucleoside samples are separated using a quartz capillary column as a separation channel. LC-MS/MS: the LC system is connected to the mass spectrometer. TOP-DOWN MS: without nuclease treatment, using a high-resolution mass spectrometer and various fragmentation patterns of ribonucleotides, RNA can be directly analyzed using MS to obtain information, including the identification of post-transcriptional modifications, relative quantification, and positional information.

Figure 2

Transcriptome sequencing methods after m⁵C modification (A) RNA-BisSeq: unmethylated cytosines of RNA fragments could be transformed into uridines when treated with bisulfite, while bisulfite does not affect m⁵C. (B) Peroxotungstate oxidation sequencing (WO-seq) and Tet-assisted WO-seq (TAWO-seq): for WO-seq, hm⁵C can be transformed into trihydroxylated thymine (^thT) by peroxotungstate, after which, the C-to-T mutation site was identified by sequencing. For TAWO-seq, m⁵C was converted to hm⁵C with NgTET1 oxidation, and hm⁵C was converted to ^thT according to the principle of WO-seq. However, the original hm⁵C of RNA was protected from being altered to ^thT by labeling with β-glucosyltransferase (βGT). The mutation of C to T was detected and identified as the original m⁵C but not the original hm⁵C. TGIRT, thermostable group II intron reverse transcriptase. (C) Nanopore-seq: a unique, scalable technology that enables direct, real-time analysis of long RNA fragments. The electrical current was monitored and recorded when nucleic acids passed through a protein nanopore. The modification signals were then decoded and identified along RNA fragments. (D) m⁵C-RIP-seq: RNA fragments containing m⁵C could be pulled down specifically by the anti-m⁵C antibody and then used to construct a sequencing library. The m⁵C peaks were identified against the input as background. (E) 5-Aza-seq: 5-azacytidine (5-Aza-C), a cytidine analog, is incorporated into the RNA molecule by RNA polymerase. RNA (cytosine-5)-methyltransferases (RCMT) can form a temporary intermediate with potential m⁵C residues; however, 5-Aza-C can inhibit the complex separation. The RCMT-RNA complex was pulled down with a specific anti-RCMT antibody, and the pulled-down RNA was used for library construction and sequencing to identify the m⁵C sites as C-to-G transversion (red underlined frame). (F) m⁵C individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP)-seq: mutating cysteine (C271) into alanine (C271A) for RCMT inhibited the separation of the enzyme-RNA complex with strong covalent bonding, which was achieved by UV cross-linking, resulting in stop position during PCR. The RCMT-RNA complex was pulled down with anti-RCMT antibody, the RNA was washed to construct the library, and the significant truncation site was considered as the m⁵C site.

Figure 3

Overview of the effectors (writers, readers, and erasers) related to m⁵C modifications of mRNA NSUN2, NSUN6, TRDMT1, TRM4B, and OsNSUN2 are RNA methyltransferases responsible for m⁵C modification. (1) m⁵C reader RAD52 recognizes m⁵C-methylated RNA to promote reactive oxygen species (ROS)-induced atypical HR repair of DSBs through the TRDMT1-m⁵C-RAD52-RAD51 axis. (2) m⁵C reader ALYREF recognizes m⁵C-methylated mRNA and transfers mRNA from the nucleus to the cytoplasm. (3) In bladder cancer, m⁵C reader YBX1 recognizes methylated heparin-binding growth factor (HDGF) mRNA and recruits ELAV-like protein 1 (ELAVL1) to stabilize HDGF, finally promoting the proliferation and metastasis of bladder cancer cells.

Figure 4

The mRNA m⁵C modification associations with tumorigenesis and metastasis in multiple cancers The red dotted line indicates that tumorigenesis and metastasis in multiple cancers are related to m⁵C modifications, whereas the purple dotted line indicates that the disease correlation with m⁵C modifications is uncertain.

Figure 5

Physiological effects of m⁵C modifications in different species