Xue et al. Biomarker Research (2020) 8:43 https://doi.org/10.1186/s40364-020-00225-0 REVIEW Open Access Advances in RNA cytosine-5 methylation: detection, regulatory mechanisms, biological functions and links to cancer Chen Xue1,2,3†, Yalei Zhao1,2,3† and Lanjuan Li1,2,3* Abstract As an important posttranscriptional modification of RNA, 5-methylcytosine (m5C) has attracted increasing interest recently, with accumulating evidence suggesting the involvement of RNA m5C modification in multiple cellular processes as well as tumorigenesis. Cooperatively, advances in m5C detection techniques have enabled transcriptome mapping of RNA methylation at single-nucleotide resolution, thus stimulating m5C-based investigations. In this review, we summarize currently available approaches for detecting m5C distribution in RNA as well as the advantages and disadvantages of these techniques. Moreover, we elucidate the regulatory mechanisms of RNA m5C modification by introducing the molecular structure, catalytic substrates, cellular distributions and biological functions of RNA m5C regulators. The functional consequences of m5C modification on mRNAs, tRNAs, rRNAs and other RNA species, including viral RNAs and vault RNAs, are also discussed. Finally, we review the role of RNA m5C modification in cancer pathogenesis and progression, in hopes of providing new insights into cancer treatment. Keywords: 5-methylcytosine, RNA modification, Epitranscriptome, Detection techniques, Biological functions Background formylcytosine (5fC), and 5-carboxylcytosine (5caC), has Epigenetic modifications, mainly including DNA methyla- been extensively studied (reviewed in [4]). In recent years, tion and histone modifications, are chemical alterations in great progress has been made in the research of RNA nucleic acids that do not change the DNA sequence but modifications, which were originally regarded as fine- play key roles in heredity, growth, longevity, aging and dis- tuning chemostructural features of non-protein-coding ease [1, 2]. DNA 5-methylcytosine (m5C) has been found RNAs but are now considered to be dynamically regu- to be the most abundant DNA modification in mamma- lated, reversible and widespread posttranscriptional regu- lian cells and is characterized by the addition of a methyl lators in diverse cellular processes (reviewed in [5]). group at the carbon-5 position of the cytosine base [3]. In Predominant mammalian RNA methylation modifications eukaryotes, the role of m5C in DNA and its oxidized de- include N6-methyladenosine (m6A), N1-methyladenosine rivatives, including 5-hydroxymethylcytosine (5hmC), 5- (m1A), pseudouridine (Ψ)andm5C, but previous studies on RNA methylation have focused on m6A[5, 6]; how- * Correspondence: [email protected] ever, emerging evidence has gradually uncovered the role 5 †Chen Xue and Yalei Zhao contributed equally to this work. of RNA m C in posttranscriptional regulation. 1State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, With the advances in RNA m5C detection techniques, The First Affiliated Hospital, College of Medicine, Zhejiang University, No. 79 5 Qingchun Road, Shangcheng District, Hangzhou 310003, Zhejiang, China including bisulfite sequencing, m C RNA immunopre- 5 2National Clinical Research Center for Infectious Diseases, The First Affiliated cipitation sequencing (m C-RIP-seq), 5-AZAcytidine- Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China mediated RNA immunoprecipitation sequencing (Aza- Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Xue et al. Biomarker Research (2020) 8:43 Page 2 of 13 IP-seq) and methylation-individual nucleotide resolution m5C patterns, including bisulfite sequencing, m5C-RIP- crosslinking immunoprecipitation sequencing (miCLIP- seq, Aza-IP-seq and miCLIP-seq. Additionally, their ad- seq), over 10,000 potential sites of m5C modification vantages and limitations are discussed to provide a more were detected within the whole human transcriptome comprehensive view of RNA m5C mapping (Table 1). [7], and the existence of m5C was found in diverse RNA species from various organisms, not only in ribosomal Bisulfite sequencing RNA (rRNA), messenger RNA (mRNA) and transfer Sodium bisulfite has been applied as a robust and reli- RNA (tRNA) but also in viral RNA, vault RNA, small able method for DNA m5C analysis since the 1990s [27]. nuclear RNA, small nucleolar RNA, pseudogenes, nat- In an acidic pH, sodium bisulfite reacts with methylated ural antisense transcripts, enhancer RNA, long noncod- and unmethylated C, resulting in deamination to uracil ing RNA (lncRNA) and microRNA (miRNA) [8–15]. sulfonate/5-methyluracil sulfonate, which can be further The distribution of m5C differs among different organ- converted to uracil/thymine at basic pH. It was not ori- isms, as it has been common to find m5C methylation ginally considered an available technique for RNA substrates in the tRNA and mRNA of eukaryotes and ar- methylation detection, as the stability of phosphodiester chaea, while no m5C methylation substrates have been bonds in RNA might be disrupted due to harsh chemical detected in bacteria [16]. It has been demonstrated that and temperature treatment, and the methylation status RNA m5C is regulated by “writers”, “erasers”, and of individual RNA sequences failed to be properly inter- “readers”, referring to RNA m5C methyltransferases rogated [28]. In 2009, a versatile bisulfite deamination (RCMTs), which are enzymes of the ten-eleven trans- protocol for RNA m5C detection was developed based location (TET) family and RNA binding proteins [16]. on PCR that allowed amplification of cDNA from low- The alteration of m5C modification as well as its regula- abundance cellular RNAs [29]. In the presence of so- tors has been proven to influence RNA stability, gene dium bisulfite, unmethylated cytosines (Cs) can be expression, and protein synthesis and thus has a great chemically deaminated to uracils (Us), which are ultim- impact on various cellular and physical processes [15, ately replaced by thymines (Ts) during subsequent PCR 17–22]. Additionally, to date, RNA m5C modification amplification, while methylated Cs remain unchanged in and its regulators have been confirmed to play crucial this process; following this mechanism, bisulfite sequen- roles in the pathogenesis of bladder cancer [23], hepato- cing is designed to differentiate unmethylated and meth- cellular carcinoma (HCC) [24], glioblastoma multiforme ylated Cs and thus investigate the prevalence of cytosine (GBM) [14] and leukemia [18], indicating the promising methylation in RNA [29]. Coupled with NGS techniques, prospect of m5C modification in cancer treatment. bisulfite conversion is now regarded as the gold standard Herein, we introduce the principles of m5C mapping for mapping modified cytosine in RNA at single nucleo- methods as well as their advantages and disadvantages. tide resolution and with high specificity. However, the Next, we summarize the mechanisms and biological broader application of bisulfite sequencing is hindered functions of m5C modification of RNAs. Finally, by dis- by the degradation of low-abundance RNAs during the cussing the role of m5C in tumorigenesis and cancer conversion process [30]. Additionally, it fails to distin- progression, we propose m5C-based approaches for can- guish m5C from other types of cytosine modifications, cer diagnosis, prognosis and clinical treatment. Although such as 5hmC [31]. Further efforts should be made to some statements in our study may briefly touch on other optimize the validity and scope of bisulfite sequencing. reviews [8, 16, 25], which discussed the detection methods and biological functions of RNA m5C methyla- m5C-RIP-seq tion in depth, we focus on literature published in the Originally applied to m6A detection in randomly frag- past five years and are the first to thoroughly discuss the mented RNA transcripts [32], m5C-RIP was later clinical prospect of RNA m5C in cancer treatment. adapted to detect RNA m5C in bacterial, archaeal, yeast and plant transcriptomes [33]. Conducted with a m5C- Approaches in detecting m5C distribution in RNA specific antibody combined with a deep sequencing tech- Although m5C was discovered in RNA almost 60 years nique, this transcriptome-wide protocol is available for ago, little progress has been made regarding the distribu- mapping m5C sites in low-abundance RNAs; it has high tion and biological roles