Characterizing 5-methylcytosine in the mammalian epitranscriptome

Hussain et al. Genome Biology 2013, 14:215 http://genomebiology.com/2013/14/11/215 REVIEW Characterizing 5-methylcytosine in the mammalian epitranscriptome Shobbir Hussain*†, Jelena Aleksic†, Sandra Blanco, Sabine Dietmann and Michaela Frye* Abstract The post-transcriptional modification 5-methylcytosine (m5C) occurs in a wide range of coding and non-coding RNAs. We describe transcriptome-wide approaches to capture the global m5C RNA methylome. We also discuss the potential functions of m5C in RNA and compare them to 6-methyladenosine modifications. Introduction Post-transcriptional changes in RNA processing are essential regulators for most, if not all, cellular responses. Although RNA modifications are more prevalent and diverse in their chemical nature than DNA modifications [1], our knowledge of their occurrence and function in RNA is generally limited. There are approximately 150 known ribonucleoside modifications: most of them have been found in tRNA and rRNA, but some occur in mRNA [1-4]. Post-transcriptional modifications are highly likely to add complexity to RNA-mediated functions. The advent of next-generation sequencing (NGS) tools has enabled the identification of RNA modifications both globally and in a substrate-specific manner. 6Methyladenosine (m6A) was the first modification to be characterized, and is now known to be present in several types of RNA and, most notably, is highly enriched around stop codons in many mRNAs [2-4]. Another known modification in RNA is 5-methylcytosine (m5C). Although m5C is a well-characterized modification in DNA, its precise regulatory functions in RNA remain unclear [5,6]. Until recently, the detection of RNA methylation involved the digestion of highly purified RNA followed by separation techniques, such as high performance liquid chromatography (HPLC) and mass spectrometry, which only allowed the identification of m5C in stable and highly * Correspondence: ; † Equal contributors Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK © 2013 BioMed Central Ltd. abundant tRNAs and rRNAs [7-10]. Labeling techniques with 3H in living cells allowed detection of m5C in mRNA and viral RNAs [11,12]. However, the deposition of m5C into mRNA remained controversial [13-17]. The recent advances in high-throughput techniques, combined with NGS, have renewed interest in the field, and have led to the identification of m5C as a widespread modification in coding and non-coding (nc)RNAs [18]. A fraction of these RNAs were found to be specifically methylated by the RNA methylase NSun2, including mRNAs, ncRNAs and several tRNAs [18]. NSun2 had been previously shown to methylate tRNAs at various positions [19-22]. Additional NSun2-methylated coding and ncRNAs were identified in two studies published in 2013 that used customized RNA immunoprecipitation approaches followed by NGS [23,24]. The regulatory functions of m5C modifications in RNA are still not fully understood. In tRNAs, in vitro cytosine-5 methylation can affect Mg2+ binding to tRNA molecules, which in turn influences the anticodon stem loop conformation and stabilizes the secondary structure [25,26]. Cytosine-5 methylation alone, or in combination with other nonessential tRNA modifications, can also protect from degradation or cleavage [22,27-29]. In rRNA, m5C is thought to play a role in translation [30]. Synthetic cytosine-5 methylated mRNAs exhibit increased stability, and loss of methylation in the 3’ UTR of p16 has been reported to reduce its stability [31,32]. The biological functions of tRNA m5C-methylation are linked to the regulation of protein translation in stress pathways and tissue differentiation in yeast, Drosophila, fish and mouse [19,22,29,33-36]. Mutations in the NSUN2 gene in humans cause an autosomal recessive syndrome characterized by intellectual disability, skin disorders and growth retardation [37-40]. These findings, together with studies carried out in NSun2-deficient mice and cell lines, suggest a wide-ranging role for m5C modifications in RNA, including cellular signaling, tissue development and differentiation, and cancer [19,21,22,36,41-43]. Hussain et al. Genome Biology 2013, 14:215 http://genomebiology.com/2013/14/11/215 Page 2 of 10 In this review, we compare the current methods to identify m5C in the mammalian transcriptome with a particular focus on NSun2-mediated methylation. We further discuss the potential of these initial studies to comprehensively determine the global but enzyme-specific cytosine-5 RNA methylome. In addition, we compare studies focusing on m6A and m5C modifications and consider the likely benefits of characterizing and elucidating the functions of the mammalian epitranscriptome. Mapping cytosine-5 methylation in the mammalian transcriptome Previous methods for detecting m5C in RNA have required exceedingly high amounts of RNA and only reproducibly identified methylated sites in highly abundant RNAs, such as tRNAs and rRNAs. Over the last four years, NGS has allowed researchers to successfully develop more sensitive techniques. Bisulfite sequencing for the detection of m5C in RNA was first described in 2009 [44]. In 2012, the method was combined with NGS and provided the first transcriptome-wide view of the human cytosine-5 RNA methylome (Figure 1A) [18]. Earlier this year, three independent studies used RNA immunoprecipitation (RIP) followed by deep sequencing to detect m5C globally (Figures 1B-D) [23,24,45]. Khoddami and Cairns, and our own study [24], developed similar but (a) (b) Bisulfite seq m5C-RIP Me T C T C technically distinct approaches to detect enzyme-specific deposition of m5C: 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP) (Figure 1C) [23] and methylation-individual nucleotide resolution crosslinking immmunoprecipitation (miCLIP) (Figure 1D) [24]. Both Aza-IP and miCLIP rely on covalent bond formation between the RNA methylase and substrate but differ in the way in which the stable covalent bond formation is achieved. We now describe these techniques in detail and discuss their specific merits and disadvantages. Bisulfite sequencing Bisulfite sequencing, a method based on the chemical deamination of cytosines and originally developed to detect m5C in DNA, was previously adapted for use with RNA (Figure 1A) [44]. The technique is based on the differential chemical reactivity of m5C compared with cytosine. Sodium bisulfite causes the deamination of unmethylated cytosines into uridines in single-stranded DNA or RNA, while m5C remains unconverted [6]. A crucial parameter is the fraction of cytosine that is converted to uridine, but a high conversion rate is only achieved by prolonged incubation under consecutive acidic and alkaline conditions, which also causes RNA degradation. This degradation can compromise the (c) (d) AzaIP = 5- azacytidine sites in RNA G Anti-m5C antibody Me Bisulfite conv (...truncated)