**1. Introduction**

RNA has been shown to play critical roles in regulating cellular functions. Comparative transcriptomics between mammals has revealed that ∼66% of human genomic DNA is transcribed. Remarkably, only ∼2% of the transcriptional production is protein‐coding messenger RNA (mRNA), while ∼98% encompasses a wide variety of non‐coding RNA (ncRNA) molecules [1, 2]. ncRNAs have been classified functionally as either housekeeping or regulatory. The

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housekeeping ncRNA genes include ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA), while examples of regulatory ncRNAs are microRNA (miRNA) and long non‐coding RNA (lncRNA) [3–5]. The complexity of RNA is further complicated by numerous post‐transcriptional modifications which alter the chemical structure of the nucleotides without changing the nucleotide sequence. Similar to the field of epigenetics which investigates the modifications of DNA and histone proteins, the study of chemical modifications of RNA is called epitranscriptomics [6, 7]. More than 140 chemically diverse and distinct modified nucleotides have been identified in both mRNA and ncRNA, including *N*<sup>6</sup> ‐methyladenosine (m<sup>6</sup> A), 5‐methyl cytidine (m<sup>5</sup> C), pseudouridine (Ѱ), adenosine (A) to inosine (I), and *N*<sup>1</sup> ‐methyladenosine (m1 A). These modifications have been identified mostly in the housekeeping ncRNAs [3, 4, 8]; however, chemical modifications have also been detected in mRNA and the regulatory ncRNAs [9–11]. Unfortunately, the knowledge about the occurrence and function of RNA modifications at transcriptome level remains scarce. Recently, the interest in RNA modifications and their functions have gained momentum owing mainly to the application of novel modifications to next‐generation sequencing (NGS) and mass spectrometry technologies, which have allowed transcriptome‐wide detection of distinct RNA modifications [12, 13]. Accurate regulation of the transcriptome is critical for gene expression and its subsequent control of cellular functions, including metabolism, proliferation, differentiation, and development. Thus, alterations in transcriptome regulation can disrupt cellular functions and lead to disease. Accumulating evidence has identified and functionally characterized several distinct types of chemical modifications of RNA nucleotides in both protein‐coding and ncRNAs, further advancing the burgeoning field of epitranscriptomics. In this chapter, we will first provide an overview of RNA modifications and then synopsize several transcriptome‐wide RNA modification mapping techniques such as m<sup>6</sup> A‐seq, m<sup>5</sup> C‐seq, pseudouridine‐seq, and NAD captureSeq. Next, we will highlight novel insights into the potential functions of RNA modifications and their disease relevance as revealed and facilitated by epitranscriptomic profiling. Finally, we will offer our perspective on how the field will progress or evolve in the near future.

U, Ψ contains an extra imino group (>C═NH), which serves as an additional hydrogen bond donor, while the carbon‐carbon (C─C) glycosidic bond linking the sugar to the base is more stable than the carbon‐nitrogen (C─N) found in U. These two chemical changes confer rigid-

modifications are reversible, suggesting that the modifications are involved in regulatory switches. Methyltransferases (METTL3, METTL14, and WTAP), termed writers, catalyze the methylation of adenosine [21–23], whereas demethylases (FTO and ALKBH5), termed eras-

An additional class of nucleotide modifications, termed RNA editing, creates an irreversible change in the nucleotide sequence. These modifications include insertions, deletions, and base substitutions and occur in all classes of RNA. When they occur in mRNA, the amino acid sequence of the protein will be altered relative to the sequence encoded by genomic DNA. RNA editing by deamination results in adenosine (A) to inosine (I) and cytosine (C) to uridine (U). A‐to‐I editing is an abundant class of RNA modifications found throughout metazoans [28]. The conversion of A‐to‐I residues by base deamination results in the synthesis of distinct proteins, which creates functional diversity and serves to enhance the response to rapid environmental changes [29]. RNA editing by deamination is mediated by two major classes of enzymes; the first class is a group of tissue‐specific and context‐dependent adenosine deaminases called ADARs [30–32]. The ADAR enzyme class (adenosine deaminases acting on RNA) catalyzes hydrolytic deamination of A‐to‐I in double‐stranded regions of RNA secondary structure [33]. The second class of enzymes, the vertebrate‐specific apolipoprotein B mRNA editing catalytic polypeptide‐like (APOBEC) family, promotes C‐to‐U editing by cytosine deamination [34]. APOBEC1, the first‐discovered member of the APOBEC family, was characterized as the zinc‐dependent cytidine deaminase which catalyzed a C‐to‐U modi-

A [20]. Unlike Ψ, m<sup>6</sup>

Epitranscriptomics for Biomedical Discovery http://dx.doi.org/10.5772/intechopen.69033

A modifications dem-

A, m6Am, m<sup>5</sup>

C, m1

A, A‐to‐I, Ѱ,

A marks are recognized by YTH domain pro-

A

281

ity to the sugar‐phosphate backbone and enhances local base stacking [19].

teins, termed readers, which regulate mRNA processing and metabolism [26, 27].

The most common internal modification in eukaryotic mRNA is m<sup>6</sup>

fication, resulting in an in‐frame stop codon in APOB mRNA [35].

The first transcriptome‐wide and NGS‐based approach for mapping m<sup>6</sup>

onstrated the feasibility of identifying RNA modifications across the entire transcriptome and established the field of epitranscriptomics [6]. The most important aspects of NGS‐based techniques are the ability to map modifications on a global scale at the single nucleotide resolution and that the modified nucleotides are analyzed within the context of the surrounding gene sequence. These features insure that the nucleotide modifications are accurately assigned to the appropriate RNA and not falsely attributed to homologous genes or RNA contaminates [6]. Now, several high‐throughput NGS‐based technologies, including RNA‐seq, have been

and NAD cap). These RNA‐seq‐based methodologies can be divided into two classes: immunoprecipitation‐based and chemical‐based methods. **Table 1** lists six representative NGS‐

**3. NGS‐based RNA modification techniques**

established to profile and quantitate RNA modifications (m<sup>6</sup>

based detection methods of RNA modifications.

ers, remove the methyl group [24, 25]. The m<sup>6</sup>

### **2. An overview of post‐transcriptional modifications of RNA**

The process of mRNA maturation involving 5ʹ‐capping, splicing, and polyadenylation has been well studied [14]. However, the more subtle post‐transcriptional modifications of epitranscriptomics, also termed RNA‐epigenetics, are now just fully coming to light. The post‐transcriptional modifications found in RNA are often called marks because they mark a region of RNA that potentially contributes to the regulation of cellular processes, including gene expression, protein translation, or RNA stability. Like mRNA maturation, enzymes are required to catalyze the reactions, which chemically modify RNA nucleotides. The most common post‐transcriptional RNA modification, Ψ, was also the first to be discovered [15]. Originally discovered in rRNA and tRNA, Ψ modifications are also present in mRNA [16, 17]. Site‐specific isomerization of uridine (U) to Ψ (5‐ribosyluracil) is irreversibly catalyzed via Ψ synthases. The family of Ψ synthases (PUS) consists of enzymes which can either function independently or those that require H/ACA ribonucleotide complexes [18]. Compared to U, Ψ contains an extra imino group (>C═NH), which serves as an additional hydrogen bond donor, while the carbon‐carbon (C─C) glycosidic bond linking the sugar to the base is more stable than the carbon‐nitrogen (C─N) found in U. These two chemical changes confer rigidity to the sugar‐phosphate backbone and enhances local base stacking [19].

The most common internal modification in eukaryotic mRNA is m<sup>6</sup> A [20]. Unlike Ψ, m<sup>6</sup> A modifications are reversible, suggesting that the modifications are involved in regulatory switches. Methyltransferases (METTL3, METTL14, and WTAP), termed writers, catalyze the methylation of adenosine [21–23], whereas demethylases (FTO and ALKBH5), termed erasers, remove the methyl group [24, 25]. The m<sup>6</sup> A marks are recognized by YTH domain proteins, termed readers, which regulate mRNA processing and metabolism [26, 27].

An additional class of nucleotide modifications, termed RNA editing, creates an irreversible change in the nucleotide sequence. These modifications include insertions, deletions, and base substitutions and occur in all classes of RNA. When they occur in mRNA, the amino acid sequence of the protein will be altered relative to the sequence encoded by genomic DNA. RNA editing by deamination results in adenosine (A) to inosine (I) and cytosine (C) to uridine (U). A‐to‐I editing is an abundant class of RNA modifications found throughout metazoans [28]. The conversion of A‐to‐I residues by base deamination results in the synthesis of distinct proteins, which creates functional diversity and serves to enhance the response to rapid environmental changes [29]. RNA editing by deamination is mediated by two major classes of enzymes; the first class is a group of tissue‐specific and context‐dependent adenosine deaminases called ADARs [30–32]. The ADAR enzyme class (adenosine deaminases acting on RNA) catalyzes hydrolytic deamination of A‐to‐I in double‐stranded regions of RNA secondary structure [33]. The second class of enzymes, the vertebrate‐specific apolipoprotein B mRNA editing catalytic polypeptide‐like (APOBEC) family, promotes C‐to‐U editing by cytosine deamination [34]. APOBEC1, the first‐discovered member of the APOBEC family, was characterized as the zinc‐dependent cytidine deaminase which catalyzed a C‐to‐U modification, resulting in an in‐frame stop codon in APOB mRNA [35].
