Deciphering Epitranscriptome: Modification of mRNA Bases Provides a New Perspective for Post-transcriptional Regulation of Gene Expression

Abstract

Gene regulation depends on dynamic and reversibly modifiable biological and chemical information in the epigenome/epitranscriptome. Accumulating evidence suggests that messenger RNAs (mRNAs) are generated in flashing bursts in the cells in a precisely regulated manner. However, the different aspects of the underlying mechanisms are not fully understood. Cellular RNAs are post-transcriptionally modified at the base level, which alters the metabolism of mRNA. The current understanding of epitranscriptome in the animal system is far ahead of that in plants. The accumulating evidence indicates that the epitranscriptomic changes play vital roles in developmental processes and stress responses. Besides being non-genetically encoded, they can be of reversible nature and involved in fine-tuning the expression of gene. However, different aspects of base modifications in mRNAs are far from adequate to assign the molecular basis/functions to the epitranscriptomic changes. Advances in the chemogenetic RNA-labeling and high-throughput next-generation sequencing techniques are enabling functional analysis of the epitranscriptomic modifications to reveal their roles in mRNA biology. Mapping of the common mRNA modifications, including N ⁶-methyladenosine (m⁶A), and 5-methylcytidine (m⁵C), have enabled the identification of other types of modifications, such as N ¹-methyladenosine. Methylation of bases in a transcript dynamically regulates the processing, cellular export, translation, and stability of the mRNA; thereby influence the important biological and physiological processes. Here, we summarize the findings in the field of mRNA base modifications with special emphasis on m⁶A, m⁵C, and their roles in growth, development, and stress tolerance, which provide a new perspective for the regulation of gene expression through post-transcriptional modification. This review also addresses some of the scientific and technical issues in epitranscriptomic study, put forward the viewpoints to resolve the issues, and discusses the future perspectives of the research in this area.

Keywords: 5-methylcytidine; N6-methyladenosine; RNA metabolism; RNA modification; central dogma; epitranscriptomics; mRNA methylation; post-transcriptional regulation.

FIGURE 1

Reversible biochemical modifications affect the transfer of genetic information (the Central Dogma). As per the central dogma, the genetic information passes from DNA, through RNA, to protein. However, epigenetic DNA base modifications [e.g., 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), N⁶-methyladenine (6-mA), and N⁶-hydroxymethyladenine (6-hmA)] and histone protein modifications [e.g., methylation (me) and acetylation (ac) of amino acids] affects RNA metabolism (including splicing, export, stability, and translation efficiency) and play crucial roles in the regulation of cellular growth, development, and protection from environmental stress. Similarly, the dynamic RNA modifications [e.g., N⁶-methyladenosine (m⁶A), N¹-methyladenosine (m¹A), and N⁶-hydroxymethyladenosine (hm⁶A)] encrypt an additional layer of information and dynamically regulate the biological processes. Small-interfering RNA (siRNA) plays important role in recruitment of DNA methyltranferase for DNA base modification, methylated mRNA bases (e.g., m⁶A) play role in protein synthesis, the histone 3 (H₃) protein trimethylated (me³) at 4th lysine of (H₃K₄me³) affects the transcription process.

FIGURE 2

Base modifications in mRNA affect post-transcriptional gene regulation. In the nucleus, RNA base modifications affect (1) pre-mRNA processing and (2) pri-miRNA maturation, and (3) their export from the nucleus. In the cytoplasm, RNA base modifications regulate (4) mRNA degradation, (5) mRNA stability, (6) RNA structure, and (7) mRNA translation efficiency.

FIGURE 3

Detection of modified bases in mRNA. (A) Bisulfite sequencing (BS-seq) for the detection of 5-methylcytosine (m⁵C). Purified mRNA is fragmented into small (100–200 nt) fragments, and subjected to bisulfite treatment. Bisulfite treatment causes converts cytosine (C) to uracil (U), but m⁵C remains unchanged. Presence of C is detected by sequencing, wherein it is replaced by T. (B) Purified mRNAs are fragmented into 100–200 nt, followed by immunoprecipitation using anti-m⁶A antibody to enrich the sample with fragments containing the modified base, library preparation, and high-throughput deep-sequencing for detection of m⁶A. (C) Purified mRNAs are fragmented followed by immunoprecipitation using anti-m⁵C antibody of the fragments containing the modified base, library preparation, and sequencing. (D) m⁵C individual-nucleotide-resolution crosslinking and immunoprecipitation (m5C-miCLIP) exploites catalytic activity of cysteine-to-alanine mutation (C271A) mutant of NSUN2 (methyltransferase) which inhibits release of the enzyme from the protein–RNA complex making stable covalent bond between NSun2 and its RNA targets. Antibody specific to the RNA bound protein is used for immunoprecipitation, followed by library preparation and sequencing. This allows detection of low-abundance methylated RNAs without the need of deep sequencing.