These are not the only ways that miRNAs inhibit expression of their targets, and scientists are still investigating their many modes of action. Improve this page Learn More. Skip to main content. Search for:. Gene expression can be regulated at various stages after an RNA transcript has been produced. Some transcripts can undergo alternative splicing. Try It. Did you have an idea for improving this content?
Carthew, R. Epitranscriptome sequencing technologies: decoding RNA modifications. Methods 14 , 23—31 PubMed Google Scholar. Yan, L. How do cells cope with RNA damage and its consequences? Oxidation and alkylation stresses activate ribosome-quality control. Singh, G. Zhao, B. Nature , — Yoon, K. Temporal control of mammalian cortical neurogenesis by m 6 A methylation.
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Cap-specific, terminal N 6 -methylation by a mammalian m 6 Am methyltransferase. Boulias, K. Identification of the m 6 Am methyltransferase PCIF1 reveals the location and functions of m 6 Am in the transcriptome. Cell 75 , — Sendinc, E. In addition to Arabidopsis, the enzymes associated with epitranscriptomic modifications have been reported in some of the agronomically important plants like Nicotiana sylvestris , maize, rice, and tomato. The methylases and demethylases have also been reported in plants, and they are evolutionarily conserved.
Any change in their expression shows a significant alteration in the m 6 A content in polyadenylated transcriptome, and drastic physiological impacts. Analysis of m 6 A landscape in rice Li D. Zhang F. Despite the progress being made in understanding m 6 A landscape in crop plants, the writers, readers, erasers for m 6 A and its functions in plant growth, development, and survival under the stress are yet to be elucidated. However, the position, pattern, and motif of m 6 A suggest that the writers, readers, and erasers might be conserved across the kingdoms.
However, the occurrence of m 1 A has also been reported in the human transcriptome Li X. Despite the progress in the detection of modified nucleosides, transcriptome-wide distributions of m 1 A in plants remain unknown. While the occurrence of some of the modified bases e. Many of these epitranscriptomic modifications like m 3 C, m 7 G, 8-oxoG, and I play important roles in animals Palladino et al. Since m 5 C is less abundant 0.
Change in m 5 C level across the tissues in Arabidopsis, with a gradual increase during vegetative growth, suggest a dynamic change in m 5 C content during plant growth and development.
More than one thousand m 5 C were detected on transcriptome-wide analysis of shoot, root, and siliques of Arabidopsis, but only a few dozen of them were commonly present among these tissues David et al. A marginal increase in expression of TRM4B an m 5 C writer was observed under cold stress in Arabidopsis, but it showed decreased expression under heat stress.
TRM4B has been further characterized in plants David et al. TRM4B loss-of-function mutants of Arabidopsis exhibited down-regulated expression of short hypocotyl 2 SHY2 and indoleacetic acid-induced protein 16 IAA16 genes involved in root development. Recently, Selmi et al. The aly mutants showed shorter primary roots, defective reproductive development including abnormal flowers and reduced seed production Pfaff et al.
Thus, m 5 C is another important epitranscriptomic mark that affects plant growth, development and adaptive responses in plants. The occurrence of ac 4 C increases mRNA half-life and promotes translation efficiency.
However, it is still not known whether ac 4 C is a reversible or not, as neither an ac 4 C reader nor its deacetylation process is known. This determines the fate of mRNA, including stability and translation Yan et al. Reversal of 8-oxoG to a normal guanosine base, as observed in the case of many other RNA base modifications, is not yet known.
Moreover, the occurrence of such modified RNA base s in plant can be expected, particularly under environmental stresses when ROS production increases significantly, but their existence has not yet been reported. Modified bases influence mRNA metabolism, including splicing, export, translation, and degradation of the transcript. The function of another modified adenosine base, m 6 Am a close homolog of m 6 A , is yet poorly understood, but it has been reported to improve translation efficiency and mRNA stability in mice by protecting the mRNA from decapping enzymes like DCP2 Mauer et al.
In Arabidopsis, a reduced ribosomal occupancy was observed in the m 5 C—marked mRNAs, indicating interfering role of m 5 C in binding of translational machinery Cui Q. A decreased m 5 C content accelerates mRNA decay, which indicates that it is another important epitranscriptomic mark affecting mRNA stability and translation efficiency in plants Cui Q.
The occurrence of 8-oxoG considerably inhibits the efficiency of peptide bond formation, which restricts translation and triggers mRNA degradation Boo and Kim, Transcription factors TFs; e. This suggests a complex interplay between the modified bases and regulatory pathways. The same signaling pathway may also activate or inactivate the synthesis of readers and erasers through post-translational modifications.
Translation efficiency was reported to be moderately increased in the METTL3-knockout mutants of mouse embryonic stem cells and embryoid bodies, which suggest a negative regulatory role of m 6 A on translation efficacy Liu et al.
However, the binding of YTHDF1 a cytoplasmic m 6 A reader cooperates with ribosomes and initiation factors to increase translation efficiency Wang et al. The presence of m 6 A in the coding region of mRNA has been reported to disrupt tRNA boarding and elongation of the translation process in vitro Choi et al.
All of these findings support the regulatory functions of m 6 A in mRNA translation. In addition to this, the presence of m 6 Am creates hindrance in the binding of mRNA-decapping enzyme DCP2, which improves the stability of the transcript Mauer et al. Similarly, m 5 C has been reported to stabilize RNA secondary structure; hence, it influences translational fidelity Helm, ; Squires and Preiss, In contrast, hm 5 C has been reported to activate translation in Drosophila melanogaster Delatte et al.
Although m 6 A has been known to promote translation efficiency in the animal system Meyer et al. In maize, m 6 A was found to be negatively correlated with translation efficiency; however, this depends on the location and content of m 6 A in the gene Luo et al. Similarly, m 5 C was also reported to be associated with reduced efficiency of translation in Arabidopsis Cui Q.
Thus, the role of different methylated bases in mRNA translation needs to be further explored to better understand the epitranscriptomic regulation of gene expression in plants. Transcripts with modified bases get easily exported, translated, and degraded, probably due to the binding of the reader at the modified base.
Pfaff et al. Reports suggest that controlling RNA modification regulates mRNA stability which ultimately fine tunes the gene expression. Geula et al. Thus, m 6 A exerts its effect through binding of the reader proteins, particularly a family of proteins containing YTH domain Xu et al. This indicates a linkage between m 6 A and mRNA degradation.
Complex cellular processes are intricately regulated by mRNA methylation. The presence of m 6 A in transcripts of pluripotent TFs prompts transcriptomic flexibility in embryonic stem cells of mouse and human Batista et al. Although cells can discriminate between self modified and non-self unmodified RNAs, epitranscriptome plays an important role in immune responses also Kariko et al.
A study on the precursor cells of neurons revealed that m 5 C regulates differentiation and motility of neural stem cells in mice and humans Flores et al. Advances in epitranscriptomics have revealed several potential biological roles of post-transcriptional mRNA modifications Zhao et al. In mouse brain, the m 6 A level was reported to increase throughout the lifespan Meyer et al. Studies have shown the role of m 6 A accumulation in learning and memory in mouse mediated by Ythdf1 binding in response to stimuli Shi et al.
Moreover, a recent study suggests the stress-mediated regulation of m 6 A accumulation in patients with depression, indicating that the dysregulation of m 6 A is associated with the development of mental disorders Engel et al.
An impaired build-up of m 6 A disrupts sex determination in Drosophila , and it causes embryonic-lethality in plants Haussmann et al. Moreover, reduced accumulation of m 6 A inhibits the differentiation of embryonic stem cells in mammals Batista et al. Some of the studies also suggest that the presence of m 6 A in mRNA plays a crucial role in spermatogenesis in mice Mus musculus L.
Hsu et al. Knockdown of MTB in Arabidopsis was reported to cause a considerable reduction in height of the plant, while hypomorphic vir allele produced defective roots and the VIR null mutants were observed to be embryo-lethal Ruzicka et al.
A distinct pattern of m 6 A accumulation was observed in different organs of Arabidopsis , which suggests that m 6 A plays a role in organogenesis and it has tissue-specific functions Wan et al. A wild-type ALKBH10B could restore the alkbh10 mutant phenotype, suggesting that m 6 A is an important regulator of flowering time in plants. The regulatory function of ECTs in leaf morphogenesis and trichome development has recently been demonstrated Arribas-Hernandez et al.
Transcripts of the genes in ect2 mutant get degraded at an accelerated rate and affect trichome branching Wei et al. Findings suggest that m 6 A might be involved in tissue-differentiation in plants. A loss-of-function mutation in OsFIP resulted in the early degeneration of microspores, irregular meiosis in prophase I. Tomato slalkbh2 mutants showed delayed fruit ripening phenotypes and increased m 6 A content compared with the wild-type plants Zhou et al. Loss of function mutation in Arabidopsis for TRM4B an m 5 C writer resulted in defective root phenotype because of the decreased content of m 5 C in the genes involved in root development Cui Q.
The m 5 C content was reported to decrease under drought and heat stress in Arabidopsis Cui Q. Similarly, m 6 A content was reported to decrease in drought stress Zhou et al. The findings indicate that m 6 A and m 5 C play important roles in post-transcriptional regulation of gene expression in plants. Several studies on the writers, readers, and erasers in plants demonstrate that mRNA modification is an important molecular mechanism for regulating plant development and environmental responses Shen et al.
Despite the progress in understanding the functions of m 6 A and m 5 C in plants, the mode of action of their writers, readers, and erasers are yet to be discovered. Post-transcriptional modifications in RNA bases have been reported to play essential roles in various functional RNAs.
These modifications alter the structure, processing, and functions of RNAs. A comprehensive understanding of the biochemical modifications in mRNA bases and the changes in accompanying non-covalent interactions is required to gain insights into the functional diversity. Although marvelous progress has been made in understanding the modified mRNA bases Liang et al. Detection of the modified RNA base helps understanding its dynamics and biological functions.
Modern high-throughput technologies together with the conventional methods Table 2 are expected to advance the field of epitranscriptomics by generating data and discoveries. However, most of the current methods of detecting modified base are specific for a particular modification but recently Khoddami et al. In this section, we present an overview of the technological advancements in the detection methods, their applications, and their limitations.
TLC separation of bases can be performed in one-dimension 1D or two-dimensions 2D using microcrystalline cellulose as a stationary phase.
The sensitivity of the method can be increased by using radioactive 32 P labeling [site-specific cleavage and radioactive labeling, ligation-assisted extraction, and thin layer chromatography SCARLET ] to detect the modification within an individual transcript Liu et al. The method has been extensively used earlier for the detection and quantification of modified RNA bases.
MS coupled to a nano-chromatography system reduces the amount to picomole of the sample required. Ross et al. Reverse transcription RT -based techniques use the primer-extension method to reveal the modified RNA bases.
However, comparative sequence analysis with unmodified RNA transcripts is necessary to eliminate structural RT-stops. The advantage of the RT-based technique includes its applicability and sensitivity to a complex mixture of mRNAs, but it requires pure and concentrated mRNA molecules.
Nevertheless, inosine cannot be detected directly by the RT-based technique, as it can base-pair with cytosine. However, inosine-specific cyanoethylation treatment, using acrylonitrile, converts inosine into N 1 -cyanoethylinosine ce 1 I which disable base pairing of inosine with cytosine.
The advances in next-generation high-throughput sequencing technologies for the detection of RNA base modifications have considerably improved the epitranscriptomic studies Li et al.
However, the read length remains shorter — nt in most of the cases. The BS-seq method combines bisulfite conversion followed by NGS to map m 5 C, which has been successfully used for epitranscriptomic analyses in several animals and plants Squires et al. Although the BS-seq method precisely identifies the site of m 5 C at single-base resolution Figure 3A , it possesses two technical disadvantages. First, the BS-seq fails to distinguish between m 5 C and other modified cytosine bases e.
Second, bisulfite treatment during sample preparation causes degradation of mRNA and thus impedes amplification of m 5 C—containing mRNA which limits the applicability of this method. Figure 3. Detection of modified bases in mRNA. A Bisulfite sequencing BS-seq for the detection of 5-methylcytosine m 5 C. Purified mRNA is fragmented into small — nt fragments, and subjected to bisulfite treatment.
Bisulfite treatment causes converts cytosine C to uracil U , but m 5 C remains unchanged. Presence of C is detected by sequencing, wherein it is replaced by T. B Purified mRNAs are fragmented into — nt, followed by immunoprecipitation using anti-m 6 A antibody to enrich the sample with fragments containing the modified base, library preparation, and high-throughput deep-sequencing for detection of m 6 A.
C Purified mRNAs are fragmented followed by immunoprecipitation using anti-m 5 C antibody of the fragments containing the modified base, library preparation, and sequencing. 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. The single-molecule sequencing approach uses either of the two NGS principles. Such technologies are useful for analyzing modified bases in mRNA, particularly in a context-specific manner, as these methods allow direct sequencing of mRNA without converting it into DNA such conversion causes the loss of modified base. However, these technologies require extensive optimization for their use in the detection of modified bases in RNA.
Further optimization of the single-molecule sequencing technologies will revolutionize epitranscriptomic research on modified bases in mRNA. Recently, Fang et al. Subsequently, they could demonstrate that this method can be successfully used to identify the regulators of other mRNA modifications such as m 1 A. RNA base modification, particularly m 6 A, is a widespread epitranscriptomic change that influences nearly every aspect of mRNA biology. Our understanding of the RNA base modification has been facilitated by the recent developments in the use of an antibody to immunoprecipitate RNAs containing modified base, and the high-throughput sequencing technologies.
However, these antibody-based detection methods cannot detect hm 5 C and ac 4 C at single-base resolution, but the success in the single-base resolution of m 6 A by sequencing might help to optimize the method Yuan et al.
This enables studying the dynamics of epitranscriptome, a post-transcriptional regulatory mechanism for gene expression. To detect m 5 C in mRNA, bisulfite-based technique cannot be used with much success.
In this method, an anti-m 5 C antibody is used to immunoprecipitate and enrich the modified base containing mRNA fragments, followed by library preparation and sequencing. This approach exploits the enzymatic activity of m 5 C methyltransferase containing a cysteine-to-alanine mutation CA in NSUN2 which inhibits release of the enzyme from protein—RNA complex.
This results in a covalent bond between the enzyme and its RNA targets. Antibody specific to the RNA-bound protein is used to immunoprecipitate the fragments containing the modified base, followed by library preparation and sequencing. Many of the RNA base modification detection methods rely on the use of antibodies for immunoprecipitation. However, an antibody may fail to distinguish between two different modified forms of a nucleobase, such as m 6 A and m 6 Am.
Moreover, the methods are dependent on the specificity of the antibody, which emphasizes the desire for the antibody-free method to draw transcriptome-wide atlas of the modified base. Hong et al. Moreover, two chemical labeling methods viz. Wang et al. In a transcriptome-wide m 6 A-SEAL-seq analysis, they could identify 8, m 6 A in human embryonic kidney and 12, m 6 A in rice leaf.
Currently, most of the epitranscriptomic studies employ a detection method with NGS technology for context-specific mapping of the modified base at single-base resolution. A major challenge in detection of the modified mRNA base has been the relatively low count of the modified base within the vast mRNA repertoire Another challenge is the precise quantification and mapping of modified RNA residues at a single-nucleotide level.
Additional challenge stems from substantial background signals often present in the maps prepared. Besides, there are many other limitations including intrinsic bias on secondary structures. For example, the m 6 A specific-antibody fails to distinguish between m 6 A and m 6 Am Schwartz et al. Moreover, current sequencing technologies have not been able to detect hm 5 C and m 1 A, particularly at the single-base resolution, which limits the functional characterization of these modified bases.
Some of the challenges in the detection of modified RNA bases at technological, experimental, and analytical levels are described here. Sequencing by synthesis approach has many restrictions in detecting the base modification. The specific antibody or chemical required for the detection of a modified base indirect detection of the modified base is known for only a limited number of modifications, which may show cross-reactivity.
The antibody-based immunoprecipitation IP sequencing method e. The location of m 6 A is inferred near the antibody crosslinking point the tyrosine residue of antibody and RNA base , but the crosslinking point might be at varying distance from the m 6 A-binding sites, which creates difficulty in precise identification of the m 6 A site, particularly when m 6 A occurs in a cluster Meyer et al.
Even the direct detection methods like SMRT face certain challenges such as the ZMW stumbles when a stretch m 6 A gets incorporated; hence, the current throughput level is too low for transcriptome-wide analysis.
Recent studies have provided unprecedented mechanistic insights into RNA base modifications, and NGS-based technologies for detecting RNA base modifications are further improving the scenario. Modern chemical biology tools would be applied to expedite the epitranscriptomic studies. High-throughput technologies to simultaneously identify different modified bases in the same RNA molecule will have considerable applicability, as modified bases may have cumulative effects on regulating biological functions.
Plants provide a unique system to elucidate the biological functions of modified RNA bases and their regulatory aspects through investigating epitranscriptomic alterations in higher eukaryotes, which are otherwise difficult to be elucidated using an animal system Shen et al.
Using a combination of techniques including genetic ablation and NGS-based mapping, the regulatory roles of the epitranscriptome in several developmental processes in plants have been demonstrated.
Compared with the writers and erasers, readers for the modified base play a more significant role in responses to environmental stresses. Therefore, it is important to characterize the role of reader proteins in the epitranscriptomic regulation of gene expression under environmental stresses Hu et al. Association between epitranscriptome and stress responses in plants indicates that such epitranscriptomic marks might be utilized in the future as important epimarks for the development of stress-tolerant crop plants Vandivier and Gregory, In the line of the success in the detection of m 6 A at single-base resolution, a similar sequencing method would be optimized for hm 5 C and m 1 A mapping.
Nevertheless, for functional characterization of epitranscriptomic modifications, quantification of the absolute stoichiometry of RNA modifications is crucial. However, several questions need to be answered before we can devise appropriate strategies to better utilize the epitranscriptomeic information.
Some of these include, why only selected mRNAs get modified? How does the modified base affect downstream mRNA processing? How do different readers recognize their targets? Even if we get answers to some of these questions, several other questions would require to be answered. Experiments designed to answer some of these questions are underway in laboratories worldwide, and we expect that the next 5 years of research in epitranscriptomics would be more exciting than the past!.
During the past few years, many RNA modifications and their functional versatility could be discovered due to the advances in chemogenetic RNA labeling techniques, high-throughput NGS, and functional validation.
Several other dynamic base modifications in mRNA are also being identified, which would require functional characterization for advances in epitranscriptomics. Numerous other epitranscriptomic modifications may be identified in the future which may show interaction with other modified bases in modulating metabolic pathways. The biological functions of several mRNA base modifications are still poorly understood, their detection at single-base resolution using technological advancements such as nanowell SMRT and nanopore Oxford Nanopore sequencing is very much promising.
However, proper experimental design with a sufficient number of replications, and inclusion of controls would be very important to rule out false-positive results and for the highest confidence level. Moreover, identifying the enzyme s involved in modification of RNA base reader , and replacing it with an unmodified base eraser is necessary for devising strategies to manipulate the expression of a gene.
However, several fundamental questions remain to be answered, including whether modified bases are conserved among plant species. Answering these questions would substantially improve our knowledge of epitranscriptomics and its effects on plant growth, fitness, and survival under environmental stress. SK and TM conceived the review. SK prepared the manuscript. SK and TM revised the manuscript and approved the final draft. Both authors contributed to the article and approved the submitted version.
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