The modifications in RNA sequence relating to the functional changes of a protein is known as the epitranscriptome. The protein synthesis involves two processes called transcription and translation. The change that occurs in RNA in the post-transcriptional stage results in alteration in function of the proteins.
There are nearly 107 recognized RNA base modifications. The study of RNA base modification is known as epitranscriptomics, which has led to wide research interest. Recently, the methods and techniques for effective description of epitranscriptome process have been progressing particularly for messenger RNA.
Mapping the epitranscriptome
The biochemical approaches combined with RNA sequencing have succeeded in mapping a particular RNA modification in a transcriptome. To attain preliminary methods for transcriptome-wide mapping of m6A, an anti-m6A antibody is utilized which immunoprecipitates the methylated RNA fragments, followed by RNA sequencing. It is very vital to acquire these maps.
The m6A has been found to be rich in mRNAs or in microRNAs, have a conserved function, and be highly dynamic. The sequences of methylated RNA immunoprecipitation (MeRIP-seq) and m6A seem alike at the earlier steps of immunoprecipitation (IP) and RNA preparation methods.
The major variations between the two methods are found in their downstream techniques than those in sample preparation. These methods are identical to the current chromatin IP-seq (ChIP-seq).
Differentiation of MeRIP-seq and m6A-seq
From these methods, the qualitative identical results showed that more m6A RNA modifications are found in introns, 5' UTRs, exons, splice junctions, ncRNAs, and intergenic regions. The study of MeRIP-seq in lincRNAs has found wide range of RNA modifications.
Through m6A-seq, it has been remarked that the genomic characteristics of m6A are specifically enriched at transcription start sites (TSSs) mostly in a single cell line. An evaluation between mouse and human data in both investigations has shown a high preservation of particular m6A sites among the two species. Finally, before MeRIP-seq, digesting the samples with several RNases has proved that m6A sites are frequently found at mRNA’s internal sites and not present at polyA tails.
Peak distribution of MeRIP-seq and m6A-seq
The distribution of the peaks is charted over gene bodies by MeRIP-seq and m6A-seq studies. Further to sequencing, the MeRIP-seq study has also applied immunoblotting to study m6A, showing that m6A appears in heart, lungs, brain, liver and kidney tissues, with specific enhancement in brain, liver, and kidney. m6A has been found in higher concentration in HepG2 and MCF7 cells than that in other cancer cell lines.
The m6A levels increase over the period of growth from embryonic tissue to an adult tissue. The distribution of m6A is altered actively based on various types of stimuli. The reduction of METTL3 subunit reliable for adding methyl groups to adenosines has been utilized in the m6A-seq study to examine the functions after modification.
The reduction leads to an increase in many alternatively spliced transcripts which in turn demonstrate the high level of m6A. From MeRIP-seq study, it has been implied that m6A does not result in total rise in the overall quantity of transcript splicing, but it may alter splicing for particular types of genes, specifically for genes with alternative and internal exons.
Site detection of epitranscriptome
While determining the m6A level in a site, it is necessary to deliberate several factors such as the coding region of a gene, mRNAs (gene isoforms), occurrence of secondary structure, read depth, and alignment pattern result in the extent of the level present. As the epitranscriptomics is a budding field, computational analysis approaches develop and the effect of these factors on detecting and quantifying m6A was investigated.
Overlap of commonly expressed genes with m6A sites
One of the challenges in site detection of epitranscriptome is overlapping of the commonly expressed genes with m6A sites. The overlap can be overcome by excluding the low expression transcription bases.
The 5' UTR peak in the m6A-seq dataset obtained by comparing the peak techniques was weakened when replacing the study's peak-caller with MeRIPPeR from the MeRIP-seq study. The different sensitivities and specificities might be possible in each of the two peak-calling techniques due to the weakness or reduction.
The numerous m6A are found at the end of the first intron and at the start of the next exon. As the occurrence of the m6A sites is not consistent, which relies on the number of exons in a gene and on the framework of an exon within a gene, a valuable technique is required to determine the presence of m6A, or any epitranscriptomic modification, and to classify the genes according to their location.
In the future, these methods will certainly provide a lot of beneficial information for therapeutic approach. The epitranscriptome analysis will help in the study of many diseases that are associated with the changes occurred in RNA. Much research has been carried out in this field.