An epitranscriptome is a set of functionally appropriate RNA modifications. There are nearly one hundred known modifications of RNA; the most common modification in internal mRNA is N6-methyladenosine (m6A). Transfer RNAs (tRNAs), mRNAs, and ribosomal RNAs (rRNAs) are the three types of RNAs that are post-transcriptionally modified.
DNA and RNA helix. Credit: Andrii Vodolazhskyi/ Shutterstock.com
In all living organisms, translation is a fundamental process. The arrangement and operation of translation process is very expensive and it consumes 40% of cellular energy, which has led to the need for strict regulation of protein production in many aspects.
The regulation of translation is generally related to the need of regulated and non-regulated proteins. Nucleotide modifications are also important for the translation process. RNA base modifications are detected by appropriate sequencing methods.
At a genome level, the study of m6A is possible with the help of recent advances in sequencing technology and the design of m6A antibodies. The sequencing technologies are listed below.
The m6A is the most available base in eukaryotic mRNA. The m6A protocol can be carried out on mRNA. The first step is to acquire a better quality of RNA sample. Polyadenylated RNA is enhanced by oligo-dT selection while the m6A-seq protocol is executed through RNA. Polyadenylated RNA is divided into small fragments with a length of 100 nt each.
Polyadenylated RNA fragmentation is performed chemically to achieve efficient fragmentation. These small fragments are used to reduce the resolution to 200 nt. Remapping of smaller fragments into the genome would become tedious because of further fragmentation.
Post-fragmentation, the sample is put through immunoprecipitation. This small amount of fragmented mRNA is retained aside to perform input control process. In the following data analysis, small fragments are retained at basic level status. The rest of the sample is immunoprecipitated by incubating it with a particular m6A antibody that pulls fragments containing multiple m6A residues.
Recombinant protein A agarose bead, which can bind with the antibodies, is used to incubate the RNA-antibody mixture in the subsequent process. Finally, the unbound fragments are eradicated, bound fragments are removed by addition of m6A to the mixture, and the bead-antibody-RNA complex deposited in solid form.
During immune precipitation, the RNA fragments and beads are combined together in the absence of antibodies. Bioanalyzer analysis and concentration measurements are used to estimate the quality of the material throughout the remaining protocol. Good result can be obtained when better quality starting material is used.
There exist some differences between m6A sequencing and MeRIP-sequencing strategies. The main difference is the category of bead: Magnetic Dynabeads are used in MeRIP sequencing strategy, which is first paired with antibodies and then combined with ribo-minus treated RNA. Here, immune precipitation process is performed twice.
Buffer composition involved in fragmentation also differs. Solvent extraction elution is used instead of competition elution. However, the fundamental concept involved in these two methods is similar to each other.
The PA-m6A sequencing name was coined from m6A sequencing strategy in combination with a PA sequencing strategy. In this method, PA-m6A has the ability of undergoing high-resolution mapping with a mammalian transcriptome m6A. It can be observed that the m6A sequence modification sites are more accurately defined when compared to conventional sequencing methods. The resolution has been reduced from 200 to 23 nt.
PA-m6A sequence is versatile when compared to the single nucleotide approaches. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PARCLIP) strategy is the base for PA-m6A strategy. In PARCLIP strategy, photoactivatable ribonucleosides are integrated into an mRNA and then they are subjected to immunoprecipitation process. It is similar to m6A protocol, but immunoprecipitation is performed with full length RNA and then this RNA is fragmented post-crosslinking through UV irradiation.
Single molecule sequencing
Single molecule sequencing has the ability to provide the base level resolution of m6A sites without the conclusion drawn on the basis of motif. The Single Molecule Real-Time (SMRT) technology is the most common platform for this method of sequencing.
This method was developed by Pacific Biosciences and Oxford Nanopore Technologies. Continuous monitoring of the process, in which DNA polymerase is integrated with fluorescent nucleotides, helps in detecting both genetic and epigenetic information at the same time.
Single molecule sequencing offers some advantages as it can be used to detect RNA modification that includes m6A sites. Single nucleotide resolution is to detect the m6A sites in RNA, which is achieved by using DNA polymerase rather than reverse transcriptase.
It can be used as an enzyme within a ZMW and in allowing direct complementary DNA (cDNA) synthesis in real time. Reverse transcription appears at standard speeds. At the instant of base incorporation, designed m6A sites exhibit important growth of interpulse duration (IPD).
Single nucleotide methods
The above techniques are not useful in the case of determining nucleotide level modification, due to a lower determination capability. Site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography method can be used to overcome this disadvantage. Therefore, the m6A modification is non-stoichiometric.