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Epitranscriptomics describes an aspect of molecular genetics, or a study thereof, that depends on biochemical modifications of RNA. By analogy to the term epigenetics, described as "functionally relevant changes to the genome that do not involve a change in the nucleotide sequence", epitranscriptomics can be defined as a functionally relevant changes to the transcriptome that do not involve a change in the ribonucleotide sequence. The epitranscriptome, therefore, is defined as the ensemble of such functionally relevant changes.

There are several types of RNA modification that impact on gene expression. These modifications happen to all types of cellular RNA including, but not limited to, ribosomal RNA(rRNA), transfer RNA(tRNA), messenger RNA(mRNA), and small nuclear RNA(snRNA). There are more than one hundred documented RNA modificaitons. A database maintained by the University of Albany details each modification. The most common and well understood mRNA modification at present is the N6-Methyladenosine (m6A), which has been observed to occur three times on average on every mRNA molecule.

The relative youth of this field means there is still much progress to be made in characterizing all modifications to  the transcriptome as well as elucidating their mechanisms of action. Once these questions are answered and biologists have a better sense of the amount of RNA modification variation the focus will turn to each modification’s biological functions. This has already been investigated in a select few proteins such as adenosine deaminase that acts on RNA (ADAR1). ADAR1 has been shown to affect antibody production and the innate immune system as well as transcripts encoding important receptors for the central nervous system. This plurality in function has caused some scientists to speculate that the epitransciptome may be even more expansive than the better defined epigenome.

N6-methyladenosine (m6A) on Alternative Splicing
The terms “eraser” and “reader” has been associated with RNA modification. “Eraser” is a general term to describe an enzyme that de-methylates m6A. Changes that mutates the gene encoding the “eraser” enzyme lead to obesity and cancer. “Reader” proteins are involved in gene expression where there are abundant m6A; “reader” proteins have a higher propensity to bind with greater affinity while de-methylated form have been reported to have less affinity.

mRNA are subject to layers of regulatory gene expression. One known mechanism involves forming a RNA stem loops. Stem Loops occur was single-stranded RNA Watson-Crick complementary base pairs within the strand forming an unpaired end or loop. Stem loops does not have one definite function but a plethora of functions however, in case of m6A regulatory mechanism, it is involved in alternative splicing. These stem and loop structures are subject to alterations regarding changes in pH, temperature, ion concentrations, etc.

m6A has been observed to be located within the loops opposite of HNRNPC binding site. HNRNPC protein binds to its site when methylated adenosine is present. HNRNPC is mostly involved in post transcriptional modification such as alternative splicing. HNRNPC binding site on such mRNA consists an abundance of uridine nucleotides. Studies have concluded that methylated adenosine residue destabilizes the hairpin structure, elongating the uridine nucleotide stretch for its site to be more accessible for HNRNPC protein to bind efficiently.

Evidence supporting this claim identified with significance that decreased m6A levels in the transcriptome lead to reduced HNRNPC binding concluding the fact that alternative splicing is co-regulated by methylation and HNRNPC binding activity. However, m6A modification does not directly bind the protein but alters the loop structure that is placed to regulate gene expression, acting more like a switch that exposes the HNRNPC region or not. It is essentially a two-step mechanism in controlling alternative splicing. Note that demethylase enzyme can indeed “erase” the methyl group thus inhibiting alternative splicing therefore, it is a two way regulation also.

Pseudo-seq and the regulation of pseudouridyltaion in yeast and human cells
Pseudouridine is a modified nucleoside found within non-coding RNAs (ncRNA). It increases the function of tRNA and rRNA by stabilizing the structure. Though mRNAs were not known for containing pseudouridine, artificial process of pseudouridylation has an affect on the function of mRNA; it changes the genetic code by making possible non-canonical base pairing in the ribosome decoding centre.

Paper looks at pseudouridylation in yeast and human RNAs using pseudo-seq. A process that utilizes a single-nucleotide-resolution method for pseudouridine identification. It identifies the known modification sites as well as other sites in ncRNAs in addition to the many pseudouridylated sites in mRNA.

There are more than 100 classes of RNA modifications have been found in mostly tRNA and rRNA and only three modified nucleotides have been discovered inside a coding sequence of mRNA (m6A,m5C, and inosine). Research has shown however that in yeast pseudouridines are quite scarce. However,much of the regulation in regards to pseudouridylation is regulated through the environment, in yeast this may be nutrient deprivation and in humans it is the serum starvation. When looking at yeast, research has utilized perturbing pseudouridine synthases deletion strains grown to high density and identified mRNA targets for each PUS protein. Results came back showing that most mRNA targets showed increase modification during post-diauxic growth. The pseudo-seq method identified 96 pseudouridines in 89 mRNAs, similar to yeast the growth of pseudouridine was regulated by cellular growth state. This approach provides an analysis of RNA pseudouridylation with single-nucleotide resolution and shows endogenous mRNAs are specifically pseudouridylated in a highly regulated manner in yeast and human cells. mRNA pseudouridyltaion could also bring a change in translation initiation efficiency, RNA localization, and other processes all cause pseuduridine  stabilizes RNA structure

Epitrancriptome and modualting sections of RNA
The concept of epitranscriptomics has been seen to have an effect not only on RNA but also on protein synthesis. RNA methylase NSun2 methylates mRNAs. This methylation has an effects the components of the postsynaptic neurons. The RNA modification sites are seen to occur at adenosine to create methyl-6-adenosine found around stop codons. Not much is known about the purpose of this methylation but it was found that human patients  lacking NSun2 are characterized by intellectual disability and neural

Engineered RNA Modification Techniques
Modifying RNA is possible using specific enzymes. AlkB is a demethylating enzyme found in E. Coli that removes methyl groups from methylated Cytosine and Adenine nucleotides. This enzyme can be modified to demethylate methyl-Guanosine as well. Modifying the RNA in this way allows for more accurate sequencing, which has medical applications. Given the more-than-100 different modified nucleotides found in nature, AlkB can be used to almost undo the complications introduced by modifying nucleotides in the epitranscriptome, thus allowing the sequencing of heavily modified mRNA strands.