After Genetics and Epigenetics comes Epitranscriptomics!

The word “epigenetics” is everywhere these days, and you might also have come across the word “epitranscriptomics”. But what is actually meant by these terms and why does it matter?

Let’s start with epigenetics: “Epigenetics”, which literally means ‘on top of genetics’, is a term that has taken on multiple meanings in the past. In my understanding, epigenetics describes (heritable) changes in gene expression that do not involve changes to the underlying DNA sequence. Certain epigenetic factors help to define and regulate gene expression in different eukaryotic cell types defining cell function and identity. This regulatory control results from altering the structure of chromatin. Chromatin is made up of nucleosomes, complexes of DNA and histone proteins. Both biomolecules within chromatin can be reversibly modified to affect the structure and accessibility of chromatin towards the transcriptional machinery: DNA can be methylated, and histones can be post-translationally modified (e.g. methylation, acetylation, phosphorylation, ….). As a consequence, genes can be switched on or off in different cell types although the underlying DNA sequence is identical.

Such a role for DNA & histone modification in gene regulation is already well established, and various human cancers are known to have perturbations in the expression of epigenetic writers, erasers and readers which contributes to tumorigenesis.

Much less is known about how RNA modifications affect RNA stability and gene expression. Interestingly, RNA modifications themselves are not new, in fact the first RNA modification m6A (N6-methyladenosine) has already been discovered in the 1970s and in total, more than 150 RNA modifications are known up to date. Six of them have been mapped to mRNA (see table below). However, it was only in 2010 when it was proposed that cells might actively control those RNA marks and might use enzymes that can add, remove or read those RNA marks to regulate gene expression. One year later, the first eraser of one of these RNA marks on mRNA was discovered (i.e. FTO for m6A) demonstrating that m6A is indeed a reversible modification. These “epigenetics of RNA” are now summarized under the term “epitranscriptomics” in analogy to the epigenetic regulation mediated by DNA methylation and histone modification. Analyzing these RNA modifications will provide key insights into whether and how epitranscriptomic regulation is involved in gene expression or disease processes.

Known internal RNA modifications in eukaryotic mRNA

RNA modificationWriterEraserReaderFunction
N6-methyladenosine (m6A)METTL3-METTL14 complexFTO/ALKBH5YTHDF1-3, YTHDC1mRNA structure & metabolism (stability, export, splicing)
5-methylcytosine
(m5C)
NSUN2, DNMT2

 

unknownunknownTranslation efficiency, mRNA structure
5- hydroxylmethylcytidine (hm5C)TETunknownunknowntranslation efficiency
pseudouridine

(Ψ)

 

pseudouridine synthase (PUS, DKC1)unknownunknownmRNA structure, genetic re-coding due to altered base pairing
N1-methyladenosine (m1A)unknownALKBH3unknowntranslation efficiency
2ʹ-O-methylnucleoside (2′OMe)unknownunknownunknownunknown

 

Technological advances about the same time made it also possible to map RNA modifications across the transcriptome. Most RNA modifications were previously invisible to sequencing technologies as the modification was simply erased during the cDNA synthesis step using the reverse transcriptase (e.g. pseudouridine or m5C are RT-silent as they don’t change the base pairing properties of the modified bases). Another challenge is the low abundance of certain RNAs, e.g. mRNA. One way to overcome these limitations is to enrich for the RNA modification using antibodies or chemical approaches before sequencing. The combination of an immunoprecipitation step using a highly specific antibody raised against m6A with high throughput sequencing has been successfully applied for the genome-wide mapping of m6A (m6A-seq and MeRIP-seq) as reported in Nature and Cell. Recently, two single-molecule methods were developed that allow to sequence RNA directly (Nanopore Technologies and SMRT sequencing). Single-molecule real-time sequencing (SMRT) uses the reverse transcriptase from HIV which incorporates fluorescent molecules across modified bases more slowly than it does across unmodified ones. Thus, each modification can be identified due to its own kinetic signature. The Toolbox for studying RNA modifications remains work in progress, but I can highly recommend one recently published review in Nature that gives a comprehensive overview about the currently available epitranscriptome sequencing methods.

The field of epitranscriptomics is still in its infancy, but we are looking forward to many exciting discoveries in the coming years!

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