An exciting time for epitranscriptomics

Epigenetics is a well-established means of gene regulatory control where chemical modifications of DNA bases or their associated histone proteins affect the expression of genes (as opposed to DNA sequence). These epigenetic marks may take the form of methylation (usually on cytosine residues) or histone acetylation/phosphorylation/ubiquitination/sumoylation etc. and can be passed down to daughter cells. Epigenetic changes are induced by the environment and provide a biological mechanism by which nurture as opposed to nature, plays a significant role in shaping our behaviours and characteristics.

Recently, scientists are uncovering the functional significance of epitranscriptomics – or the chemical modification of RNA. The first report of mRNA modification, specifically,  methylation of the N6 position of adenosine (m6A), occurred in 1974 by Fritz Rottman et al.. The m6A modification is the most common eukaryotic mRNA modification however its functional significance remained unclear until the 2010s. In 2012, Dominissini et al. using m6A-antibody enriched RNA-seq, discovered 12000 methylated sites in 7000 coding genes and 250 non-coding genes. Typically highly concentrated around stop codons, within long internal exons and at transcription start sites, it became evident that genes without these modifications tend to be more highly expressed. He writes about it in a Science essay here.

There are several players involved in epitranscriptomics, and they are often referred to as writers, readers and erasers. A methyltransferase METTL3 (“writer“) produces the m6A modification, the YT521-B homology (YTH) domain family of proteins are “readers” that bind to m6A modified RNA and regulate functions that affect protein expression such as RNA degradation, translation and splicing. Finally the “erasers” such as enzyme FTO, implicated in various diseases such as cancer and obesity, removed these m6A marks. Thereby completing the set of actors required for establishing epitranscriptomics as a regulatory mechanism for RNA expression.

Samie Jaffery’s group recently published a controversial paper that identified another epitranscriptomic modification N6,2O-dimethyladenosine (m6Am), located near the 5′ caps of mRNA, that is the main substrate of eraser FTO instead of m6A. m6Am is correlated with increased mRNA stability as it makes 5′ caps harder to remove, which thereby increases protein expression. This is in contrast with m6A modification, which is associated with suppression of protein expression. This highlights distinct functional roles of these RNA methylation marks.

There are already studies demonstrating m6A regulation is utilized also by non-coding RNA. The simple self-made schema below shows how m6A modification was used by Xist to carry out its transcriptional repression of the X chromosome, demonstrated by Patil et al. in a recent Nature publication.

xistm6a

Epitranscriptomics is a relatively young field and opens up new possibilities to study how RNAs are regulated and in particular, how non-coding RNAs may carry out their functions. This signals yet more exciting times ahead for RNA researchers!

Two man-made nucleotide bases produces never before seen semi-synthetic organism

As if altering the genetic code was not enough, now there might be a possibility to completely change the way it was written. Two new nucleotide bases have been created that were stably replicated in bacteria, expanding the possible number of base-pairs to 3 and increasing the coding capacity of DNA exponentially.

In his initial study published in Nature, Dr. Floyd E. Romesberg, a Chemistry Professor at the Scripps Research Institute in California, produced two nucleoside triphosphates called dNaMTP and d5SICSTP, which basepair via hydrophobic interactions instead of hydrogen bonding. A nucleotide transporter had to be expressed in the bacteria in order for their uptake, but once inside, DNA polymerases had no problem incorporating and amplifying the synthetic nucleotides, making copies of an introduced plasmid containing a single base-pair of the unnatural bases.

There were concerns initially that DNA repair mechanisms may work to remove these foreign bases, but that did not seem to be the case. The unnatural bases persisted for more than 3 days after he stopped supplying the bacteria with nucleotides at a retention of 45% (at 0 days, retention was in excess of 95% as suggested by termination of reads by Sanger sequencing) and 6 days at 15%. This led him to guess that their decay over time was due to replication-mediated mispriming instead of DNA repair.

In his most recent study in PNAS, Dr. Romesberg and his graduate student Yorke Zhang made improvements to the nucleotide transporter used, which previously induced some toxicity. They also further optimized one of the unnatural bases to be better incorporated by DNA polymerases, increasing copying efficiency. Finally, to prevent the decay of the unnatural bases over time, they used CRISPR to eliminate the DNA that had lost the unnatural bases. The resulting bacteria was able to indefinitely retain the unnatural bases under various sequence context.

romesberg

Image taken from News Medical Life Sciences‘ interview with Professor Romesberg

The so-called semi-synthetic organism produced therefore uses 3 base-pairs and has 6 nucleotide types instead of 4. It remains to be seen whether its DNA can be transcribed into RNA and then more importantly into protein. But it holds the potential to greatly change the way things are currently done – imagine what we could do with 172 amino acids instead of 20!