mRNA Stability Controlled by Dynamic Methylation at Transcription Start Site

February 2017

TriLink had the honor of hosting Dr. Samie Jaffrey of the Weill Cornell Medical College on January 27th. Dr. Jaffrey traded the chill of New York City for the San Diego sun and delivered a captivating talk at TriLink on his Lab’s pivotal research supporting his recently published Nature paper on epitranscriptomics.

The idea that heredity is influenced by more than just the genetic code has been of interest to scientists for decades. Though epigenetics was coined in 1942 to describe a theoretical model of how genes may interact with the environment to produce a phenotype, it wasn’t until the 1990s that epigenetics was linked to post-transcriptional modification of DNA, thanks to advancing biological techniques.

Epigenetics, which literally means "on top of genetics", is now a relatively well-established field that has led to important insights into heredity; indeed, many have come to consider epigenetics as the link between nature and nurture.

However, research in epitranscriptomics, which refers to the post-transcriptional modification of mRNA, has significantly lagged in comparison. In fact, proof that mRNA is systematically modified post-transcriptionally, has only come to light in recent years.

Early studies indicated that as much as 25% of mRNAs contain adenosine that is methylated at the N6 position (N6-methyladenosine [m⁶A]). More recent studies have highlighted various enzymes with demethylation activity at these sites, suggesting that the epitranscriptome is highly dynamic. However, the functions and mechanisms are still unclear.

In a recent paper published in Nature, researchers from the Jaffrey Lab sought to better understand epitranscriptomics by focusing on the function of methylation sites within the 5 cap, a structure important for initiation of transcription and the stability of the resulting mRNA.

The 5 cap structure contains 5-5 triphosphate linkage between the 5 end of the RNA and a methylated guanosine nucleotide (m⁷G). The first nucleotide adjacent to the cap and sometimes the second nucleotide are also often methylated at the 2 hydroxyl site.

In previous studies, it was reported that if the first nucleotide adjacent to m⁷G is 2-O- methyladenosine (Am), the nucleotide is often methylated at the N6 position as well (m^6.Am). However, the function and mechanism of this methyl group is unknown.

In this study, led by Jan Mauer, the researchers sought to understand the function of m^6.Am at the transcription start site and the mechansims of (de)methylation. Initial experiments indicated that demethylation of m^6.Am to Am at this site occurs through fat mass and obesity–associated protein (FTO), which is an enzyme previously reported to demethylate A⁶m.

Furthermore, they found that m^6.Am is a preferred FTO substrate, but only at transcription start sites, suggesting a specialized function for m^6.Am at this site. Internal m^6.Am was not demethylated by FTO, even at high concentrations. This was confirmed in vitro and in vivo.

The authors then noted that mRNA containing m^6.Am display increased stability when compared with mRNA that start with Am Gm Cm Um. Indeed, the average half-life increased by 2.5 hours. In line with this observation, they detected increased levels of mRNA with m^6.Am when compared to other mRNAs

Final experiments showed that m^6.Am may provide stability because it confers resistance to decapping by enzyme DCP2. Because decapping of mRNA is essential to microRNA-mediated degradation, the group then tested whether presence of m^6.Am impacts this process. Consistent with their hypothesis, mRNAs containing m^6.Am were less susceptible to microRNA-mediated degradation in HeLa and Hek293 cells.

Featured Product: Custom oligonucleotide containing internal m⁶A. TriLink specializes in manufacturing custom, highly modified mRNA, RNA, DNA. Contact us for a custom quote!