Hidden information in mRNA

Published March 01 2017

Modifications of the extended mRNA cap. IMAGE PROVIDED BY SAMIE JAFFREY

Just before the translation step of protein synthesis, the cell protects its messenger RNA with a 5′ methylguanosine triphosphate cap and a 3′ polyadenosine tail. Other modifications can occur within the message itself. One of these, called m6Am, appears on adenosine nucleotides at the start of transcripts and has a methyl group on both its sugar and nitrogenous base. This modification was discovered in the 1970s but received little attention, and its role was unknown.

In a paper published Dec. 21 in the journal Nature, Samie Jaffrey of the Weill Cornell Medical College and colleagues explored m6Am in depth. They determined which enzyme specifically removes this modification and the profound effects this mark has on mRNA stability.

Jaffrey says that the m6Am modification largely was overlooked after its initial discovery. The m6Am modification was present at less than 10 percent of the level of the m6A modification, which also is found in mRNA. The adenosine is modified only at its nitrogenous base and occurs internally rather than at the beginning of the transcript. In addition, FTO, the fat mass and obesity-associated protein, was identified as the “eraser” of the m6Am mark. The m6Am modification fell by the wayside.

But by using analytical chemistry techniques, the Jaffrey group discovered that FTO actually prefers the m6Am modification over m6A. FTO showed a higher catalytic efficiency toward m6Am. In cells, FTO overexpression decreased m6Am levels specifically, and its knockdown increased the ratio between m6Am and Am.

Next the Jaffrey group looked at this modification’s role. While the cap and tail are known both to protect and aid in translation initiation, modifications to the nucleotides themselves have been studied less. Jaffrey calls these marks “hidden information sitting inside RNA molecules” and sought to uncover their significance.

To figure out what m6Am is doing, the group analyzed previously compiled translation efficiency data. This data reveal how well a large group of mRNA transcripts is translated at a given point, explains Jaffrey. The investigators sorted transcripts based on the first nucleotide identity, Am, Cm, Gm or Um, and used specialized mapping techniques to identify those beginning with m6Am. From this analysis, m6Am transcripts showed longer half-life and higher expression levels, an indication of stability.

Next, the group collaborated with Mergerditch Kiledjian of Rutgers University to study the decapping of mRNA transcripts. The 5′ methylguanosine triphosphate cap at the beginning of messenger RNA can get “decapped” by DCP2, the putative enzyme in a large complex. By using a radioactivity assay where the release of methylguanosine diphosphate was detected if DCP2 succeeded in decapping the mRNA, researchers found that transcripts beginning with the m6Am modification were not as easily decapped and destabilized.

Finally, the Jaffrey group explored the phenomenon of microRNA-mediated degradation of mRNA transcripts. This degradation is important, as some transcripts are mysteriously resistant to its effects.

The group again looked at previously compiled translation efficiency data, this time in DICER knockdown cells, a key component of the degradation machinery. Transcripts beginning with m6Am did not change significantly, while others increased, an indication of lowered susceptibility.

Jaffrey marvels at how the stability of these mRNAs was encoded: “It was actually in the nucleotides, something that was invisible to standard methods,” he notes. Next steps from this work include studying how levels of this modification are affected in disease states as well as identifying the methyl-transferase that adds this mark.

For the time being, m6Am is back in the spotlight.

Dawn Hayward Dawn Hayward is a graduate student at the Johns Hopkins University School of Medicine.