Unlike DNA or histone methylation, knowledge about N6-methyladenosine (m6A) modification of RNA is a little cloudy. It’s involved in some cancers and other diseases, but we don’t know as much about its expression in living cells. Now, a new method called GEMS makes m6A shine bright like a diamond for drug discovery and delivery of therapeutics.
With GEMS (“genetically encoded m6A sensor”), Kate Meyer’s lab at Duke University School of Medicine brought some sparkle to their DART-seq approach by adding fluorescence. This new twist on DART-seq is compatible with high-throughput screening (HTS) techniques and living cells. It doesn’t require RNA isolation or specialized equipment. Here are the rich details of the approach:
- There are two parts: a fusion protein and a reporter sequence
- Like DART-seq, GEMS relies on a fusion protein called APO1-YTH that directly binds to m6A sites and edits nearby cytosines to uracils
- With GEMS, there’s also a reporter sequence that includes EGFP, then a special sensor sequence, and finally a tweaked destabilization domain from the E. coli dihydrofolate reductase (DHFR) gene
- When the reporter isn’t methylated, the fusion protein doesn’t bind
- So, EGFP-DHFR is made, but it degrades
- When the reporter is methylated with m6A in the sensor sequence, the C-to-U changes produce two stop codons between the EGFP and DHFR sequences
- Only the fluorescing EGFR protein, which is stable, is made
Human HEK293T cells expressing both the fusion protein and the reporter sequence were blinged out with fluorescence, whereas those with only the reporter were dark. In addition, reporter methylation and fluorescence levels depended on the levels of METTL3, the transferase responsible for m6A, and the system worked in various human and mouse cell types.
METTL3 is a potential therapeutic target, but it’s been difficult to study its inhibition in living cells with HTS approaches like fluorescence methods. The researchers showed that a small molecule inhibitor of METTL3 reduced GEMS fluorescence, but it was difficult to see dynamic changes because the EGFP was so stable. There could also be some unwanted side effects because the fusion protein edits endogenous m6A RNA, as well as the reporter. So, the team made a few brilliant tweaks:
- Adding a PEST degradation sequence to the EGFP coding sequence reduced the sparkly protein’s half-life a bit, making it easier to detect changes in methylation
- Adding dCas13 to the fusion protein and expressing a guide RNA specific for the reporter sequence made the system much more targeted
Instead of reducing METTL3 levels, another way to potentially treat disease would be to specifically express therapeutic proteins. The team showed that GEMS could do just that by replacing EGFP with the coding sequences for tumor suppressor proteins in human Huh-7 carcinoma cells. Cell growth slowed, and the cells migrated less than cells with the regular GEMS system.
The team concludes that GEMS is a real showstopper, perhaps even a “loud” luxury, that shines a colorful light on m6A modification in living cells, and it could someday be used to develop new therapies.
Check out this luxurious advance at Nature Biotechnology, January 2024.