Methods and compositions to sense m6A RNA modifications using a genetically encoded sensor
Unmet Need
Gene expression is highly regulated through the addition of chemical modifications to RNA molecules. One such modification is methylation of adenosine residues to form m6A, a modified nucleotide which is found in thousands of cellular RNAs and which plays critical roles in cellular function. m6A is deposited in RNAs by methyltransferase enzymes, and recent studies have revealed that dysregulation of m6A and m6A methyltransferase proteins contributes to a variety of human diseases, including cardiovascular disease, infection, and several cancers. Consistent with this, m6A methyltransferase proteins have emerged as promising therapeutic targets for cancer. However, identifying drugs or small molecules that interact with these enzymes is challenging since there are no methods available for directly assessing the effects of candidate drugs on m6A methyltransferase activity at high throughput. Thus, there is a need for an easy to use, high-throughput assay for measuring RNA methylation in cells.
Technology
Duke inventors have developed a novel sensor system that is intended to be used to screen for m6A methylation of mRNAs in living cells. This system contains two components: 1) a modified deaminase enzyme that binds to m6A sites in and catalyzes C-to-U mutations at nearby cytidine residues, and 2) a reporter mRNA. The reporter mRNA contains the coding sequence for a fluorescent protein followed by a short linker region and then the coding sequence for a destabilizing domain. The linker region contains sequences that are susceptible to m6A modification. When this linker region is methylated, the deaminase enzyme binds to m6A and converts nearby cytidines to uridines, which results in in-frame stop codons being introduced into the reporter mRNA. This prevents translation of the destabilizing domain and results in stable fluorescent protein production. In the absence of methylation, the reporter mRNA produces a destabilized fluorescent protein and cells do not fluoresce. Thus, this system provides a simple readout for the presence of m6A (methylation = cellular fluorescence; no methylation = no fluorescence). This technology has been demonstrated to effectively sense m6A cultured cells and to respond to both pharmacological inhibition and genetic disruption of the m6A methyltransferase enzyme.
Other Applications
This technology could also be used to screen for and identify endogenous cellular proteins involved in RNA methylation and demethylation as well as in high-throughput screens for small molecules or other inhibitors of m6A methyltransferase or demethylase enzymes for drug development.
Advantages
- Can be used in a variety of tissues, cells, and animal models to detect the presence of mRNA methylation
- Compatible with current fluorescence-based sorting and screening equipment
- Provides a readout for m6A in living cells