DEVELOPMENT OF A REPORTER CONSTRUCT FOR RNA STABILITY MEASUREMENT IN HEK 293 CELLS

Methyltransferase-like 3 (METTL3) is an enzyme associated with mRNA stability and regulation of translation through N⁶-methyladenosine (m⁶A) modification. METTL3 has been implicated in cancer cell growth and survival. We established a strong reporter for RNA methylation that we intend to use to demonstrate the genes responsible for the regulation of METTL3. Our reporter consists of a green fluorescent protein (GFP) control gene and mCherry gene with the m⁶A target sequence in its 3′ UTR. Here, we introduced short hairpin RNA (shRNA) using a lentiviral vector to knock down METTL3 expression in Flp-In 293 cells (Flp-In cells). We found that decreasing METTL3 expression in the Flp-In cells increased the stability of mCherry compared to the GFP control; this suggests that our reporter is viable. We plan to use this reporter in a genome-wide screening to uncover the regulators of METTL3. These data will offer additional insight into the m⁶A methylation pathway and provide the foundation for more thorough investigation of the interaction between METTL3 and other proteins.