Decoding the interplay between m6A modification and stress granule stability by live-cell imaging

We present a spatiotemporal m6A imaging system (SMIS) that can monitor the m6A modification and the translation of mRNAs with high specificity and sensitivity in a single live cell. The SMIS system contains a m6A-modified reporter mRNA and fluorescence resonance energy transfer (FRET) biosensors. To quantify the methylation viariation achieved at m6A sites in the reporter mRNA after STM2457 treatment, a classical METTL3 inhibitor, we performed m6ACE-seq to measure the relative m6A level with single-base-resolusion. HeLa cells ovexpressing SMIS-WT system, namely SMIS-WT cells, were preteated with DMSO or STM2457 for 3 hours and incubated with DMSO or STM2457 (60μM) and DOX (1μg/mL) for 12 hours. Then, the cells was harvested and lyzed for total RNA extraction and mRNA purification. The purified mRNA was further handled according to the protocol of m6ACE-seq that is previously published on nature communication (PMID: 31822664).