TimeLapse-seq after amino acid starvation in Mettl3 knockout MEF

Degradation of mRNA containing N6-methyladenosine (m6A) is essential for cell growth, differentiation, and stress responses. Here, we show that m6A markedly alters ribosome dynamics and that these alterations mediate the degradation effect of m6A on mRNA. We find that m6A is a potent inducer of ribosome stalling, and these stalls lead to ribosome collisions that form a unique conformation unlike those seen in other contexts. We find that the degree of ribosome stalling correlates with m6A-mediated mRNA degradation, and increasing the persistence of collided ribosomes correlates with enhanced m6A-mediated mRNA degradation. Ribosome stalling and collision at m6A is followed by recruitment of YTHDF m6A reader proteins to promote mRNA degradation. We show that mechanisms that reduce ribosome stalling and collisions, such as translation suppression during stress, stabilize m6A-mRNAs and increase their abundance, enabling stress responses. Overall, our study reveals the ribosome as the initial m6A sensor for beginning m6A-mRNA degradation. Overall design: TimeLap-seq was performed as previously described. Briefly, cells were grown to 80% confluency. Cells were washed with PBS for three times, and then incubated in amino acid replete or amino acid-depleted media for 3 h with 500 µM 4-thiouridine (4sU, SigmaAldrich, T4509). After 3 h, total RNA was collected using Qiagen RNeasy Mini kit RLT buffer (Qiagen, 74104) supplemented with 1% ??-mercaptoethanol (BME). Cells were lysed by passing through a 22-gauge needle, and RNA was isolated following the manufacturer's instruction with RPE buffer supplemented with 1% BME. 10 µg total RNA was treated with 2,2,2-trifluoroethylamine (TFEE) and sodium periodate (NaIO4) at 45 °C for 1 h to convert 4sU to N4-trifluoroethylcytosine. RNA was then purified twice using RNAClean beads (Beckman Coulter, A63987). 1 µg of treated RNA was used for RNA-seq library preparation using NEBNext®rRNA Depletion Kit(NEB, E6310X) and NEBNext®Ultra™II Directional RNA Library Prep Kit (NEB, E7760L). Library quality was assessed on Agilent TapeStation4200 using High Sensitivity DNA ScreenTape (Agilent, 5067-5584). Library was quantified on Qubit4 Fluorometer using Qubit 1X dsDNA HS Assay Kit (ThermoFisher, Q33231). Libraries were sequenced with pair-end 2x100 cycles on Illumina NovaSeq 6000.