OTTR ribosome profiling in Mettl14 knockout mESC
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: OTTR ribosome profiling was performed using OTTR cDNA library construction kit (Karnateq, R2201001S). Briefly, cells were grown in a 150 mm dish to 70% confluency. Cells were washed with ice-cold PBS supplemented with 100 µg/mL cycloheximide and 100 µg/mL tigecycline. Cells were lysed in 300 µL lysis buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM, MgCl2, 1 mM DTT, 1 % Triton X, 100 µg/mL cycloheximide, 100 µg/mL tigecycline, and 15 U/mL DNaeI (ThermoFisher, 89836)]. Lysates were passed through a 25-gauge needle five times, incubated on ice for 20 min, and clarified by centrifugation for 10 min at 20,000 x g at 4 °C. RNA in the lysate was quantified using Qubit™ RNA Broad Range kit (ThermoFisher, Q10211) on Qubit4 Fluorometer. 30 µg RNA was digested using 600 U P1 nuclease (NEB, M0660S) in 400 µL supplemented with 42 µL 0.2 M Bis-Tris, pH 6.0 (Life Technologies, J62686.AE) at 37 °C for 1 h. OTTR ribosome profiling libraries were prepared using mirRICH small RNA enrichment method according to the manufacturer's instruction. Final library quality was assessed on Agilent TapeStation4200 using D1000 ScreenTape (Agilent, 5067-5582). Library was quantified on Qubit4 Fluorometer using Qubit 1X dsDNA HS Assay Kit (ThermoFisher, Q33231). Libraries were sequenced with single-end 100 cycles on Illumina NovaSeq 6000.