Figure 3.

Incorporation of reactive sulfur atom in ms² modification. (A) Experimental design to identify the sulfur source for ms² in intact cells. The red letters represent the stable isotope-labeled reactive sulfur atoms of CysSSH. (B) HeLa cells were treated with 100 μM of CysSSH or the CysS³⁴SH donor. Subsequently, total RNA was purified and subjected to mass spectrometry. Representative mass chromatograms of ms²t⁶A and m³⁴s²t⁶A are shown. The CysS³⁴SH donor markedly increased the m³⁴s²t⁶A level. (C) The isotopic distribution (relative abundance of m³⁴s²t⁶A to ms²t⁶A) of ms²t⁶A in cells treated with vehicle (CysSSH donor) or the CysS³⁴SH donor. n = 4 each, ****P < 0.0001. The isotopic distribution of ms²t⁶A in response to different (D) doses or (E) periods of treatment with 100 μM of the CysS³⁴SH donor. **P < 0.01, ****P < 0.0001, n = 4 each. (F) Pulse-chasing of m³⁴s²t⁶A showing the relatively slow rate of decrease in the amount of m³⁴s²t⁶A in cells. Data points represent the relative change in the isotopic distribution of ms²t⁶A from four replicates.