S. pombe trm140Δ and trm141Δ are each partially deficient in m3C and can be rescued by trm140+ or trm141+. (A–H) Mass chromatograms of m3C and m5C from total RNA from WT (wild type), trm140Δ, trm141Δ and the double mutant, trm140Δ trm141Δ, cells transformed by empty plasmid pRep4X (A–D), or with Rep4X expressing trm140+ or trm141+ (E–H). The y-axes indicate abundance and the numbers associated with the peaks indicate quantities for m3C and m5C, which were used to calculate the m3C/m3C value given in each panel; the value of 0.2091 in the WT cells was set as 100%. (The high levels of both m3C and m5C in G reflect a generally higher concentration of free nucleosides in that digest as indicated by its adenosine content relative to others samples [not shown]). (I) Bar plot showing C32 misincorporations for individual tRNAsSer and tRNAsThr in trm140Δ, trm141Δ, and WT cells. Error bars indicate standard deviation of four replicates for WT and two replicates each for trm140Δ and trm141Δ.
