An XGU anticodon and t6A37 are important for m3C modification of tRNAThr(CGU). (A) G35 and U36 of tRNAThr(CGU) are required for m3C formation. Bulk RNA from strains containing integrated tT(CGU)28,50 variants with mutations as indicated were analyzed for m3C by primer extension, using a primer complementary to residues 51–37 of tRNAThr(CGU)28,50. The major read-through band on the top of the gel corresponds to the full-length tRNA. Note that for variants with G35 and U36, the primer extension is slightly displaced, presumably due to a different primer extension sequence. (B) Summary of primer extension results of m3C modification in A. +++, WT levels of m3C; +, low but detectable m3C; ND, m3C not detected. (C) t6A37 is important for m3C formation of tRNAThr species. Bulk RNA from SUA5+ and sua5Δ cells was analyzed by primer extension with primers as indicated, to evaluate the importance of t6A for m3C modification of tRNAThr(CGU), tRNAThr(UGU), and tRNAThr(IGU).