A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop

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FIGURE 2.
FIGURE 2.

ABP140 is required for m3C modification of tRNAThr and tRNASer. (A) Lack of the m3C primer extension block is due to lack of ABP140. ORFs YOR239W and/or YOR240W were deleted by transformation of a wild-type strain (BY4741), and 2 μg RNA from these strains was assayed for the m3C primer extension block after transformation with a CEN ABP140 plasmid (lanes a,c,e), or with an empty vector (lanes b,d,f), as indicated. (Lane g) No RNA. Note that the m3C and m2,2G arrows point to the residue immediately preceding the modified residue that causes the primer extension block. (B) ABP140 is required for m3C modification of other tRNAThr species. Bulk RNA (2 μg) from a wild-type strain (lanes b,e,h) and an abp140-Δ strain (a,d,g) was assayed by primer extension using 5′-labeled primers designed against tRNAThr(IGU) (a–c), tRNAThr(UGU) (d–f), and tRNAThr(CGU) (g–i). (Lanes c,f,i) no RNA controls; *, unexplained primer extension stop or pause in tRNAThr(CGU). (C) ABP140 is required for m3C modification of tRNASer(CGA), tRNASer(UGA) and tRNASer(GCU). Bulk RNA (2 μg) from a wild-type strain (lanes a,d) and an abp140-Δ strain (b,e) was assayed by primer extension using 5′-labeled primers designed against tRNASer(CGA) (a–c), tRNASer(UGA) (d–f), and tRNASer(GCU) (g–i). (D) HPLC analysis of tRNAThr(IGU) purified from an abp140-Δ (trm140-Δ) strain. tRNAThr(IGU) prepared from wild-type and abp140-Δ strains was digested to nucleosides and chromatographed on an HPLC column, as described in Materials and Methods.

This Article

  1. Published in Advance April 25, 2011, doi: 10.1261/rna.2652611 RNA 17: 1100-1110

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