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. 2017 Mar;23(3):406–419. doi: 10.1261/rna.059667.116

FIGURE 9.

FIGURE 9.

Ses1 stimulates m3C modification of tRNASer(CGA) and tRNASer(UGA) in vivo and the unique tRNASer V-loop that Ses1 recognizes is important for m3C modification. (A) Ses1 stimulates m3C formation in vivo. Bulk RNA from mod5Δ cells expressing SES1, ORF240, vector, or MOD5 as indicated was analyzed for m3C of tRNASer(CGA)/(UGA) or tRNAThr(IGU) by primer extension in the presence of ddTTP, as described in Materials and Methods. (B) Schematic of tRNALeu(UAA) M1 and M2 variants. The secondary structure of tRNALeu(UAA) M1 is shown, with anticodon loop mutations as indicated (circled). Nucleotides in the V-loop that were changed to the V-loop of tRNASer(GCU) to make the tRNALeu(UAA) M2 variant are boxed. (C) The variable loop of tRNASer(GCU) is sufficient to confer m3C modification in vivo of tRNALeu(UAA) with G35 and A37 mutations. Variants described in B were integrated into a trm732Δ strain, to eliminate the possibility of 2′-O-methylation at C32 (Guy et al. 2012), and bulk RNA from strains containing either variants M1 or M2 was analyzed for m3C by primer extension, using ddTTP and primers extending from residue e8 to 36 of M1 and from residue 47 to 36 of M2.