Fig 5. TRM5-mediated m¹I modifications in two-step reaction.

(A) Proposed two-step modification model of TRM5-mediated m¹G modification of tRNA-Ala. (B) Relative nucleoside level of modification m¹I with varying conditions of tRNA-Asp-A37, AtTAD1, AtTRM5 mutant, AtTAD1 mutant, and tRNA-Ala-C37. + indicates presence, ++ indicates two-fold increase,—indicates absence. (C) Qualitative analysis of tRNA-Ala^(TGC) and tRNA-Ala^(CGC) modifications. tRNAs were enriched, deep sequenced, aligned using segemehl to tRNA references and the modifications present were inferred from observed base substitutions between wild type and trm5-1. Position 37 in the gDNA, labelled as consensus, is an adenine. Sequence logo shows the proportion of nucleotides and hence inferred modifications at positon 37. Anticodons TGC and CGC are shown in the sequence logos for Ala^(TGC) and tRNA-Ala^(CGC), respectively. Base substitutions observed from 3 biological replicates are shown. (D) Qualitative analysis of tRNA-Ala^(AGC) modifications by Sanger sequencing. tRNA-Ala^(AGC) was PCR amplified from wild type, tad1-2 and trm5-1 and Sanger sequenced. Position 37 in the gDNA is an adenine. In wild-type cDNA, a thymine was detected, in tad1-2 cDNA an adenine was observed and in trm5-1 a guanine was detected.