Fig 1.
Schematic of approaches to identify tRNA modification enzymes.
(A) Expression of tRNA and candidate genes in S. cerevisiae. An exogenous tRNA known or expected to receive the modification of interest, and a candidate modification gene are co-expressed in a yeast strain lacking the modification. RNA is extracted from the yeast and analyzed by primer extension with a fluorescent oligonucleotide specific for the tRNA to determine if the modification is present. (B) Analysis of D. melanogaster tRNA in dsRNA-treated S2R+ cells. S2R+ cells are treated with dsRNA complementary to the gene of interest, and tRNA is analyzed using fluorescent primer extension to detect the modification.
Table 1.
Yeast strains used in this study.
Table 2.
Plasmids used in this study.
Table 3.
Tye665-labeled oligonucleotides used for primer extension.
Table 4.
Oligonucleotides used for qRT-PCR.
Table 5.
Oligonucleotides used for D. melanogaster dsRNA generation.
Fig 2.
Detection of m2,2G26 by fluorescent primer extension in yeast cells.
(A) Schematic of S. cerevisiae tRNATyr. Location of primer binding is shown in blue. Selected nucleotides are numbered in red. (B) Detection of m2,2G26 by fluorescent primer extension in yeast cells. Left, bulk RNA was extracted from indicated strains and analyzed by primer extension to yeast tRNATyr. Selected nucleotides from the sequencing reactions are provided on the left of the gel. Right, bulk RNA was extracted from culture of indicated individual strains or culture from mixtures of strains in indicated ratios, and then analyzed by primer extension to yeast tRNATyr.
Fig 3.
Identification of TRM1 genes from A. thaliana in yeast cells.
(A) Schematic of A. thaliana tRNALeu(AAG)-G37C. Location of primer binding is shown in blue. G37C mutation is boxed. Predicted location of m2,2G26 (cyan) and acp3U20b (magenta) modifications are circled. Selected nucleotides are numbered in red. (B) A. thaliana tRNALeu(AAG)-G37C is expressed in yeast cells. RNA was extracted from yeast expressing the indicated plasmid (vertical label) and analyzed by Northern blot to the indicated probe (horizontal label) as described in Materials and methods. (C) TRM1a or TRM1b expression results in a primer extension block consistent with m2,2G26 on A. thaliana tRNA. Bulk RNA was extracted from a trm1Δ yeast strain expressing the indicated plasmids and then analyzed by primer extension to A. thaliana tRNALeu(AAG)-G37C.
Table 6.
Relative mRNA levels of A. thaliana TRM1 homologs expressed in yeast.
Fig 4.
CG6388 encodes the TRM1 enzyme in D. melanogaster.
(A) Schematic of D. melanogaster tRNATyr. Location of primer binding is shown in blue. Selected nucleotides are numbered in red. (B) TRM1 mRNA levels significantly decrease in dsRNA-treated S2R+ cells. Cells were treated with dsRNA to TRM1 as indicated and mRNA levels were measured by qRT-PCR. RNA levels are expressed relative to actin (ACT42a). (C) Knockdown of CG6388 by RNAi results in loss of a primer extension block consistent with m2,2G26. S2R+ cells were treated twice over 6 days with dsRNA to indicated gene as described in Materials and methods. After harvest of cells, RNA was extracted and primer extension to tRNATyr was performed. (D) Time course of CG6388 knockdown by RNAi. S2R+ cells were treated as indicated, RNA was extracted, and primer extension to tRNATyr was performed.
Fig 5.
CG10050 encodes the DTWD2A enzyme in D. melanogaster.
(A) Sequence alignment of DTWD2 proteins. Green box represents the DTW domain, and arrows denote predicted active site residues. (B) Schematic of D. melanogaster tRNAVal(CAC). Location of primer binding is shown in blue. Selected nucleotides are numbered in red. (C) Knockdown of CG10050 by RNAi results in loss of a primer extension block consistent with acp3U20b on tRNA. S2R+ cells were treated three times over 8 days with dsRNA to indicated genes. After harvest of cells, RNA was extracted and primer extension to tRNAVal(CAC) was performed.
Fig 6.
A. thaliana DTW2A expression results in a primer extension block consistent with acp3U20b on A. thaliana tRNA.
Bulk RNA was extracted from a trm1Δ met22Δ yeast strain expressing the indicated plasmids and then analyzed by primer extension to A. thaliana tRNALeu(AAG)-G37C.
Table 7.
Relative mRNA levels of A. thaliana DTWD2 genes expressed in yeast.