Evolving specificity of tRNA 3-methyl-cytidine-32 (m3C32) modification: a subset of tRNAsSer requires N6-isopentenylation of A37

Abstract

Post-transcriptional modifications of anticodon loop (ACL) nucleotides impact tRNA structure, affinity for the ribosome, and decoding activity, and these activities can be fine-tuned by interactions between nucleobases on either side of the anticodon. A recently discovered ACL modification circuit involving positions 32, 34, and 37 is disrupted by a human disease-associated mutation to the gene encoding a tRNA modification enzyme. We used tRNA-HydroSeq (-HySeq) to examine (3)methyl-cytidine-32 (m(3)C32), which is found in yeast only in the ACLs of tRNAs(Ser) and tRNAs(Thr) In contrast to that reported for Saccharomyces cerevisiae in which all m(3)C32 depends on a single gene, TRM140, the m(3)C32 of tRNAs(Ser) and tRNAs(Thr) of the fission yeast S. pombe, are each dependent on one of two related genes, trm140(+) and trm141(+), homologs of which are found in higher eukaryotes. Interestingly, mammals and other vertebrates contain a third homolog and also contain m(3)C at new sites, positions 32 on tRNAs(Arg) and C47:3 in the variable arm of tRNAs(Ser) More significantly, by examining S. pombe mutants deficient for other modifications, we found that m(3)C32 on the three tRNAs(Ser) that contain anticodon base A36, requires N(6)-isopentenyl modification of A37 (i(6)A37). This new C32-A37 ACL circuitry indicates that i(6)A37 is a pre- or corequisite for m(3)C32 on these tRNAs. Examination of the tRNA database suggests that such circuitry may be more expansive than observed here. The results emphasize two contemporary themes, that tRNA modifications are interconnected, and that some specific modifications on tRNAs of the same anticodon identity are species-specific.

Keywords: anticodon loop; isopentenylation; tRNA-HySeq.

FIGURE 1.

The S. pombe tRNAs for serine and threonine have m³C32 modification. (A) A cartoon of schematized tRNA anticodon loop with different nucleotide positions indicated as referred to in the text. (B) The tRNA^SerUGA and tRNA^ThrUGU track profiles from IGV display software showing misincorporations as colored bars (Arimbasseri et al. 2015). IGV introduces color by default if ≥15% mismatch is detected relative to the reference gene sequence. Color key: green, A; red, T; orange, G; blue, C. Sequence read counts are indicated on the y-axis. (C) Plot showing C32 misincorporation levels for all S. pombe tRNAs that have C at position 32. Error bars indicate standard deviation of four replicates. The tRNAs^Ser and tRNAs^Thr are further grouped according to base identity at position 36; note that three of the four tRNAs^Ser contain A36 while the other tRNA^Ser contains U36 as do all of the tRNAs^Thr. (D) Heat map showing fractions of each nucleotide misincorporated at C32 for the seven tRNA anticodons to the right.

FIGURE 2.

Two sequence homologs for TRM140 in S. pombe. (A) Cartoon showing the general domain architecture of S. cerevisiae TRM140 and the two S. pombe homologs. Position of +1 frame shift in S. cerevisiae gene is indicated as +1 FS, which corresponds to amino acid 301; amino acid numbering is further indicated in panel B. (B) Alignment of the two S. pombe homologs of TRM140 with the S. cerevisiae protein. Only the C-terminal domain of the S. cerevisiae protein (starting from 301) is shown. Asterisks indicate amino acids D466, D547, and the horizontal line indicates the C-terminal 20 amino acids important for catalytic activity of TRM140 (Noma et al. 2011). (C) Phylogenetic tree of trm140⁺ and trm141⁺ homologs in different species generated from an alignment created using MUSCLE in Jalview (Edgar 2004; Waterhouse et al. 2009).

FIGURE 3.

S. pombe trm140Δ and trm141Δ are each partially deficient in m³C and can be rescued by trm140⁺ or trm141⁺. (A–H) Mass chromatograms of m³C and m⁵C from total RNA from WT (wild type), trm140Δ, trm141Δ and the double mutant, trm140Δ trm141Δ, cells transformed by empty plasmid pRep4X (A–D), or with Rep4X expressing trm140⁺ or trm141⁺ (E–H). The y-axes indicate abundance and the numbers associated with the peaks indicate quantities for m³C and m⁵C, which were used to calculate the m³C/m³C value given in each panel; the value of 0.2091 in the WT cells was set as 100%. (The high levels of both m³C and m⁵C in G reflect a generally higher concentration of free nucleosides in that digest as indicated by its adenosine content relative to others samples [not shown]). (I) Bar plot showing C32 misincorporations for individual tRNAs^Ser and tRNAs^Thr in trm140Δ, trm141Δ, and WT cells. Error bars indicate standard deviation of four replicates for WT and two replicates each for trm140Δ and trm141Δ.

FIGURE 4.

m³C32 modification in mouse and human cells. (A) Heat map of 121 unique mouse embryonic fibroblast (MEF) tRNA sequences with C at position 32, 34 of which have significant, ≥0.1, misincorporation at position 32. (B) Bar plot of the 34 tRNAs from A that have ≥0.1 misincorporation at C32. (C) Bar plot for all unique tRNAs that show significant C32 misincorporation in human A549 cells. (D) Bar plots for all unique tRNAs from MEFs and A549 cells that show significant misincorporation at C47:3. Error bars indicate standard deviation of two replicates each for MEFs and A549.

FIGURE 5.

Loss of i⁶A37 from tRNAs^Ser causes their loss of m³C32. (A,B) IGV display tracks for tRNA^SerUGA (A) and tRNA^ThrUGU (B); colors as in Figure 1; note the absence of C32 misincorporations from the tit1Δ cells of A but not B. (C) Bar plot of C32 misincorporations in the mutants indicated. Brackets with asterisks indicate the three Tit1 substrates, all of which have A at position 36 and differ from the four other m³C32-containing tRNAs which contain U at position 36. Error bars indicate standard deviations of four replicates each for WT and tit1Δ, and two replicates each for tit1Δ+tit1-T12A and tit1Δ+tit1⁺. (D) Mass chromatograms of m³C and m⁵C in WT and tit1Δ cells; numbers indicate abundances. (E) Pie chart showing the A37 modification status of tRNAs that carry m³C32; compiled from data in the tRNA modification database (UK, unknown) (Jühling et al. 2009).