Evolving specificity of tRNA 3-methyl-cytidine-32 (m³C32) modification: a subset of tRNAs^Ser 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.

Factors That Shape Eukaryotic tRNAomes: Processing, Modification and Anticodon–Codon Use

“…This suggested that the t 6 A 37 found on the tRNAs Thr and tRNA Ser GCU, might be required for m 3 C 32 formation [133]. Dependency of m 3 C 32 on t 6 A 37 was indeed reported for tRNAs Thr in S. cerevisiae [148] (also see below).…”

“…We highlight two recent cases that involve position 37 of different tRNAs, the yW37 and i 6 A 37 of tRNA Phe and tRNAs Ser , respectively and their interdependent modifications elsewhere in their anticodon stem loop (ASL) [133,134] (Figure 5C). Regarding the position 37 modifications of these tRNAs, while bacterial tRNAs Phe and other bacterial tRNAs that decode UNN codons contain i 6 A 37 , the tRNAs Phe in eukaryotes almost without exception contain the hypermodified G nucleotide, yW37.…”

“…For a subset of tRNAs Ser , the interdependency of positions 37 and 32 is such that i 6 A 37 is prerequisite for formation of m 3 C 32 [133], the latter of which is a eukaryote-specific tRNA modification. It was noted that the i 6 A37-m 3 C 32 ASL modification circuit may have implications for disease as mutations in TRIT1, the gene responsible for i 6 A 37 formation on cytosolic and mitochondrial tRNAs, cause human pathology due in large part to mitochondrial dysfunction [145,146].…”

“…In humans i 6 A 37 is found on cytosolic tRNAs Ser and tRNA Ser[Sec] in addition to several mitochondrial tRNAs that contain i 6 A 37 or ms 2 i 6 A 37 among which is the major species tRNA Ser(UGA) [130] that also contains m 3 C 32 (also see [102,131]). Although yeast S. cerevisiae and S. pombe mutants lacking m 3 C 32 exhibit no growth phenotype under various conditions [133,147], the S. cerevisiae double mutant trm140Δ trm1Δ that also lacks m 2 2 G 26 on multiple overlapping tRNAs exhibits slow growth in the presence of the translation inhibitor cycloheximide [147]. …”

“…It was interesting that m 3 C 32 modification occurs in the absence of i 6 A 37 on tRNAs Thr and the tRNA Ser GCU that is not modified with i 6 A 37 [133] but are instead modified with t 6 A 37 . This suggested that the t 6 A 37 found on the tRNAs Thr and tRNA Ser GCU, might be required for m 3 C 32 formation [133].…”