Depletion of Saccharomyces cerevisiae tRNAHis Guanylyltransferase Thg1p Leads to Uncharged tRNAHis with Additional m5C

tRNAs from all organisms are extensively modified, with numerous alterations of base and ribose moieties at different positions (47). In the yeast Saccharomyces cerevisiae, 25 different chemical modifications of tRNA have been described. These occur at 34 positions, and each tRNA bears ∼11 modifications (22). Although many of these modifications and the corresponding modification enzymes are highly conserved in different organisms, only three modification enzymes are known to be essential in yeast. Gcd10p/Gcd14p catalyzes formation of m1A58, which occurs in numerous tRNA species and is essential for tRNAiMet (3). Tad2p/Tad3p catalyzes adenosine deaminase activity to form I34, which affects seven yeast tRNAs (13). Thg1p catalyzes tRNAHis guanylyltransferase activity to add an extra guanine nucleotide to the 5′ end of tRNAHis at position −1, called G−1 (17). A unique feature of all tRNAHis species is the presence of an extra G−1 residue opposite the discriminator base at position 73. Among more than 500 characterized tRNA species, only one other tRNA species, tRNAPhe from Tetrahymena pyriformis mitochondria, has an additional residue at its 5′ end (43). The extra G−1 residue of tRNAHis is generated in two very different ways. In prokaryotes, chloroplasts, and plant mitochondria, G−1 is genome encoded and retained in the mature tRNAHis because RNase P cleaves at an unusual position to form an 8-bp aminoacyl stem containing G−1-C73 (7, 34). By contrast, for eukaryotic cytoplasmic tRNAHis and at least one animal mitochondrial tRNAHis, G−1 is not genome encoded (27) but instead is added posttranscriptionally. We demonstrated previously that the essential protein Thg1p has tRNAHis guanylyltransferase activity in vitro and is required for G−1 addition to tRNAHis in vivo (17). We used a biochemical genomics screen (30) to show that glutathione S-transferase-Thg1p copurifies with yeast tRNAHis guanylyltransferase activity and then showed that yeast Thg1p expressed and purified from Escherichia coli has the correct activity in vitro. This reaction is highly specific for tRNAHis, and the 3′→5′ extension of a polynucleotide chain catalyzed by Thg1p is highly unusual. The only other similar 3′→5′ polynucleotide extension activity is that found in the mitochondria of the amoeboid protist Acanthamoeba castellani (36) and the chytridiomycete fungus Spizellomyces punctatus (6), which are likely responsible for editing the 5′ ends of tRNAs (28). Thg1p adds G−1 to monophosphorylated tRNAHis (p-tRNAHis) by a ligase-like mechanism, involving formation of an Ap-p-tRNAHis-adenylylated intermediate, and removal of the AMP moiety during subsequent G−1 addition (17); however, Thg1p lacks any of the six characteristic motifs found in the superfamily of ligases/mRNA capping enzymes (45). We also demonstrated that Thg1p is required for tRNAHis guanylyltransferase in vivo, by showing that as cells are depleted of Thg1p they accumulate tRNAHis lacking G−1 (17). Here we investigate the cellular events that occur as cells stop growing when depleted of Thg1p, with the objective of explaining the essential function of Thg1p and of probing the physiological responses of loss of Thg1p and of G−1 from tRNAHis. Four results are described. First, we show that the accumulation of tRNAHis lacking G−1 is accompanied by coincident appearance of uncharged tRNAHis, which may explain why Thg1p is essential. Second, we show that Thg1p-depleted cells appear to activate the GCN4 pathway, which normally responds to amino acid or purine starvation by increased expression of a number of genes involved in amino acid biosynthesis (21). Third, we show by fluorescence in situ hybridization (FISH) that tRNAHis accumulates in the nucleus in Thg1p-depleted cells. Fourth, we found unexpectedly that the tRNAHis of Thg1p-depleted cells accumulates additional 5-methylcytidine (m5C) modifications, well after the loss of G−1 and aminoacylation. The m5C modification requires the tRNA m5C-methyltransferase Trm4p, which catalyzes all other known m5C modifications of yeast tRNAs (31), and we have localized the additional m5C by development of a new RNA mapping technique. This accumulation of m5C in tRNAHis is, to our knowledge, the first example of additional tRNA modifications observed during a cellular response in eukaryotes, and its timing suggests that the accumulation of m5C is an indirect consequence of lack of Thg1p. The additional m5C might be caused by a prolonged presence of uncharged tRNA in the nucleus, or it might be part of a regulatory response.