The biological role of many nonessential tRNA modifications outside of the anticodon remains elusive despite their evolutionary conservation. We show here that m7G46 methyltransferase Trm8p/Trm82p acts as a hub of synthetic interactions with several tRNA modification enzymes, resulting in temperature-sensitive growth. Analysis of three double mutants indicates reduced levels of tRNA(Val(AAC)), consistent with a role of the corresponding modifications in maintenance of tRNA levels. Detailed examination of a trm8-delta trm4-delta double mutant demonstrates rapid degradation of preexisting tRNA(Val(AAC)) accompanied by its de-aminoacylation. Multiple copies of tRNA(Val(AAC)) suppress the trm8-delta trm4-delta growth defect, directly implicating this tRNA in the phenotype. These results define a rapid tRNA degradation (RTD) pathway that is independent of the TRF4/RRP6-dependent nuclear surveillance pathway. The degradation of an endogenous tRNA species at a rate typical of mRNA decay demonstrates a critical role of nonessential modifications for tRNA stability and cell survival.
ABSTRACT7-methylguanosine (m 7 G) modification of tRNA occurs widely in eukaryotes and bacteria, is nearly always found at position 46, and is one of the few modifications that confers a positive charge to the base. Screening of a Saccharomyces cerevisiae genomic library of purified GST-ORF fusion proteins reveals two previously uncharacterized proteins that copurify with m 7 G methyltransferase activity on pre-tRNA Phe . ORF YDL201w encodes Trm8, a protein that is highly conserved in prokaryotes and eukaryotes and that contains an S-adenosylmethionine binding domain. ORF YDR165w encodes Trm82, a less highly conserved protein containing putative WD40 repeats, which are often implicated in macromolecular interactions. Neither protein has significant sequence similarity to yeast Abd1, which catalyzes m 7 G modification of the 59 cap of mRNA, other than the methyltransferase motif shared by Trm8 and Abd1. Several lines of evidence indicate that both Trm8 and Trm82 proteins are required for tRNA m 7 G-methyltransferase activity: Extracts derived from strains lacking either gene have undetectable m 7 G methyltransferase activity, RNA from strains lacking either gene have much reduced m 7 G, and coexpression of both proteins is required to overproduce activity. Aniline cleavage mapping shows that Trm8/Trm82 proteins modify pre-tRNA Phe at G46, the site that is modified in vivo. Trm8 and Trm82 proteins form a complex, as affinity purification of Trm8 protein causes copurification of Trm82 protein in approximate equimolar yield. This functional two-protein family appears to be retained in eukaryotes, as expression of both corresponding human proteins, METTL1 and WDR4, is required for m 7 Gmethyltransferase activity.
We show that Saccharomyces cerevisiae strains lacking Trm8p/Trm82p tRNA m 7 G methyltransferase are temperature-sensitive in synthetic media containing glycerol. Bacterial TRM8 orthologs complement the growth defect of trm8-⌬, trm82-⌬, and trm8-⌬ trm82-⌬ double mutants, suggesting that bacteria employ a single subunit for Trm8p/Trm82p function. The growth phenotype of trm8 mutants correlates with lack of tRNA m 7 G methyltransferase activity in vitro and in vivo, based on analysis of 10 mutant alleles of trm8 and bacterial orthologs, and suggests that m 7 G modification is the cellular function important for growth. Initial examination of the roles of the yeast subunits shows that Trm8p has most of the functions required to effect m 7 G modification, and that a major role of Trm82p is to maintain cellular levels of Trm8p. Trm8p efficiently cross-links to pretRNA Phe in vitro in the presence or absence of Trm82p, in addition to its known residual tRNA m 7 G modification activity and its SAM-binding domain. Surprisingly, the levels of Trm8p, but not its mRNA, are severely reduced in a trm82-⌬ strain. Although Trm8p can be produced in the absence of Trm82p by deliberate overproduction, the resulting protein is inactive, suggesting that a second role of Trm82p is to stabilize Trm8p in an active conformation.
We developed a method to co-express protein pairs from collections of otherwise identical Escherichia coli plasmids expressing different ORFs by incorporating a 61-nucleotide sequence (LINK) into the plasmid to allow generation of tandem plasmids. Tandem plasmids are formed in a ligation-independent manner, propagate efficiently, and produce protein pairs in high quantities. This greatly facilitates co-expression for structural genomics projects that produce thousands of clones bearing identical origins and antibiotic markers.
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