Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA

Abstract

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.

RNA recognition mechanism of eukaryote tRNA (m7G46) methyltransferase (Trm8–Trm82 complex)

“…This eubacterial enzyme is composed of only a TrmB subunit [17]. In contrast, the yeast enzyme is a heterodimer composed of two protein subunits (Trm8 and Trm82) [15]. In this report, we demonstrate differences in tRNA recognition mechanisms between eubacterial and eukaryote tRNA (m 7 G46) methyltransferases.…”

“…In the case of the yeast enzyme, amino acid sequence analysis strongly suggests that the Trm8 protein functions as a catalytic subunit [15]. However, the role of the Trm82 subunit is still unclear.…”

“…Cuvettes with a 1 mm path length were used. Each sample in the annealing buffer was pre-incubated at 15,20,25,30,35,40,45, 50, 55, 60, 65 or 70°C for 15 min and then the spectrum was recorded from 300 to 200 nm at each temperature. The scan was repeated three times.…”

“…The methyl acceptance activities of these truncated transcripts by A. aeolicus tRNA (m 7 G46) methyltransferase were previously investigated: the transcripts F, G, and H in Fig 2 were well methylated and transcripts C, E and I were slowly methylated [17]. After the annealing, we measured CD-spectra of the transcripts at various temperatures (15,20,25,30,35, 40, 45, 50, 55, 60, 65 and 70°C) to check the conformation. For example, transcript C had a clear peak (260-280 nm) derived from high order structures at 15-40°C (data not shown).…”

“…More recently, bacterial genes have also been identified as trmB (classical name, yggH) from E. coli [16] and Aquifex aeolicus [17]. A gene disruption mutant of yeast [15] or E. coli [16] has revealed that the tRNA (m 7 G46) methyltransferase activity is not essential for cell viability. However, it has been reported that a yeast double mutant strain lacking both trm8 and trm4 genes showed rapid degradation of tRNA Val [18].…”