Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA.
7-methylguanosine (m7G) 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 m7G 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 m7G modification of the 5' 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 m7G-methyltransferase activity: Extracts derived from strains lacking either gene have undetectable m7G methyltransferase activity, RNA from strains lacking either gene have much reduced m7G, and coexpression of both proteins is required to overproduce activity. Aniline cleavage mapping shows that Trm8/Trm82 proteins modify pre-tRNAPhe 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 m7G-methyltransferase activity.
Alexandrov
Andrei
A
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, New York 14642, USA.
Martzen
Mark R
MR
Phizicky
Eric M
EM
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D030562:Databases, Protein; D005057:Eukaryotic Cells / Q000502:physiology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D012322:RNA Precursors / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012359:tRNA Methyltransferases / Q000235:genetics / Q000378:metabolism*
D012322:RNA Precursors; D012360:RNA, Transfer, Phe; D029701:Saccharomyces cerevisiae Proteins; C417912:WDR4 protein, human; D006151:Guanosine; C016578:7-methylguanosine; D008780:Methyltransferases; D012359:tRNA Methyltransferases; C036814:tRNA (guanine-N7-)-methyltransferase; C055097:mRNA (guanine(N7))-methyltransferase; D019204:GTP-Binding Proteins