Yeast Nfs1p is involved in thio-modification of both mitochondrial and cytoplasmic tRNAs.
The IscS protein is a pyridoxal phosphate-containing cysteine desulfurase involved in iron-sulfur cluster biogenesis. In prokaryotes, IscS is also involved in various metabolic functions, including thio-modification of tRNA. By contrast, the eukaryotic ortholog of IscS (Nfs1) has thus far been shown to be functional only in mitochondrial iron-sulfur cluster biogenesis. We demonstrate here that yeast Nfs1p is also required for the post-transcriptional thio-modification of both mitochondrial (mt) and cytoplasmic (cy) tRNAs in vivo. Depletion of Nfs1p resulted in an immediate impairment of the 2-thio-modification of 5-carboxymethylaminomethyl-2-thiouridine at the wobble positions of mt-tRNA(UUU)(Lys) and mt-tRNA(UUG)(Gln). In addition, we observed a severe reduction in the 2-thio-modification of 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) of cy-tRNA(UUU)(Lys2) and cy-tRNA(UUC)(Glu3), although the effect was somewhat delayed compared with that seen in mt-tRNAs. Mass spectrometry analysis revealed an increase in 5-methoxycarbonylmethyluridine concomitant with a decrease in mcm(5)s(2)U in cy-tRNAs that were prepared from Nfs1p-depleted cells. These results suggest that Nfs1p is involved in the 2-thio-modification of both 5-carboxymethylaminomethyl-2-thiouridine in mt-tRNAs and mcm(5)s(2)U in cy-tRNAs.
Nakai
Yumi
Y
Department of Biochemistry, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan. med004@art.osaka-med.ac.jp
Umeda
Noriko
N
Suzuki
Tsutomu
T
Nakai
Masato
M
Hayashi
Hideyuki
H
Watanabe
Kimitsuna
K
Kagamiyama
Hiroyuki
H
D015152:Blotting, Northern; D003593:Cytoplasm / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D013058:Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008956:Models, Chemical; D010663:Phenylmercury Compounds / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D013466:Sulfurtransferases; D013891:Thiouridine / Q000031:analogs & derivatives* / Q000737:chemistry; D013997:Time Factors; D014529:Uridine / Q000737:chemistry
D007506:Iron-Sulfur Proteins; D024101:Mitochondrial Proteins; D010663:Phenylmercury Compounds; D029701:Saccharomyces cerevisiae Proteins; D013891:Thiouridine; C056922:((N-acryloylamino)phenyl)mercuric chloride; C030546:5-(carboxymethylaminomethyl)-2-thiouridine; D012343:RNA, Transfer; D013466:Sulfurtransferases; C115912:NFS1 protein, S cerevisiae; D014529:Uridine