Depletion of Saccharomyces cerevisiae tRNA(His) guanylyltransferase Thg1p leads to uncharged tRNAHis with additional m(5)C.
The essential Saccharomyces cerevisiae tRNA(His) guanylyltransferase (Thg1p) is responsible for the unusual G(-1) addition to the 5' end of cytoplasmic tRNA(His). We report here that tRNA(His) from Thg1p-depleted cells is uncharged, although histidyl tRNA synthetase is active and the 3' end of the tRNA is intact, suggesting that G(-1) is a critical determinant for aminoacylation of tRNA(His) in vivo. Thg1p depletion leads to activation of the GCN4 pathway, most, but not all, of which is Gcn2p dependent, and to the accumulation of tRNA(His) in the nucleus. Surprisingly, tRNA(His) in Thg1p-depleted cells accumulates additional m(5)C modifications, which are delayed relative to the loss of G(-1) and aminoacylation. The additional modification is likely due to tRNA m(5)C methyltransferase Trm4p. We developed a new method to map m(5)C residues in RNA and localized the additional m(5)C to positions 48 and 50. This is the first documented example of the accumulation of additional modifications in a eukaryotic tRNA species.
Gu
Weifeng
W
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Hurto
Rebecca L
RL
Hopper
Anita K
AK
Grayhack
Elizabeth J
EJ
Phizicky
Eric M
EM
D044503:5-Methylcytosine / Q000378:metabolism*; D000107:Acetylation; D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D012331:RNA, Fungal / Q000032:analysis / Q000378:metabolism; D012354:RNA, Transfer, His / Q000032:analysis / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012359:tRNA Methyltransferases / Q000378:metabolism
D004268:DNA-Binding Proteins; D012331:RNA, Fungal; D012354:RNA, Transfer, His; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; C476348:Thp1 protein, S cerevisiae; D006147:Guanine; D044503:5-Methylcytosine; D012359:tRNA Methyltransferases; C024845:NCL1 protein, S cerevisiae; D011494:Protein Kinases; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases