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Depletion of Saccharomyces cerevisiae tRNA^His Guanylyltransferase Thg1p Leads to Uncharged tRNA^His with Additional m⁵C

Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Box 712, Rochester, New York 14642,¹ Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033²

Received 20 May 2005/ Returned for modification 15 June 2005/ Accepted 24 June 2005

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 m⁵C modifications, which are delayed relative to the loss of G_–1 and aminoacylation. The additional modification is likely due to tRNA m⁵C methyltransferase Trm4p. We developed a new method to map m⁵C residues in RNA and localized the additional m⁵C to positions 48 and 50. This is the first documented example of the accumulation of additional modifications in a eukaryotic tRNA species.

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