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

Abstract

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.

ACKNOWLEDGMENTS

We are grateful to A. Alexandrov and J. Jackman for valuable advice.

This research was supported by grants GM52347 to E.M.P. and GM27930 to A.K.H. from the National Institutes of Health.