Precise analysis of modification status at various stage of tRNA maturation in Saccharomyces cerevisiae

Transfer RNAs (tRNAs) are decorated with various post-transcriptional modifications which are enzymatically introduced at various stages of maturation. It is known that eukaryotic tRNAs are modified both in nucleus and cytoplasm. However, the order of tRNA modifications remains to be investigated. To unveil the precise timing of each modification associated with tRNA processing, we isolated precursor forms of Saccharomyces cerevisiae tRNAs at different stages of maturation, and analyzed their primary structures including modifications by mass spectrometry. The primary transcript of tRNA(Ile) was isolated from the tRNA fraction co-precipitated with Lhp1p, a yeast homolog of La protein. The precursor tRNA(Ile) without 3'-trailer sequence was isolated from the expression controllable strain when RNase P was transiently inactivated. Mass spectrometric analyses of these tRNAs revealed that many modifications were fully introduced at the stage of primary transcript, however, some modifications were partially introduced.