Abstract

The tRNA nucleotidyltransferase enzyme is crucial for normal tRNA maturation in eukaryotic cells. Sequence analysis revealed a possible nuclear localization signal (KDTKGKSIPVVNHIFKFSMKRK) in the Arabidopsis thaliana tRNA nucleotidyltransferase. We set out to examine the role of this sequence by expressing wild-type and variant Arabidopsis tRNA nucleotidyltransferase proteins in Saccharomyces cerevisiae. Viability assays implementing plasmid shuffling and complementation of a temperature-sensitive strain revealed that while the wild-type enzyme could replace the nucleocytosolic role of the yeast tRNA nucleotidyltransferase the variant enzymes could not. Subsequent enzyme activity assays revealed that enzyme activity was significantly reduced for the variant enzymes. Localization studies utilizing tRNA nucleotidyltransferase-green fluorescent protein fusion proteins showed a pattern of distribution inconsistent with the role of the modified region as a nuclear localization signal. In fact, the patterns observed seem to point to a possible stability-modulating function for our candidate region with a marked augmentation in fluorescence in the cytosol and nucleus in the variant proteins. Future experiments will be required to accurately define the role of this sequence in tRNA nucleotidyltransferase structure and function. Furthermore, additional studies are needed to define any nuclear localization signals that may be present in the Arabidopsis tRNA nucleotidyltransferase.