TRNA nuclear transport: Defining the critical regions of human tRNA imet by point mutagenesis

Abstract

We recently described a carrier-mediated nuclear transport system for tRNA in Xenopus laevis oocytes. A natural human tRNA i met variant with a G to T transversion in position 57 is defective in transport across the nuclear membrane. In addition, processing of the primary transcript of the variant gene is much less efficient than the wild type. We now describe the nuclear transport and processing phenotypes of 30 different point mutants generated by in vitro mutagenesis of a wild-type human tRNA i met gene. The effects of each nucleotide change on processing and transport were analyzed in X. laevis oocytes following nuclear microinjection of each mutant gene. Mutants exhibiting transport-defective behavior were further characterized by measuring transport kinetics of the purified mature tRNA. Our studies demonstrate that many mutations affect tRNA i met nuclear transport, although those with the most deleterious effects are clustered in the highly conserved D stem-loop and T stem-loop regions.