Impaired nuclear transport of a human variant tRNA_i^Met

Abstract

Recently, a human variant tRNA_i^Met (initiator tRNA^Met) gene has been cloned from a recombinant library of fetal liver DNA¹. This gene contains a G to T transversion within the highly conserved TCGA sequence in loop 4 (‘Tψ loop’), a sequence position occupied exclusively by a purine (usually G) in almost 200 prokaryotic and eukaryotic tRNAs². Recent studies with the cloned gene, both in vitro³ and when expressed in an intact cell on a simian virus 40 viral vector⁴, have shown that one functional consequence of this base substitution is a reduction in the rate of processing of the primary transcript of the gene. This reaction involves sequential excision of 5′- and 3′-terminal sequences³. Here we show that following microinjection of the variant tRNA_i^Met gene into the germinal vesicle of the intact Xenopus laevis oocyte, the primary transcript is slowly but accurately processed to a mature variant tRNA_i^Met species which remains trapped within the oocyte nucleus, its escape apparently being prevented by the nuclear membrane. This is the first evidence that the transport of a tRNA molecule can be blocked by a point mutation. The results suggest that a selective tRNA nuclear transport mechanism exists in the eukaryotic cell and identify a critical role for the highly conserved Tψ loop in this process.