Impaired nuclear transport of a human variant tRNAiMet.

Abstract

Recently, a human variant tRNAiMet (initiator tRNAMet) 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 tRNAiMet gene into the germinal vesicle of the intact Xenopus laevis oocyte, the primary transcript is slowly but accurately processed to a mature variant tRNAiMet 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.