摘要：

Cloned yeast tRNA Tyr genes are transcribed and processed after micro-injection into Xenopus laevis oocyte nuclei (De Robertis & Olson, 1979; Melton et al., 1980). The early transcript, about 104 nucleotides long, has a 5′ leader, two or three U residues at the 3′ end and an intervening sequence. The termini are then matured, giving rise to a molecule 92 nucleotides long, which is subsequently spliced to produce a 78 nucleotides long tRNA by a mechanism similar to that found in yeast. Here we analyse the base modifications in these three RNA molecules to test whether the extra RNA segments, and especially the intervening sequence, influence the introduction of base modifications into tRNA. The 104, 92 and 78 RNAs were isolated from gels and analysed by two-dimensional chromatography after complete digestion with nuclease P 1 into 5′-mononucleotides (direct labelling) or ribonuclease T 2 into 3′-mononucleotides (nearest neighbour labelling). By analysing oocytes labelled separately with [α- 32P]ATP, -GTP, -UTP or -CTP it was possible to examine all the base modifications added by the oocyte. The results show that the tRNA Tyr base modifications occur in a sequential order: (1) the early 104 precursor already has five base modifications on it, including all the modifications in the TΨ loop; (2) 11 additional modifications only become apparent in the 92 precursor; and (3) finally, two base modifications, Q base and isopentenyladenosine, are introduced in the anticodon loop only after splicing of the intervening sequence. No modifications were observed in the 5′ leader, 3′ trailer or intervening sequences. Some base modifications added by the oocyte are not normally found in yeast (e.g. Q base), and it seems as if the Xenopus oocyte processes this yeast tRNA as one of its own tRNAs. However, all the modifications normally present in yeast were added by the oocyte, with the exception of a Gm. All base modifications were introduced in a sequential fashion. The splicing pathway found in vitro with yeast extracts (Knapp et al., 1979; Peebles et al., 1979), which has an unusual 3′ phosphate-ended intermediate, was confirmed in vivo in the frog oocyte by analysing the nearest neighbour labelling of the isopentenyladenosine that is located adjacent to the splicing point.

展开