All human tRNATyrgenes contain introns as a prerequisite for pseudouridine biosynthesis in the anticodon

Abstract

Two synthetic oligonucleotides, one specific for the 5′ exon, the other spanning the splice junction, were used to show that (a) the human haploid genome contains at least 12 independent gene loci for tRNA_Tyr (b) that all of them carry an intron. From one of the cloned human tRNA_Tyr genes (pHtT1) the 20 bp intron was deleted to generate pHtT1δ. Homologous in vitro transcription, fingerprint analyses of the products and elucidation T their nucleoside composition revealed that the pseudouridine (ψ₃₅) in the center of the anticodon of tRNA_Tyr was synthesized in the intron-containing precursor where as this U to ψ modification did not take place in precursors or mature tRNA_Tyr derived from pHtT1δ. On the basis of these results and of studies from other laboratories we suggest that the evolutionary pressure for maintaining introns in eukaryotic tRNAs_Tyr is this strict intron-requirement for ψ synthesis. Taking into account that all eukaryotic cytoplasmic tRNAs_Tyr contain a ψ₃₅ we discuss here a special need for this modified nucleoside in stabilizing codon-anticodon interactions involving (a) classical base pairing upon translation of tyrosine codons and (b) unconventional interactions during UAG amber codon suppression by tRNAG^Tyr_GψA in eukaryotic cells.