Characterization of nuclear tRNA(Tyr) introns: their evolution from red algae to higher plants

We have previously isolated numerous intron-containing nuclear tRNA(Tyr) genes derived from either monocotyledonous (Triticum) or dicotyledonous (Arabidopsis, Nicotiana) plants by screening the corresponding genomic phage libraries with a synthetic tRNA(Tyr)-specific oligonucleotide. Here we have characterized additional tRNA(Tyr) genes from phylogenetically divergent plant species representing red algae (Champia), brown algae (Cystophyllum), green algae (Ulva), stonewort (Chara), liverwort (Marchantia), moss (Polytrichum), fern (Rumohra) and gymnosperms (Ginkgo) using amplification of the coding sequences from the corresponding genomic DNAs by polymerase chain reaction (PCR). All novel tRNA(Tyr) genes contain intervening sequences of variable sequence and length ranging in size from 11 to 21 bp. However, two features are conserved in all plant pre-tRNA(Tyr) introns: they possess a uridine and less frequently an adenosine at the 5' boundary and can adopt similar intron secondary structures in which an extended anticodon helix of 4-5 bp is formed by base-pairing between nucleotides of the intron and the anticodon loop. In order to elucidate the potential role of the highly conserved uridine at the first intron position, we have replaced it by all other nucleosides in an Arabidopsis pre-tRNA(Tyr) and have studied in wheat germ extract its effect on splicing and on conversion of U to psi in the GpsiA anticodon. Furthermore, we discuss the putative acquisition of tRNA(Tyr) introns at an early step of evolution after the separation of Archaea and Eucarya.