Characterization of nuclear tRNATyr introns: their evolution from red algae to higher plants

We have previously isolated numerous intron-containing nuclear tRNATyr genes derived from either monocotyledonous (Triticum) or dicotyledonous (Arabidopsis, Nicotiana) plants by screening the corresponding genomic phage libraries with a synthetic tRNATyr-specific oligonucleotide. Here we have characterized additional tRNATyr 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 tRNATyr 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-tRNATyr 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-tRNATyr and have studied in wheat germ extract its effect on splicing and on conversion of U to Ψ in the GΨA anticodon. Furthermore, we discuss the putative acquisition of tRNATyr introns at an early step of evolution after the separation of Archaea and Eucarya.