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

Abstract

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 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 (Chard), 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 pretRNA Tyr and have studied in wheat germ extract its effect on splicing and on conversion of U to 4* in the G*PA anticodon. Furthermore, we discuss the putative acquisition of tRNA Tyr introns at an early step of evolution after the separation of Archaea and Eucarya.

Analyses of Genomic tRNA Reveal Presence of Novel tRNAs in Oryza sativa

“…These conserved structural features of introns are important to in-vitro intron splicing (Stange et al, 1992; Akama et al, 2000). Mutation in the cytosine residue at either the 5′ or 3′ end of the intron border leads to deleterious effects on maturation of tRNA and involvement of the D-stem and variable loop (Stange et al, 1992; Akama et al, 1997, 2000). The intron sequence of pre-tRNA Met was conserved more than the intron sequence of pre-tRNA Tyr .…”

“…In tRNA Tyr , only T was found to be conserved at the first or second position, T-G at the fifth and sixth position and G nucleotide at 0–4 nucleotides upstream of the 3′ end of the intron (Figure 3). The T residue of the intron at the first or second position is most important for formation of pseudouridine at the second position of the anti-codon loop (Akama et al, 1997). The intron in tRNA Ala was found in between position 39 and 40 while the position of the intron in tRNA Cys , tRNA Met , and tRNA Tyr was present in between nucleotides 38 and 39.…”

Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

“…Subsequently, a great number of intron-containing tRNA genes coding either for tRNA Tyr or elongator tRNA Met were characterized in lower and higher plant (including Triticum ) nuclear genomes (28–30). Utilizing an intron-containing plant pre-tRNA Tyr as substrate, we have developed a cell-free processing and splicing system from wheat germ that supports efficient removal of 5′ and 3′ flanking sequences, accurate excision of the intervening sequence and ligation of the resulting tRNA halves (31).…”

Characterization of a 1-aminocyclopropane-1-carboxylate synthase gene from loblolly pine (Pinus taeda L.)

“…Very little information is available on splicing patterns in gymnosperms, so it is unknown whether the noncanonical sequence might be more commonly used in gymnosperms or may represent a unique feature of this particular transcript. Likewise, the 5′-splice junction (UU) sequence is non-canonical, although one exception in gymnosperms, the tRNA Tyr for Ginkgo biloba, has previously been reported (Akama et al, 1997). However, as a tRNA transcript the G. biloba gene product would likely be spliced via different cellular machinery.…”