Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain

Abstract

It has been proposed that yeast and Xenopus splicing endonucleases initially recognize features in the mature tRNA domain common to all tRNA species and that the sequence and structure of the intron are only minor determinants of splice-site selection. In accordance with this postulation, we show that yeast endonuclease splices heterologous pre-tRNATY' species from vertebrates and plants which differ in their mature domains and intron secondary structures. In contrast, wheat germ splicing endonuclease displays a pronounced preference for homologous pre-tRNA species; an extensive study of heterologous substrates revealed that neither yeast pre-tRNA species specific for leucine, serine, phenylalanine and tyrosine nor human and Xenopus pre-tRNATYr species were spliced. In order to identify the elements essential for pre-tRNA splicing in plants, we constructed chimeric genes coding for tRNA precursors with a plant intron secondary structure and with mature tRNATy' domains from yeast and Xenopus, respectively. The chimeric pre-tRNA comprising the mature tRNATyr domain from Xenopus was spliced efficiently in wheat germ extract, whereas the chimeric construct containing the mature tRNATyr domain from yeast was not spliced at all. These data indicate that intron secondary structure contributes to the specificity of plant splicing endonuclease and that unique features of the mature tRNA domain play a dominant role in enzyme-substrate recognition. We further investigated the influence of specific nucleotides in the mature domain on splicing by generating a number of mutated pre-tRNA species. Our results suggest that nucleotides located in the D stem, i.e. in the center of the pre-tRNA molecule, are recognition points for plant splicing endonuclease.

The tRNASer-isoacceptors and their genes in Nicotiana rustica: genome organization, expression in vitro and sequence analyses

“…In vitro processing of pre-tRNAs in yeast nuclear extract Yeast nuclear extract was prepared from S. cerevisiae strain 20B-12 (MAT e pep 4e-3 trpl) as described by Stange et al [40]. In vitro processing assays were carried out under the same conditions as established for the in vitro splicing of pre-tRNAs [40].…”

“…In vitro processing assays were carried out under the same conditions as established for the in vitro splicing of pre-tRNAs [40].…”

Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid

“…Further, we were unable to splice TRL-78 transcripts in vitro either under self-splicing conditions or using mitochondrial lysates. Finally, the TRL-78 in vitro transcript could not be cut or spliced using yeast tRNA splicing enzymes (M.Steiger and E.Phizicky, personal communication), which have been shown to splice heterologous introncontaining tRNA species from vertebrates and plants (30). We conclude that there is little if any TRL-64 transcript present in Varkud mitochondria and that the 14-nt insert is not recognized as an intron by nuclear or mitochondrial enzymes.…”

The far side of auxin signaling: fundamental cellular activities and their contribution to a defined growth response in plants

“…Perhaps even more surprising is the observation that defects in auxin-controlled growth responses appear to represent the major consequence of this mutation. Unfortunately, we still lack detailed information on tRNA expression control in plants (Lin et al 1992; Stange et al 1992; Carneiro et al 1994; Ulmasov and Folk 1995). So, it remains a mystery as to how a single tRNA anticodon mutation could produce highly defined growth defects, as those observed by Perry and colleagues (Perry et al 2005).…”