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Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNATyr GΨ A and elongator tRNAMet CmAU contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNATyr. Here we have studied the expression of an Arabidopsis elongator tRNAMet gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNAMet precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNAMet to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3′ and 5′ splice sites and of a structured intron for pre-tRNAMet splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNAMet splicing and that a highly structured intron is indispensable for pre-tRNAMet splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNAMet gene, is efficiently processed and spliced in both plant extracts.