Splicing of Arabidopsis tRNA(Met) precursors in tobacco cell and wheat germ extracts

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA(G PsiA)(Tyr) and elongator tRNA(M)et(C)mAU contain intervening sequences. We have previo usly investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA(Tyr). Here we have studied the expression of an Arabidopsis elongator tRNA(Met) gene in two plant extracts of different origin. This gene was first transcr ibed either in HeLa or in tobacco cell nuclear extract and splicing of intr on-containing tRNA(Met) precursors was then examined in wheat germ S23 extr act and in the tobacco system. The results show that conversion of pre-tRNA (Met) to mature tRNA proceeds very efficiently in both plant extracts. In o rder to elucidate the potential role of specific nucleotides at the 3' and 5' splice sites and of a structured intron for pre-tRNA(Met) splicing in ei ther extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair p re-tRNA(Met) splicing and that a highly structured intron is indispensable for pre-tRNA(Met) splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat an d tobacco splicing endonuclease, whereas pre-tRNA molecules that can form a n extended anticodon stem of only two putative base pairs are not spliced a t all. An amber suppressor, generated from the intron-containing elongator tRNA(Met) gene, is efficiently processed and spliced in both plant extracts .