Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae.

Abstract

To evaluate the role of exon domains in tRNA splicing, the anti-codon stem of proline pre-tRNAUGG from Saccharomyces cerevisiae was altered by site-directed mutagenesis of the suJ8 gene. Sixteen alleles were constructed that encode mutant pre-tRNAs containing all possible base combinations in the last base pair of the anticodon stem adjacent to the anticodon loop (positions 31 and 39). The altered pre-tRNAs were screened by using an in vitro endonucleolytic cleavage assay to determine whether perturbations in secondary structure affect the intron excision reaction. The pre-tRNAs were cleaved efficiently whenever secondary structure in the anticodon stem was maintained through standard base pairing or G-U interactions. However, most of the pre-tRNAs with disrupted secondary structure were poor substrates for intron excision. We also determined the extent to which the suJ8 alleles produce functional products in vivo. Each allele was integrated in one to three copies into a yeast chromosome or introduced on a high-copy-number plasmid by transformation. The formation of a functional product was assayed by the ability of each allele to suppress the + 1 frameshift mutation his4-713 through four-base codon reading, as shown previously for the SUF8-1 suppressor allele. We found that alleles containing any standard base pair or G U pair at position 31/39 in the anticodon stem failed to suppress his4-713. We could not assess in vivo splicing with these alleles because the tRNA products, even if they are made, would be expected to read a normal triplet rather than a quadruplet codon. However, all of the alleles that contained a disrupted base pair at position 31/ 39 in the anticodon stem altered the structure of the tRNA in a manner that caused frameshift suppression. Suppression indicated that splicing must have occurred to some extent in vivo even though most of the suppressor alleles produced pre-tRNAs that were cleaved with low efficiency or not at all in vitro. These results have important implications for the interpretation of in vitro cleavage assays in general and for the potential use of suppressors to select mutations that affect tRNA splicing.Splicing of transfer RNA in Saccharomyces cerevisiae requires three separate enzymes: a detergent-extractable endonuclease, a soluble ligase, and a 2'-phosphatase (26). The endonuclease cleaves the pre-tRNA, releasing the intron as a linear molecule (25). The ligase joins the half molecules through an ATP-dependent, three-step mechanism involving cyclic phosphodiesterase, polynucleotide kinase, and RNA ligase activities (12, 28). Finally, the residual 2'-phosphate at the splice site is removed to produce a mature, primary tRNA sequence.The splicing enzymes of S. cerevisiae process all introncontaining intermediates in the synthesis of 10 of the 46 cytoplasmic tRNA isoacceptors (14, 23). The enzymes form an in vitro complex composed minimally of the endonuclease and the ligase (11). The ligase, and by extension probably the complex as well, is localized in vivo in associatio...

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

“…luevis (Mattoccia et al, 1988), HeLa cells (Filipowicz and Shatkin, 1983), wheat germ (Stange et al, 1988) and in the archaebacterium Halobacterium volcanii (Thompson and Daniels, 1988). The HeLa and Xenopus endonucleases were shown to excise introns from quite a number of heterologous pre-tRNA species.…”

“…It has been shown that Xenopus and mammalian splicing endonucleases are capable of excising introns from heterologous pre-tRNA species (Melton et al, 1980;Standring et al, 1981;Filipowicz and Shatkin, 1983;van To1 et al, 1987;Mattoccia et al, 1988). Little information exists about the species specificity of the yeast splicing endonuclease until now.…”

“…Mutations that alter pretRNA secondary structure have revealed that the anticodon stem plays a critical role in cleavage-site selection. Extending the length of the anticodon stem by one or two base pairs produces introns which are two and four nucleotides longer, respectively, than the wild-type intron (Greer et al, 1987;Mattoccia et al, 1988;Reyes and Abelson, 1988). Furthermore, removal of introns is impaired in yeast extract if pretRNA species carry mutations that disrupt the bottom base pair of the anticodon stem (Mathison et al, 1989).…”

“…The ability of Xenopus (Melton et al, 1980;Mattoccia et al, 1988) and mammalian cell extracts (Standring et al, 1981 ;Filipowicz and Shatkin, 1983) to process yeast pre-tRNA species suggests that features required for splicing are conserved across species boundaries. This, however, is not the case for the Halohacterium volcanii endonuclease, which is Table 1..…”

“…The endonuclease apparently recognizes features that are shared by all of its substrates; (a) the IVS is always located one nucleotide 3' to the anticodon (Ogden et al, 1984), (b) the 3' splice site is present in a single-stranded loop and the 5'junction is located either in a single-stranded or labile double-stranded region, (c) the precursors probably form a tRNA-like tertiary structure in the mature domain of the molecule as suggested by chemical and enzymic structurespecific probes (Lee and Knapp, 1985;Swerdlow and Guthrie, 1984). Presumably the splicing endonuclease initially interacts with conserved nucleotides in this portion of the tRNA precursor which are common to all tRNA species (Mattoccia et al, 1988;Reyes and Abelson, 1988). Mutations that alter pretRNA secondary structure have revealed that the anticodon stem plays a critical role in cleavage-site selection.…”