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We have used an in vitro reconstitution system to determine the effects of a large number of mutations in the highly conserved 5' terminal domain of the yeast U2 snRNA on pre-mRNA splicing. Whereas many mutations have little or no functional consequence, base substitutions in two regions were found to have drastic effects on pre-mRNA splicing. A previously unrecognized function for the U2 snRNA in the second step of splicing was found by alteration of the absolutely conserved sequence AGA upstream of the branch point recognition sequence. The effects of these mutations suggest the formation of a structure involving the U2 snRNA similar to the guanosine-binding site found in the catalytic core of group I introns.

We have altered the TACTAAC sequence in the yeast CYH2m gene intron to TACTACC. This mutation changes the nucleotide at the normal position of the branch in intron RNA lariats produced during pre-mRNA splicing, and it prevents splicing in vivo. In a yeast pre-mRNA splicing system, CYH2m pre-mRNA carrying the TACTACC mutation is not specifically cut or rearranged in any way. Substitution of an A for the first G of the CYH2m intron, converting the highly conserved GTATGT 5' splice site sequence to ATATGT, also blocks intron excision in vivo and in vitro: pre-mRNA carrying this mutation was still cut normally at the mutant 5' splice site in vitro, to give authentic exon 1 and an intron-exon 2 lariat RNA with an A-A 2'-5' phosphodiester linkage at the branch point. This lariat RNA is a dead-end product. The subsequent cleavage at the 3' splice site is therefore sensitive to the sequence of the 5' end of the intron attached at the branch point.

Splicing of transfer RNA precursors containing intervening sequences proceeds in two distinct stages: endonucleolytic cleavage, followed by ligation. We have physically separated endonuclease and ligase activities from extracts of yeast cells, and we report properties of the partially purified endonuclease preparation. The endonuclease behaves as an integral membrane protein: it is purified from a membrane fraction from which it can be solubilized with nonionic detergents, and the activity of the endonuclease in the membrane fraction is stimulated by nonionic detergents. The endonuclease cleaves precursor tRNAs at two sites to excise the intervening sequence precisely. Both the extent and the accuracy of cleavage are enhanced by the presence of spermidine; the degree of stimulation varies with the pre-tRNA substrate. The cleavage products possess 5'-hydroxyl and 2',3'-cyclic phosphodiester termini. The cyclic phosphodiester termini can be opened to 2'-phosphates by a cyclic phosphodiesterase activity in the preparation.

Ligase activity was detected in extracts of Escherichia coli, Clostridium tartarivorum, Rhodospirillum salexigens, Chromatium gracile, and Chlorobium limicola. Ligase was measured by joining of tRNA halves produced from yeast IVS-containing tRNA precursors by a yeast endonuclease. The structure of tRNATyr halves joined by an E. coli extract was examined. The ligated junction is resistant to nuclease P1 and RNAase T2 but sensitive to venom phosphodiesterase and alkaline hydrolysis, consistent with a 2',5' linkage. The nuclease-resistant junction dinucleotide comigrates with authentic (2',5') APA marker in thin-layer chromatography. The phosphate in the newly formed phosphodiester bond is derived from the pre-tRNA substrate. The widespread existence of a bacterial ligase raises the possibility of a novel class of RNA processing reactions.

Transcripts from an ADC1-actin gene fusion containing the yeast actin intron are efficiently spliced in vivo. Four distinct forms of the excised actin intron are found in poly(A)- RNA from yeast carrying this transcription unit on a multicopy plasmid. Two of them migrate abnormally slowly in polyacrylamide gels, and one of these contains a block to reverse transcription at the conserved UACUAAC sequence (TACTAAC box). We also detect a larger RNA in the poly(A)+ fraction with abnormal gel mobility and the same reverse transcriptase block. Analysis of the major species of excised actin intron, labeled with 32P in vivo, reveals the presence of a branch at the third A in the UACUAAC sequence. This A is linked via a 3'-5' phosphodiester bond to the downstream C, and via a 2'-5' phosphodiester bond to a G, presumably from the 5' end of the intron. Thus this intron RNA is in the form of a lariat.

The intermediates of the yeast tRNA splicing reaction have been characterized. The intervening sequence is excised as an unique linear molecule. It has 5'-hydroxyl and 3'-phosphate termini. Correspondingly, the half-tRNA molecules are shown to have a 3'-phosphate terminus on the 5' half and 5'-hydroxyl terminus on the 3' half. These isolated halves have been shown to be active in the ligation step of tRNA splicing. Removal of the 3'-phosphate from the 5' half eliminates the ability of the 5' half to participate in ligation.

Genes for yeast tRNATyr and tRNAPhe have been sequenced (Goodman, Olson and Hall, 1977; Valenzuela et al., 1978) which contain additional nucleotides (intervening sequences) within the middle of the gene that are not present in the mature tRNA. We have isolated precursors to rRNATyr and tRNAPhe from a yeast temperature-sensitive mutant (at the rna1 locus) which accumulates only certain precursor tRNAs at the nonpermissive temperature. The tRNATyr and tRNAPhe precursors were analyzed by oligonucleotide mapping; they each contain the intervening sequence and fully matured 5' and 3' termini. Furthermore, these precursors were used as substrates to search for an enzymatic activity which can remove the intervening sequences and religate the ends. We have shown that wild-type yeast contains such an activity, and that this activity specifically removes the intervening sequences to produce mature-sized RNAs.

S. cerevisae tRNA introns interrupt the gene at a constant position in the anticodon loop. Pre-tRNAs are matured by an endonuclease and a ligase. The endonuclease alone can accurately release the intron from the pre-tRNA. Here, we investigate the mechanism of splice site selection by the endonuclease. We propose that it initially recognizes features in the mature domain common to all tRNAs. Once positioned on the enzyme, the splice sites are recognizable because they are a fixed distance from the mature domain. To test this hypothesis, we developed a system for synthesizing pre-tRNA by bacteriophage T7 RNA polymerase. To search for recognition sites, we made several mutations. Mutations of C56 and U8 strongly affect endonuclease recognition of pre-tRNA. With insertion and deletion mutations, we show that the anticodon stem determines splicing specificity. The sequence and structure of the intron are not strong determinants of splice site selection.

A gene for Saccharomyces cerevisiae tRNATrp has been sequenced which contains an intervening sequence of 34 bp (H. S. Kang and J. Abelson, unpublished results). The mutant yeast strain ts-136 accumulates a precursor to tRNATrp which contains mature ends and is colinear with the tRNATrp gene. A nuclear extract from Xenopus oocytes is capable of supporting transcription of the tRNATrp gene contained on plasmid pBR313. The products are precursor tRNAs which contain the intervening RNA sequence. The Xenopus extract accurately splices the precursor transcript to mature-sized tRNATrp.

The yeast SUP53 gene encodes a leucine-inserting amber suppressor tRNA. We have introduced specific base substitutions into both 5' and 3' elements of the intragenic promoter of this tRNA gene. The influence of these sequence changes on promoter function has been investigated by transcription of the mutant genes in a homologous cell-free system. Our results do not support the idea that tertiary intragenic structure is important in tRNA gene transcription. For one of the SUP53 mutants we are able to suggest a plausible molecular basis for defective transcription: a single base substitution in the 3' element of the intragenic promoter prevents the interaction of this element with a putative transcription factor.

The yeast rna mutations (rna2-rna11) are a set of temperature-sensitive mutations that result in the accumulation of intron-containing mRNA precursors at the restrictive temperature. We have used the yeast in vitro splicing system to investigate the role of products of the RNA genes in mRNA splicing. We have tested the heat lability of the in vitro mRNA splicing reaction in extracts isolated from mutant and wild-type cells. Extracts isolated from seven of the nine rna mutants demonstrated heat lability in this assay, while most wild-type extracts were stable under the conditions utilized. We have also demonstrated that heat inactivation usually results in the specific loss of an exchangeable component by showing that most combinations of heat-inactivated extracts from different mutants complement one another. In three cases (rna2, rna5, and rna11), the linkage of the in vitro defect to the rna mutations was ascertained by a combination of reversion, tetrad, and in vitro complementation analyses. Furthermore, each heat-inactivated extract was capable of complementation by at least one fraction of the wild-type splicing system. Thus many of the RNA genes are likely to code for products directly involved in and essential for mRNA splicing.