John Abelson

The yeast Saccharomyces cerevisiae is known to contain the highly conserved and unbiquitous protein actin. We have used cloned actin sequences from Dictyostelium discoideum to identify and clone the actin gene in yeast. Hybridization to genomic fragments of yeast DNA suggest that there is a single actin gene in yeast. We have determined the nucleotide sequence of that gene and its flanking regions. The sequence of the gene reveals an intervening sequence of 309 base pairs in the coding sequences at the 5' end of the gene. The existence and location of the intervening sequence was verified by using the dideoxy chain termination technique to determine the sequence at the 5' terminus of the actin mRNA. The similarity of the splice junction sequences in this gene to those found in higher eukaryotes suggests that yeast must possess a similar splicing enzyme.

The nucleotide sequence of the lac promoter-operator region has been determined. The 122 base pairs comprising this region include the recognition sites for RNA polymerase, the positive regulatory protein, CAP, and the negative regulatory protein, the repressor. Identification of mutant variants of the sequence combined with the in vitro biochemical studies of others has allowed us to tentatively identify the recognition site for each of these proteins, and to suggest how CAP might act at a distance to affect the interaction of RNA polymerase with the promoter.

The in vitro splicing reactions of pre-messenger RNA (pre-mRNA) in a yeast extract were analyzed by glycerol gradient centrifugation. Labeled pre-mRNA appears in a 40S peak only if the pre-mRNA undergoes the first of the two partial splicing reactions. RNA analysis after extraction of glycerol gradient fractions shows that lariat-form intermediates, molecules that occur only in mRNA splicing, are found almost exclusively in this 40S complex. Another reaction intermediate, cut 5' exon RNA, can also be found concentrated in this complex. The complex is stable even in 400 mM KCl, although at this salt concentration, it sediments at 35S and is clearly distinguishable from 40S ribosomal subunits. This complex, termed a "spliceosome," is thought to contain components necessary for mRNA splicing; its existence can explain how separated exons on pre-mRNA are brought into contact.

Precursors to mRNA become substrates for splicing by being assembled into a complex multisubunit structure, the spliceosome. To study the assembly of the yeast spliceosome, intermediate complexes were separated by electrophoresis on nondenaturing polyacrylamide gels. Four splicing-dependent complexes, A1, A2-1, A2-2, and B, were observed. The order of assembly of these complexes was determined to be B----A2-1----A1----A2-2. The assembly process can be blocked at complex A1 by addition of 5 mM EDTA or by carrying out the assembly process in heat-inactivated rna2 extracts. The snRNA composition of the complexes was determined by hybridization with probes for five yeast snRNAs. snR14 (U4) was only found in complex A2-1, snR6 (U6) and snR7 (U5) were in complexes A1, A2-1, and A2-2, whereas snR20 (U2) was in all four of the complexes. snR19 (U1) was not present in any of the complexes. Hybridization with these probes was also employed to detect snRNPs present in yeast splicing extracts. We found that snR6, snR7, and snR14 were present together in a large complex. This complex underwent an ATP-dependent dissociation to give snR7 and snR6-snR14 complexes. snR19 and snR20 are present in distinct RNPs but the mobility of these is not affected by ATP. A mechanism for spliceosome assembly is proposed.