The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and proteinprotein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The antiCef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.The eukaryotic spliceosome is a multicomponent ribonucleoprotein particle composed of five small nuclear RNAs, U1, U2, U4/U6, and U5, and a number of protein factors (for reviews, see Refs. 1-6). Spliceosome assembly is a stepwise process involving sequential binding of small nuclear RNAs and protein factors (7-13). During spliceosome assembly, U1 first binds to the 5Ј splice site followed by binding of U2 to the branch site through base pair interactions between the small nuclear RNAs and the intron sequences. U4/U6 and U5 are then added to the spliceosome as a preformed three-small nuclear RNP particle. This triggers a conformational rearrangement of the spliceosome in which base pairing of U1 with the 5Ј splice site is replaced by U6, and base paired U4/U6 unwinds to form new base pairings between U6 and U2 (14 -17). U1 and U4 thus become only loosely associated with the spliceosome, which is now activated and ready for catalytic reactions. It is believed that such structural rearrangements of the spliceosome are mediated by protein factors. Although several proteins containing the DEX(D/H) box motif have been shown RNA unwindase activity (6, 18 -21), no substrate specificity could be demonstrated. It remains a question what dictates the substrate specificity and mediates conformational rearrangement of the spliceosome during spliceosome assembly.We have previously shown that the yeast Saccharomyces cerevisiae Prp19p protein is essential for pre-mRNA splicing and is required before the first step of the splicing reaction. Prp19p is not tightly associated with small nuclear RNAs but is associated with the spliceosome immediately after or concomitant with dissociation of U4 from the spliceosome, s...

SummaryCarbon metabolites suppress the expression of a-amylase genes in germinating seeds and in suspension-cultured cells of rice. We have used suspension cell culture as a model system to study the mechanisms of metabolic regulation of a-amylase gene expression in rice. Both transcription rate and mRNA stability increased as cells were starved of sucrose; the transcription rate of a-amylase genes in cells starved of sucrose for 24 h was seven times greater than in cells provided with sucrose. The half-life of a-amylase mRNA was less than 1 h in cells provided with sucrose, but increased to 12 h in cells starved of this sugar. A protein synthesis inhibitor, cycloheximide (CHX), induced massive accumulation of a-amylase mRNA in cells provided with sucrose. The longer half-life of mRNA induced by sucrose starvation and the massive accumulation of mRNA caused by CHX were specific to the a-amylase genes, since actin genes were not similarly affected. Our findings suggest that both transcriptional and post-transcriptional control mechanisms are important in the metabolic regulation of a-amylase gene expression and de n o w synthesized proteins are involved in these mechanisms. The expression of a-amylase and actin genes is regulated in an opposite manner by sugars, which also suggests the operation of a differential regulatory mechanism under different growth conditions.

The yeast protein Prp19p is essential for pre-mRNA splicing and is associated with the spliceosome concurrently with or just after dissociation of U4 small nuclear RNA. In splicing extracts, Prp19p is associated with several other proteins in a large protein complex of unknown function, but at least one of these proteins is also essential for splicing (W. Splicing of pre-mRNA occurs on a large ribonucleoprotein particle (RNP) called the spliceosome, which consists of five small nuclear RNAs (snRNAs) and a number of protein factors (for reviews, see references 18, 38, 39, 47, and 49). The roles of snRNAs in spliceosome assembly have been extensively studied in both Saccharomyces cerevisiae and mammals. Base-pairing-mediated interactions between small nuclear RNPs (snRNPs), and between snRNPs and the pre-mRNA, appear to play important roles in assembly of the spliceosome (for reviews, see references 18, 47, and 49). Additional proteins are also required for proper assembly and function of the spliceosome (for reviews, see reference 4, 42, 44, and 47).-Assembly of the spliceosome is a multistep process that involves sequential binding of snRNAs to the pre-mRNA in the order U1, U2, and then U4-U6 plus U5 as a preformed tri-snRNP (8,12,28,40). After all five snRNAs are associated with the pre-mRNA, U4 becomes only loosely associated with the spliceosome and does not participate in the subsequent splicing reaction (61). A large conformational rearrangement of the spliceosome occurs, accompanying U4 dissociation as the mode of interactions between pre-mRNA and snRNAs changes. New base-pairings between U5 and the pre-mRNA and between U6 and the 5Ј-splice site region of the pre-mRNA are detected (56). It is generally believed that an RNA helicase activity is involved in this step of the assembly process to unwind base-pairings between U4 and U6 snRNAs and between pre-mRNA and snRNAs. Nevertheless, no RNA helicase activity has been demonstrated. Factors mediating such conformational change have also not yet been identified.Identification of protein factors involved in pre-mRNA splicing has been greatly facilitated by yeast genetics. A large number of PRP (precursor RNA processing) genes that encode protein splicing factors have been identified by screening temperature-sensitive mutants defective in pre-mRNA splicing (55). Other genes have been identified through genetic interactions with introns, PRP genes, or snRNA genes. They include suppressors of temperature-sensitive alleles of PRP genes (37), suppressors of snRNA mutations (46,57), and mutants with a synthetic lethal phenotype for mutations in snRNA or protein factors (17,32,50). Biochemical and genetic studies reveal that many of these genes encode snRNP-associated proteins. The SNP1, MUD1, PRP39, and PRP40 genes encode protein components of the U1 snRNP (16,25,26,32,35). Snp1p is the yeast homolog of human U1-70K protein (16, 25), and Mud1p is a U1A-like protein (32). Prp8p and Prp18p are components of the U5 snRNP (21, 36), while Prp4p and Prp6p are part of the U...