No abstract

Pre-mRNA binding to the yeast U2 small nuclear ribonucleoprotein (snRNP) during prespliceosome formation requires ATP hydrolysis, the highly conserved UACUAAC box of the branch point region of the pre-mRNA, and several factors. Here we analyzed the binding of a radiolabeled 2-O-methyl oligonucleotide complementary to U2 small nuclear RNA to study interactions between the UACUAAC box, U2 snRNP, and Prp5p, a DEAD box protein necessary for prespliceosome formation. Binding of the 2-O-methyl oligonucleotide to the U2 snRNP in yeast cell extract was assayed by gel electrophoresis. Binding was rapid, enhanced by ATP, and dependent on the integrity and conformation of the U2 snRNP. It was also stimulated by Prp5p that was found to associate physically with U2 snRNP. In vitro heat inactivation of the temperature-sensitive prp5-1 mutant extract decreased oligonucleotide binding to U2 and the ATP enhancement of binding by 3-fold. Furthermore, the temperature-sensitive prp5-1 mutation maps to the ATP-binding motif I within the helicase-like domain. Thus the catalytic activity of Prp5p likely promotes a conformational change in the U2 snRNP.The spliceosome is a large, dynamic ribonucleoprotein particle that catalyzes nuclear pre-mRNA splicing. It is composed of multiple factors including five snRNPs 1 and numerous nonsnRNP proteins, which assemble on the pre-mRNA (1, 2). It undergoes a number of conformational changes during its transit through a splicing cycle. At least seven such changes are disruptions of RNA base pairings (3-5), whereas other changes involve protein-protein and RNA-protein interactions. Some of these changes are probably catalyzed by DEXD/H-box proteins, eight of which are known to be required for the splicing pathway in the yeast Saccharomyces cerevisiae (6).DEXH/D-box proteins are numerous in both prokaryotes and eukaryotes and are involved in diverse biological processes, but they all have a similar domain with six or seven signature motifs (7,8). In the archetypal DEAD box family member, EIF4a, these motifs encode RNA-dependent ATPase and RNA unwinding activities (9, 10). To date the spliceosomal DEXH/D box proteins are fulfilling the prediction that they catalyze the RNA rearrangements in the spliceosome. They are required at steps in which ATP hydrolysis and spliceosomal structural changes both occur; most have been shown to have RNA-dependent ATPase activity; and some have even been found to have RNA unwinding activity (6). Nevertheless, the actual targets of the spliceosomal DEAD box proteins are unknown. Some of the proteins may not even unwind RNA but instead act as an RNPase to remove protein bound to RNA (11,12).Two of the first DEAD-box proteins required for yeast prespliceosome formation on the pre-mRNA are Sub2p (13-15) and Prp5p (16). The prespliceosome is an early intermediate in spliceosome formation. It immediately succeeds the binding of the U1 snRNP and at least two proteins (Bbp and Mud2p) to the pre-mRNA. During prespliceosome formation, Bbp and Mud2p are displaced from the pre-mR...

The mammalian 70K protein, a component of the U1 small nuclear ribonucleoprotein involved in pre-mRNA splicing, interacts with a number of proteins important for regulating constitutive and alternative splicing. Similar proteins that interact with the yeast homolog of the 70K protein, Snp1p, have yet to be identified. We used the two-hybrid system to find four U1-Snp1 associating (Usa) proteins. Two of these proteins physically associate with Snp1p as assayed by coimmunoprecipitation. One is Prp8p, a known, essential spliceosomal component. This interaction suggests some novel functions for Snp1p and the U1 small nuclear ribonucleoprotein late in spliceosome development. The other, Exo84p, is a conserved subunit of the exocyst, an eight-protein complex functioning in secretion. We show here that Exo84p is also involved in pre-mRNA splicing. A temperaturesensitive exo84 mutation caused increased ratios of pre-mRNA to mRNA for the Rpl30 and actin transcripts in cells incubated at the non-permissive temperature. The mutation also led to a defect in splicing and prespliceosome formation in vitro; an indication that Exo84p has a direct role in splicing. The results elucidate a surprising link between splicing and secretion.The U1 snRNP 1 has an early, hierarchic role in pre-mRNA splicing in the yeast Saccharomyces cerevisiae (1-3). It must be bound to pre-mRNA for subsequent stable association of the other four spliceosomal snRNPs with the pre-mRNA. Once U1 snRNP is bound, U2 snRNP binds, and the prespliceosome is formed. The tri-snRNP complex, U4/U6.U5, then binds to form the spliceosome. The spliceosome next undergoes a number of coordinated rearrangements (4). The duplexes between the U4 and U6 snRNAs and between U1 snRNA and the 5Ј-splice site (SS) of the pre-mRNA are disrupted, whereas new pairings between U2 and U6 snRNAs, U6 snRNA and the 5Ј-SS, and U5 snRNA and the pre-mRNA exons 1 and 2 are formed. These rearrangements lead to the formation of the active catalytic site of the spliceosome. Splicing of the pre-mRNA then ensues by two transesterification reactions.The yeast U1 snRNP recognizes both the 5Ј-SS and the branchpoint region of the pre-mRNA (3, 5). From 5 to 7 nucleotides of the 5Ј end of the U1 snRNA base pair with the

This study was designed to compare the efficacy of three insulinotropic agents in the control of postprandial hyperglycemia in type 2 diabetes. Fifteen subjects with noninsulin-requiring type 2 diabetes were admitted to the General Clinical Research Center on four separate occasions. During the control study and following 7-10 d on each study medication, daylong glucose profiles were performed to investigate the effects of the assigned medication on postprandial hyperglycemia. During each admission, placebo or study medications were administered before three isocaloric meals as follows: immediate-release glipizide 30 min before breakfast and 30 min before supper, glipizide gastrointestinal therapeutic system (GITS) 30 min before breakfast, or nateglinide 120 mg 10 min before breakfast, before lunch, and before supper. Blood was drawn for analysis of glucose, insulin, and C-peptide at -0.05, 0, 0.25, 0.5, 1, 2, 3, and 4 h relative to each test meal. Immediate-release glipizide, nateglinide, or glipizide GITS administration resulted in significantly lower integrated daylong (glucose area under the curve) and peak glucose levels, compared with placebo. There were no significant differences in the daylong integrated glucose levels among the three study medications. The peak postbreakfast glucose level (but not glucose area under the curve) was lower with nateglinide, compared with either immediate-release glipizide or glipizide GITS. Postlunch and postdinner integrated glucose levels were significantly lower with immediate-release glipizide or glipizide GITS, compared with nateglinide. C-peptide levels were significantly higher with immediate-release glipizide, compared with glipizide GITS. Insulin levels did not differ among the three study medications. Once-daily glipizide GITS, twice-daily immediate-release glipizide, or three-times-a-day administration of nateglinide results in equivalent control of postmeal hyperglycemia in type 2 diabetes. The decision to prescribe one of these three insulinotropic agents should be based on factors such as the patient's ability to comply with complex dosing regimens, the need to control fasting hyperglycemia, the risk of interprandial hypoglycemia, and pharmacoeconomic considerations, rather than postprandial glucose-lowering efficacy.