In Vitro Studies of the Prp9·Prp11·Prp21 Complex Indicate a Pathway for U2 Small Nuclear Ribonucleoprotein Activation

Abstract

Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly. This paper describes a study of interactions between these proteins and their role in spliceosome assembly. The proteins were expressed in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and present direct evidence of a Prp9·Prp11·Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9·Prp11·Prp21 complex were further investigated using proteins synthesized in vitro. In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide- directed RNaseH cleavage. We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome. Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly. This paper describes a study of interactions between these proteins and their role in spliceosome assembly. The proteins were expressed in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and present direct evidence of a Prp9·Prp11·Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9·Prp11·Prp21 complex were further investigated using proteins synthesized in vitro. In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide- directed RNaseH cleavage. We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome. INTRODUCTIONEukaryotic pre-mRNA splicing is a dynamic process involving a complex array of RNA-RNA and RNA-protein interactions (reviewed in Refs. 1Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Crossref PubMed Scopus (553) Google Scholar and 2Moore M.J. Query C.C. Sharp P.A. The RNA World. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 303-357Google Scholar). These interactions take place on a large, multi-protein RNA complex called the spliceosome. Assembly of the spliceosome occurs by ordered association of five snRNPs 1The abbreviations used are: snRNPsmall nuclear ribonucleoproteinPCRpolymerase chain reactionPASprotein A-SepharosePAGEpolyacrylamide gel electrophoresis. with the pre-mRNA substrate. Initially, the U1 snRNP associates with the pre-mRNA, followed by the U2 snRNP and finally the U4/U6·U5 tri-snRNP. The two trans-esterification reactions leading to intron removal and exon ligation are thought to be catalyzed by the RNA components of the spliceosome (reviewed in Ref. 3). The spliceosomal proteins are thought to facilitate modulation and stabilization of RNA structures critical for the splicing reaction.A large number of proteins required for pre-mRNA splicing have been identified in yeast (4Vijayraghavan U. Company M. Abelson J. Genes Dev. 1989; 3: 1206-1216Crossref PubMed Scopus (268) Google Scholar, 5Ruby S.W. Abelson J. Trends Genet. 1991; 7: 79-85Abstract Full Text PDF PubMed Scopus (184) Google Scholar). Some of these proteins can be found stably associated with specific snRNPs. Others are components of spliceosome complexes. Yet another class of splicing proteins acts transiently to facilitate spliceosome formation.The initial step in spliceosome assembly is the stable association of the U1 snRNP with the pre-mRNA (reviewed in Ref. 2). This step requires sequence elements in the pre-mRNA at the 5′ splice site and the branch point. Pre-spliceosome formation occurs when the U2 snRNP adds to the U1·pre-mRNA complex. U2 binding also requires sequences in the branch point pairing region of the pre-mRNA. These sequences become paired to the complementary branch point region of the U2 snRNA (2Moore M.J. Query C.C. Sharp P.A. The RNA World. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 303-357Google Scholar). The branch point region of U2 is situated between two highly conserved secondary structural elements (6Guthrie C. Patterson B. Annu. Rev. Genet. 1988; 22: 387-419Crossref PubMed Scopus (368) Google Scholar). One of these is the 5′ stem-loop structure and the other is a set of stem-loops with pseudoknot characteristics (7Ares Jr., M. Igel A.H. Genes Dev. 1990; 4: 2132-2145Crossref PubMed Scopus (108) Google Scholar, 8Miraglia L. Seiwert S. Igel A.H. Ares M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7061-7065Crossref PubMed Scopus (22) Google Scholar). U2 cold-sensitive mutations and revertants in this pseudoknot-like region have suggested potential structural transitions that may be important for U2 snRNP activities, including pre-spliceosome formation (9Zavanelli M.I. Ares M. Genes Dev. 1991; 5: 2521-2533Crossref PubMed Scopus (44) Google Scholar, 10Zavanelli M.I. Britton J.S. Igel A.H. Ares M. Mol. Cell. Biol. 1994; 14: 1689-1697Crossref PubMed Scopus (42) Google Scholar).Four proteins known to be required for stable association of the U2 snRNP with the pre-mRNA are Prp5, Prp9, Prp11, and Prp21 (reviewed in Ref. 11). Prp5 is a member of the DEAD family of putative RNA helicases (12Dalbadie-McFarland G. Abelson J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4236-4240Crossref PubMed Scopus (92) Google Scholar), and Prp9 and Prp11 contain amino acid homology to the TFIIIA type zinc finger RNA binding domains (13Legrain P. Choulika A. EMBO J. 1990; 9: 2775-2781Crossref PubMed Scopus (64) Google Scholar, 14Chang T.-W. Clark M.W. Lustig A.J. Cusick M.E. Abelson J. Mol. Cell. Biol. 1988; 8: 2379-2393Crossref PubMed Scopus (73) Google Scholar). Prp21 is a member of a family of proteins related in sequence to the Drosophila gene, suppressor of white apricot (15Chou T.-B. Zachar Z. Bingham P.M. EMBO J. 1987; 6: 4095-4104Crossref PubMed Scopus (82) Google Scholar). The finding that mutant alleles of the PRP5, PRP9, PRP11, and PRP21 genes show synthetic lethal genetic interactions suggested a tightly coupled role for the corresponding proteins in splicing (16Ruby S.W. Cheng T.-W. Abelson J. Genes Dev. 1993; 7: 1909-1925Crossref PubMed Scopus (114) Google Scholar, 17Wells S.E. Ares M. Mol. Cell. Biol. 1994; 14: 6337-6349Crossref PubMed Scopus (55) Google Scholar). That these proteins also interact genetically with the cold-sensitive U2 mutations described above suggested that changes in the structure of the U2 snRNA may be critical to the activities of Prp5, Prp9, Prp11, and Prp21 (16Ruby S.W. Cheng T.-W. Abelson J. Genes Dev. 1993; 7: 1909-1925Crossref PubMed Scopus (114) Google Scholar, 17Wells S.E. Ares M. Mol. Cell. Biol. 1994; 14: 6337-6349Crossref PubMed Scopus (55) Google Scholar). Pre-spliceosome formation requires ATP hydrolysis. Because Prp5 is an RNA-dependent ATPase (18O'Day C.L. Dalbadie-McFarland G. Abelson J. J. Biol. Chem. 1996; 271: 33261-33267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), it is tempting to speculate that Prp5 is responsible, at least in part, for the ATP dependence of pre-spliceosome formation. Could such an ATP-dependent reaction by Prp5 be coupled to U2 snRNA structural changes and the activity of Prp9, Prp11, and Prp21? To better understand these genetic interactions, we have purified the Prp9, Prp11, and Prp21 proteins using recombinant Escherichia coli expression systems. Purification of these proteins has allowed us to investigate their biochemical characteristics and their interactions with Prp5 and the U2 snRNP.DISCUSSIONThere are several lines of evidence to suggest that Prp9, Prp11, and Prp21 interact with each other to form a protein complex. First, SF3A, the mammalian splicing factor consisting of the Prp9, Prp11, and Prp21 homologs (SAP61, SAP62, and SAP114, respectively) (28Bennett M. Reed R. Science. 1993; 262: 105-108Crossref PubMed Scopus (74) Google Scholar, 29Brosi R. Groning K. Behrens S.-E. Luhrmann R. Kramer A. Science. 1993; 262: 102-105Crossref PubMed Scopus (118) Google Scholar, 30Behrens S.-E. Galisson F. Legrain P. Luhrmann R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8229-8233Crossref PubMed Scopus (48) Google Scholar, 31Kramer A. Legrain P. Mulhauser F. Groning K. Brosi R. Bilbe G. Nucleic Acids Res. 1994; 22: 5223-5228Crossref PubMed Scopus (35) Google Scholar), behaves as a tight protein complex in vitro when subjected to extensive column chromatography (32Brosi R. Hauri H.-P. Kramer A. J. Biol. Chem. 1993; 268: 17640-17646Abstract Full Text PDF PubMed Google Scholar). In addition, far Western type experiments testing SAP61 and SAP62 with SAP114 showed a direct interaction between these components of SF3A (28Bennett M. Reed R. Science. 1993; 262: 105-108Crossref PubMed Scopus (74) Google Scholar, 33Chiara M.D. Champion-Arnaud P. Buvoli M. Nadel-Ginard B. Reed R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6403-6407Crossref PubMed Scopus (33) Google Scholar). Second, genetic analysis of yeast strains carrying any two of the temperature-sensitive mutations of the prp9-1, prp11-1, or prp21-1 alleles exhibited a synthetic lethal phenotype, suggesting a tightly coupled role for these proteins (16Ruby S.W. Cheng T.-W. Abelson J. Genes Dev. 1993; 7: 1909-1925Crossref PubMed Scopus (114) Google Scholar, 17Wells S.E. Ares M. Mol. Cell. Biol. 1994; 14: 6337-6349Crossref PubMed Scopus (55) Google Scholar). Finally, two-hybrid experiments performed with the coding regions of Prp9, Prp11, and Prp21 revealed that these proteins interact in vivo in a manner consistent with the formation of a Prp9·Prp11·Prp21 complex (22McPheeters D.S. Fabrizio P. Abelson J. Genes Dev. 1989; 3: 2124-2136Crossref PubMed Scopus (61) Google Scholar, 23Legrain P. Chapon C. Galisson F. Genes Dev. 1993; 7: 1390-1399Crossref PubMed Scopus (42) Google Scholar).Although these lines of evidence are compelling, there had been no biochemical investigation of the yeast Prp9·Prp11· Prp21 complex. We have presented here direct physical evidence for the yeast Prp9·Prp11·Prp21 complex and have begun to characterize the biochemistry of this complex in vitro. First, we demonstrated a direct interaction between Prp11 and Prp9 and Prp21 by demonstrating that the entire complex is inactivated by heat in prp11-1 or prp21-1 extracts. All three proteins must be added to regain activity. The formation of a Prp9·Prp11·Prp21 complex in the absence of any other yeast proteins was studied by analyzing proteins made in a rabbit reticulocyte in vitro translation system. Prp9·Prp11·Prp21 formed in vitro is highly stable even at salt concentrations greater than 0.5 M NaCl. Interestingly, Legrain and colleagues (23Legrain P. Chapon C. Galisson F. Genes Dev. 1993; 7: 1390-1399Crossref PubMed Scopus (42) Google Scholar) demonstrated formation of a Prp9 homodimer using the yeast two-hybrid assay system. We were unable to detect homodimers of either Prp9, Prp11, or Prp21 in our in vitro experimental system. It is possible that observation of homodimer formation by Prp9 requires an additional factor(s) not present in our in vitro system.Previous genetic experiments also demonstrated synthetic lethal interactions of prp9-1, prp11-1, and prp21-1 with prp5-1 mutants (16Ruby S.W. Cheng T.-W. Abelson J. Genes Dev. 1993; 7: 1909-1925Crossref PubMed Scopus (114) Google Scholar, 17Wells S.E. Ares M. Mol. Cell. Biol. 1994; 14: 6337-6349Crossref PubMed Scopus (55) Google Scholar). We were unable to detect a direct interaction between Prp9·Prp11·Prp21 and Prp5, so we investigated the possibility that the observed genetic interaction between these proteins was mediated through a functional interaction. In the accompanying paper (18O'Day C.L. Dalbadie-McFarland G. Abelson J. J. Biol. Chem. 1996; 271: 33261-33267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), we demonstrated that Prp5 mediated an ATP-dependent structural change in the U2 snRNP, leading to exposure of its branch point region. We hypothesized that this exposure of the branch point region facilitated interaction of the U2 snRNP with the complementary region of the pre-mRNA. In this paper, we demonstrated that Prp9·Prp11· Prp21 is necessary for stabilization of a U2 structure that Prp5 can utilize as a substrate.Based on differences in RNaseH sensitivity of U2 RNA in mutant extracts, we propose a model in Fig. 7 for changes in U2 snRNP conformation in the activation of the U2 snRNP during spliceosome assembly. This model describes two “open” (RNaseH-accessible) states. These two states are differentiated by whether or not they require ATP to form and by their ability to assemble into a pre-spliceosome. In the absence of ATP, there are two (or more) conformational states that are in dynamic equilibrium. The open form of the equilibrium is inactive in pre-spliceosome assembly. In the absence of Prp9, Prp11, and Prp21 (e.g. in a heat-treated prp9-1 extract), this equilibrium favors the inactive, open state. Addition of Prp9·Prp11·Prp21 in the absence of ATP favors a shift to the closed state. In the presence of ATP and PRP5 this closed complex is transformed to an active open state poised for interaction with the branch point sequence of the pre-mRNA.Ares and colleagues (9Zavanelli M.I. Ares M. Genes Dev. 1991; 5: 2521-2533Crossref PubMed Scopus (44) Google Scholar, 10Zavanelli M.I. Britton J.S. Igel A.H. Ares M. Mol. Cell. Biol. 1994; 14: 1689-1697Crossref PubMed Scopus (42) Google Scholar) have shown that in the U2 cold-sensitive mutants G53A and C62U, the U2 snRNA favors an altered secondary structure or misfolded form that is in equilibrium with competent snRNP (9Zavanelli M.I. Ares M. Genes Dev. 1991; 5: 2521-2533Crossref PubMed Scopus (44) Google Scholar, 10Zavanelli M.I. Britton J.S. Igel A.H. Ares M. Mol. Cell. Biol. 1994; 14: 1689-1697Crossref PubMed Scopus (42) Google Scholar). These mutants are synthetically lethal with prp5-1, prp9-1, prp11-1, and prp21-1 (16Ruby S.W. Cheng T.-W. Abelson J. Genes Dev. 1993; 7: 1909-1925Crossref PubMed Scopus (114) Google Scholar, 17Wells S.E. Ares M. Mol. Cell. Biol. 1994; 14: 6337-6349Crossref PubMed Scopus (55) Google Scholar). Our investigation of the U2 snRNP in these mutant extracts indicated that virtually all of the snRNP was in an open conformation even in the absence of ATP. Thus the synthetic lethality could be explained by the lack of a significant population of Prp9·Prp11·Prp21-associated U2 snRNP for Prp5 to activate. The G53A and C62U mutations affect the equilibrium between different forms of the stem II structure of U2 RNA. It is not clear how different forms of this structure could affect the accessibility of the branch point interaction sequence; however, recent work by Yan and Ares (34Yan D. Ares M. Mol. Cell. Biol. 1996; 16: 818-828Crossref PubMed Scopus (54) Google Scholar) has identified U2 mutants very close to the branch point pairing sequence that interact synergistically with mutants in PRP5, PRP9, PRP11, and PRP21. The mutants are changes in U40, C41, U45, C46, and U47 just downstream from the branch point interaction sequence between residues 34 and 39. How changes in the 40-47 region affect U2 function is not clear, but one possibility is that the mutants enhance pairing with a sequence in U2 between 25 and 30. Such a helical interaction might prevent the pairing between U2 and the intron. It will be very interesting to find out how mutants in the 40-47 region score in the RNase H assay.Other members of the DEAD family of RNA helicases show characteristic low binding affinities for their RNA substrates (35Goss D. Biochemistry. 1987; 26: 1551-1556Crossref PubMed Scopus (34) Google Scholar, 36Abramson R.D. Dever T.E. Merrick W.C. J. Biol. Chem. 1988; 263: 6016-6019Abstract Full Text PDF PubMed Google Scholar). Although the detailed mechanism for specific RNA-dependent ATP hydrolysis and RNA unwinding by these proteins is not well understood, a possible mechanism could be the use of auxiliary proteins to facilitate activity and/or specificity. In the case of eIF-4A, eIF-4B is necessary to stimulate helicase activity (37Ray B.K. Lawson T.G. Kramer J.C. Cladaras M.H. Grifo J.A. Abramson R.D. Merrick W.C. Thach R.E. J. Biol. Chem. 1985; 260: 7651-7658Abstract Full Text PDF PubMed Google Scholar). eIF-4B contains an RNP motif suggestive of an RNA binding function (38Milburn S.C. Hershey J.W.B. Davies M.V. Kelleher K. Kaufman R. EMBO J. 1990; 9: 2783-2790Crossref PubMed Scopus (86) Google Scholar). Thus, a possible role for eIF-4B may be to or enhance RNA structures for use as by it has been shown that Prp9·Prp11·Prp21 to the U2 snRNP Legrain P. M. Mol. Cell. Biol. 1990; PubMed Scopus (82) Google Scholar) and the RNaseH form of the U2 snRNP that is a for Prp5 The of purified Prp9·Prp11·Prp21 protein complex facilitate investigation of potential Prp5 RNA helicase activity in for Prp9·Prp11·Prp21 Pre-spliceosome have described evidence that Prp9·Prp11·Prp21 associates with U2 and with the pre-spliceosome. possible of these proteins in the spliceosome assembly has been a highly snRNA based on the Abelson J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar), we have the presence of Prp9·Prp11·Prp21 in snRNA spliceosomal complexes. Although we of with to Prp11 and Prp21, we the that interactions with any one of the components of Prp9·Prp11·Prp21 the presence of the complex. This is Prp9·Prp11·Prp21 is a highly stable complex the in vitro of the splicing we out the possibility that the complex is during spliceosome assembly. of the spliceosome assembly is shown in Fig. We found that Prp21 could be identified as an of an complex. In experiments (18O'Day C.L. Dalbadie-McFarland G. Abelson J. J. Biol. Chem. 1996; 271: 33261-33267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) we not detect a in the in a Prp21 This was of complex is in these and the that is in that form may on the experimental We were unable to detect Prp9·Prp11·Prp21 in the other and It that Prp9·Prp11·Prp21 the complex and at the so we the to to conformational differences in these leading to of Prp9·Prp11·Prp21 or to conformational differences that to of the for INTRODUCTIONEukaryotic pre-mRNA splicing is a dynamic process involving a complex array of RNA-RNA and RNA-protein interactions (reviewed in Refs. 1Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Crossref PubMed Scopus (553) Google Scholar and 2Moore M.J. Query C.C. Sharp P.A. The RNA World. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 303-357Google Scholar). These interactions take place on a large, multi-protein RNA complex called the spliceosome. Assembly of the spliceosome occurs by ordered association of five snRNPs 1The abbreviations used are: snRNPsmall nuclear ribonucleoproteinPCRpolymerase chain reactionPASprotein A-SepharosePAGEpolyacrylamide gel electrophoresis. with the pre-mRNA substrate. Initially, the U1 snRNP associates with the pre-mRNA, followed by the U2 snRNP and finally the U4/U6·U5 tri-snRNP. The two trans-esterification reactions leading to intron removal and exon ligation are thought to be catalyzed by the RNA components of the spliceosome (reviewed in Ref. 3). The spliceosomal proteins are thought to facilitate modulation and stabilization of RNA structures critical for the splicing reaction.A large number of proteins required for pre-mRNA splicing have been identified in yeast (4Vijayraghavan U. Company M. Abelson J. Genes Dev. 1989; 3: 1206-1216Crossref PubMed Scopus (268) Google Scholar, 5Ruby S.W. Abelson J. Trends Genet. 1991; 7: 79-85Abstract Full Text PDF PubMed Scopus (184) Google Scholar). Some of these proteins can be found stably associated with specific snRNPs. Others are components of spliceosome complexes. Yet another class of splicing proteins acts transiently to facilitate spliceosome formation.The initial step in spliceosome assembly is the stable association of the U1 snRNP with the pre-mRNA (reviewed in Ref. 2). This step requires sequence elements in the pre-mRNA at the 5′ splice site and the branch point. Pre-spliceosome formation occurs when the U2 snRNP adds to the U1·pre-mRNA complex. U2 binding also requires sequences in the branch point pairing region of the pre-mRNA. These sequences become paired to the complementary branch point region of the U2 snRNA (2Moore M.J. Query C.C. Sharp P.A. The RNA World. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 303-357Google Scholar). The branch point region of U2 is situated between two highly conserved secondary structural elements (6Guthrie C. Patterson B. Annu. Rev. Genet. 1988; 22: 387-419Crossref PubMed Scopus (368) Google Scholar). One of these is the 5′ stem-loop structure and the other is a set of stem-loops with pseudoknot characteristics (7Ares Jr., M. Igel A.H. Genes Dev. 1990; 4: 2132-2145Crossref PubMed Scopus (108) Google Scholar, 8Miraglia L. Seiwert S. Igel A.H. Ares M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7061-7065Crossref PubMed Scopus (22) Google Scholar). U2 cold-sensitive mutations and revertants in this pseudoknot-like region have suggested potential structural transitions that may be important for U2 snRNP activities, including pre-spliceosome formation (9Zavanelli M.I. Ares M. Genes Dev. 1991; 5: 2521-2533Crossref PubMed Scopus (44) Google Scholar, 10Zavanelli M.I. Britton J.S. Igel A.H. Ares M. Mol. Cell. Biol. 1994; 14: 1689-1697Crossref PubMed Scopus (42) Google Scholar).Four proteins known to be required for stable association of the U2 snRNP with the pre-mRNA are Prp5, Prp9, Prp11, and Prp21 (reviewed in Ref. 11). Prp5 is a member of the DEAD family of putative RNA helicases (12Dalbadie-McFarland G. Abelson J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4236-4240Crossref PubMed Scopus (92) Google Scholar), and Prp9 and Prp11 contain amino acid homology to the TFIIIA type zinc finger RNA binding domains (13Legrain P. Choulika A. EMBO J. 1990; 9: 2775-2781Crossref PubMed Scopus (64) Google Scholar, 14Chang T.-W. Clark M.W. Lustig A.J. Cusick M.E. Abelson J. Mol. Cell. Biol. 1988; 8: 2379-2393Crossref PubMed Scopus (73) Google Scholar). Prp21 is a member of a family of proteins related in sequence to the Drosophila gene, suppressor of white apricot (15Chou T.-B. Zachar Z. Bingham P.M. EMBO J. 1987; 6: 4095-4104Crossref PubMed Scopus (82) Google Scholar). The finding that mutant alleles of the PRP5, PRP9, PRP11, and PRP21 genes show synthetic lethal genetic interactions suggested a tightly coupled role for the corresponding proteins in splicing (16Ruby S.W. Cheng T.-W. Abelson J. Genes Dev. 1993; 7: 1909-1925Crossref PubMed Scopus (114) Google Scholar, 17Wells S.E. Ares M. Mol. Cell. Biol. 1994; 14: 6337-6349Crossref PubMed Scopus (55) Google Scholar). That these proteins also interact genetically with the cold-sensitive U2 mutations described above suggested that changes in the structure of the U2 snRNA may be critical to the activities of Prp5, Prp9, Prp11, and Prp21 (16Ruby S.W. Cheng T.-W. Abelson J. Genes Dev. 1993; 7: 1909-1925Crossref PubMed Scopus (114) Google Scholar, 17Wells S.E. Ares M. Mol. Cell. Biol. 1994; 14: 6337-6349Crossref PubMed Scopus (55) Google Scholar). Pre-spliceosome formation requires ATP hydrolysis. Because Prp5 is an RNA-dependent ATPase (18O'Day C.L. Dalbadie-McFarland G. Abelson J. J. Biol. Chem. 1996; 271: 33261-33267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), it is tempting to speculate that Prp5 is responsible, at least in part, for the ATP dependence of pre-spliceosome formation. Could such an ATP-dependent reaction by Prp5 be coupled to U2 snRNA structural changes and the activity of Prp9, Prp11, and Prp21? To better understand these genetic interactions, we have purified the Prp9, Prp11, and Prp21 proteins using recombinant Escherichia coli expression systems. Purification of these proteins has allowed us to investigate their biochemical characteristics and their interactions with Prp5 and the U2