ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable ..... 5) or in the presence of control (C) peptide (lane 6). Asterisks (*) indicate.
.=j 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 20 5483 -5489
The yeast PRP8 protein interacts directly with pre-mRNA Erica Whittaker and Jean D.Beggs* Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK Received August 28, 1991; Accepted September 19, 1991
ABSTRACT The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATPdependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.
INTRODUCTION The two transesterification reactions of nuclear pre-mRNA splicing occur within a large RNA-protein complex termed the spliceosome. The spliceosome is a dynamic structure in which multiple interactions among splicing factors mediate the removal of introns from nuclear pre-mRNA molecules. It is composed of U1, U2, U4, U5 and U6 snRNPs, plus other non-snRNP proteins (for reviews see refs 1-5). Self-splicing group II introns produce equivalent reaction intermediates and products to those of pre-mRNA splicing, suggesting that nuclear pre-mRNA splicing may be primarily an RNA-catalysed reaction (6,7); however, proteins also contribute to the catalysis, and probably play structural and regulatory roles, influencing the specificity and efficiency of the reaction. Yeast genetics in combination with biochemical analysis has proven to be a powerful means for dissecting the protein components of the splicing apparatus. Identification of splicing factors has been facilitated by the isolation of prp (pre-mRNA *
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processing) mutants of S. cerevisiae that have conditional lethal phenotypes due to defects in pre-mRNA splicing. At least 30 PRP genes have been identified to date (8-10). In vitro studies demonstrated that the PRP gene products PRP3, PRP4, PRP5, PRP7, PRP8 and PRP1 1 proteins are required for spliceosome assembly, since heat-inactivated extracts from these strains do not form spliceosomes (11). Immunological analyses revealed that several of these PRP proteins are associated with snRNPs and/or spliceosomes. PRP8 protein, the subject of this paper, was shown to be a stable component of both U5 snRNPs and U4/U5/U6 multi-snRNP particles (12). In addition, affinity purification of yeast spliceosomes identified the 280 kD PRP8 protein as an integral component of spliceosomes (13). PRP4 and PRP6 are protein components of the U4/U6 snRNP complex (14-16), while PRP24 protein is associated with the U6 snRNP and interacts only transiently with the U4/U6 complex (17). PRP9 protein is required for stable association of the U2 snRNP with pre-mRNA (16), and PRP1 is probably a non-snRNP protein that associates with spliceosomes (18). Other PRP gene products appear to have only a transient association with the spliceosome, at different stages of the splicing reaction. PRP2 protein interacts with the assembled spliceosome immediately prior to and throughout the first cleavage-ligation reaction (19), whereas PRP16 protein interacts with spliceosomes at the second step (20). PRP18 protein is necessary for initiation of the second step of the splicing reaction (21), and PRP22 protein for release of the spliced mRNA (22). A number of putative RNA helicases or RNA-dependent ATPases termed DEAD or DEAH box proteins have been identified which participate in RNA splicing in yeast (reviewed in ref. 23). PRP5 and PRP28 are DEAD box proteins required early in splicing (24, 25). Another DEAD box protein is encoded by the SPP81 gene which was isolated as a suppressor of a prp8 mutation (26). In addition, a prp8 allele has been isolated that suppresses a prp28 mutation (25). The implication of interactions between PRP8 protein and RNA helicases within the splicing apparatus will be discussed later. To date PRP8 protein is the only yeast splicing factor for which homologues have been identified in mammals and in other higher organisms. Antibodies raised against several distinct regions of PRP8 protein react with a protein in HeLa cell nuclear extracts that is similar in size (>200kD) to the yeast protein and is associated with purified HeLa U5 snRNPs (27). The same protein
5484 Nucleic Acids Research, Vol. 19, No. 20 was detected in HeLa U41U5/U6 multi-snRNP particles and in affinity purified HeLa spliceosomes (28). In addition, the putative mammalian homologue of PRP8 protein was reported to contact pre-mRNA in the spliceosome (29). PRP8 protein is also conserved with respect to size and immunological epitopes as a snRNP protein in Drosophila melanogaster (T. Paterson, J.D.B., D. Finnegan and R.Luhrmann, manuscript submitted) and in tobacco and pea nuclear extracts (H. Kulesza, G. Simpson, J.W.S. Brown and J.D.B., unpublished results). The conservation of PRP8 protein across such a wide phylogenetic spectrum suggests that it plays a fundamental role in splicing. Here we use UV-crosslinking (30) combined with immunological techniques to investigate interactions between PRP8 protein and pre-mRNA. We show that PRP8 protein has a specific interaction with splicing substrate RNA; this interaction is ATP-dependent, and the RNA must be capable of participating in the first cleavage-ligation reaction to be contacted by PRP8 protein. We discuss the possibility that the unusually large PRP8 protein may have important structural and/or regulatory functions in the spliceosome.
MATERIALS AND METHODS Plasmids and preparation of RNA substrates Plasmids pSPrpS IA, pSPrp5 1A(5 '-0) and pSPrp5 IA(A3B) (3 1) were transcribed in vitro with SP6 RNA polymerase (32) to generate wild-type and mutant rp5 lA pre-mRNAs. The BglIIEcoRI fragment from pBMCY44 (33), containing the intron and most of exons 1 and 2 of rp28, was subcloned into pSPT 19, yielding plasmid pT7rp28. Plasmid p(T7)GEM287 contains the same region of the rp28 gene but possesses a single base substitution (A to C) at the branchpoint nucleotide. Plasmids pT7rp28 and p(T7)GEM287 were linearised with EcoRI, and transcribed in vitro with T7 RNA polymerase to produce rp28 pre-mRNAs. Oligodeoxynucleotide-directed cleavage of RNA substrates utilising the RNase H activity endogenous to yeast splicing extracts, was performed as described by Rymond and Rosbash (34). Oligodeoxynucleotides 1 and 2 (5' to 3': ATCAGTACGACCTAAA and GAATGGAGCATCAGTA, respectively) and A and C (35) were synthesised by the OSWEL DNA Service, Edinburgh. Full-length and RNase H-cleaved transcripts were purified by electrophoresis and electroelution from the gel with a UEA Unidirectional Electroelutor (IBI, New Haven, Connecticut, USA). For UV-crosslinking experiments, RNA transcripts of higher specific activity were produced by using 60 /ACi[a-32P]UTP (800 Ci/mmol, Amersham UK) in IOrI transcription reaction mixtures containing a total concentration of 25 ItM UTP. High specific activity transcripts were not gel purified. In vitro splicing reactions Yeast whole cell extracts were prepared and in vitro splicing reactions were performed as described previously (36, 37). Splicing reactions of 0.005 ml (for native gel electrophoresis), of 0.01 ml (for immunoprecipitation of splicing reaction products) or 0.05 ml (for immunoprecipitation from UV-irradiated samples) were composed of 50%(v/v) extract, which was a mix of 3 parts whole cell extract (36) to 2 parts 35% saturated ammonium sulphate precipitate of whole cell extract (38). Splicing reaction mixtures were incubated at 25°C for 7 minutes, then quenched on ice.
Native gel electrophoresis of splicing reactions Non-denaturing gel electrophoresis was performed essentially as described previously (39), except that 10 mM EDTA was present in both the electrophoresis buffer and the composite acrylamide/agarose gel.
UV-Crosslinking assay The method for UV-induced crosslinking of RNA to protein was modified from refs. 29, 40. Splicing reaction mixtures containing approximately 20 nM high specific activity radiolabelled substrate RNA were quenched on ice. Escherichia coli tRNA was included at a concentration of 0.2 mg/ml to dissociate weakly bound proteins. Aliquots (0.01 ml for analysis of total UV-crosslinked proteins or 0.05 ml for immunoprecipitations) were transferred to a 96 U-well microtiter plate (Cel-Cult, Sterilin Ltd, UK), and kept on ice. The samples were irradiated for 15 minutes with a short-wave germicidal lamp; the energy density at the bottom of each well was 3.7 mW/cm2, as measured by a Blak-Ray Ultra-Violet Meter, Model J-225 (Ultra-Violet Products, Inc., USA). UV-irradiated samples were digested with RNase Ti (2.5 U/pd; Boehringer Mannheim, UK) for 30 minutes at 37°C, and then either subjected to immunoprecipitation or stored at -70°C before protein gel electrophoresis. In some instances, UV-irradiated samples were subjected to denaturing conditions prior to immunoprecipitation. After RNase digestion, denaturation buffer was added to the samples, giving final conditions of 1 %(w/v) SDS (Serva), 1 % (v/v) Triton X-100 (BDH) and 100 mM DTT (Sigma) in 100 Mi1 total volume. The samples were heated to 100°C for one minute, then placed on ice. The samples were diluted 10-fold to reduce the concentration of denaturants before immunoprecipitation. Crosslinked yeast extract proteins or proteins immunoprecipitated from UV-irradiated samples were fractionated by electrophoresis in 8.5% (w/v) SDS-polyacrylamide gels (41) and
autoradiographed. Antiserum to PRP8-,B-galactosidase fusion protein 8.4 has been described (12). Anti-8.6 antibodies were raised against an aminoterminal peptide of PRP8 protein (G.J. Anderson and J.D.B., to be published elsewhere). Antibodies were bound to Protein A-Sepharose (PAS; Sigma) beads in NTN buffer (150 mM NaCl; 50 mM Tris-HCl, pH 7.5; 0.05% (v/v) Nonidet P40) and washed three times with the same buffer. UV-irradiated and standard splicing reactions were adjusted to contain 300 mM K+, 1 mM Mg2+, 11 mM EDTA, 9 mM Tris-HCl (pH 8.0), and 0.6 mg/ml E. coli tRNA. (tRNA was not added to UVirradiated samples which already contained competitor RNA.) The samples were incubated at 4°C with PAS-bound antibodies for 2 hours with mixing. The antibody complexes were washed twice with NTN buffer and once with NT buffer (150 mM NaCl; 50 mM Tris-HCl, pH 7.5). For antigen competition experiments with anti-8.4 antiserum, purified fusion protein or f3-galactosidase was added (2 ltg protein/pl antiserum) during adsorption of antibodies to PAS beads. For antigen competition experiments with anti-8.6 antiserum, the control or test peptide (0.2 ikg peptide/pl antiserum) was present with PAS-bound anti-8.6 antibodies and sample during immunoprecipitation. Immunoprecipitated RNA was recovered by treatment with
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Nucleic Acids Research, Vol. 19, No. 20 5485 proteinase K, extraction with phenol and chloroform, and precipitation with ethanol. The RNA species were separated by electrophoresis through a 6% polyacrylamide/8 M urea gel, and 32P-labelled species were detected by autoradiography.
RESULTS PRP8 protein can be UV-crosslinked to pre-mRNA In vitro splicing reactions containing radiolabelled pre-mRNA were irradiated with short-wave UV light. After digestion with RNase TI, the extract proteins were analysed by SDSpolyacrylamide gel electrophoresis. UV-crosslinking identified many yeast proteins that had interacted with splicing substrate RNA (Figure la, lane 1), although most of these interactions occurred under conditions that are not compatible with splicing (lanes 2 to 4). Immunoprecipitation from UV-irradiated splicing reactions with anti-PRP8 antibodies resulted in the recovery of radiolabelled PRP8 protein (Figure la, lane 5), indicating that PRP8 was in direct contact with pre-mRNA during the splicing reaction. The specificity of these antibodies for PRP8 protein has been reported (12, 13), and the specificity of immunoprecipitation of the radiolabelled protein was confirmed in an antigen competition experiment. Pre-incubation of anti-8.4 antibodies with purified FP8.4 (the PRP8 fusion protein to which the anti-8.4 antibodies were raised) inhibited precipitation of radiolabelled PRP8 (lane 9), due to successful competition by the fusion protein for the antigen binding sites, whereas incubation with purified f-galactosidase (the carrier polypeptide) did not (lane 10). The interaction between PRP8 protein and pre-mRNA was not detected in control reaction mixtures with no added ATP, (lanes 2 and 6), nor in reaction mixtures assembled with mutant substrate RNAs, rp5lA 5'-0 and rp5lA A3B, that have deletions at the 5' splice site and UACUAAC (lariat branchpoint) box, respectively (lanes 3, 4, 7 and 8). These mutant substrate RNAs do not form splicing complexes and therefore are not spliced in vitro (31). Similar results were obtained with anti-8.6 antibodies, raised against an amino-terminal peptide of PRP8 protein. Figure lb, lane 3 shows precipitation with anti-8.6 antibodies of radiolabelled PRP8 protein from UV-irradiated splicing reactions assembled with a different splicing substrate, rp28 pre-mRNA. Immunoprecipitation of PRP8 protein was inhibited by aminoterminal PRP8 peptide (lane 5) but not by a control peptide (lane 6), indicating the specificity of the antibody interaction. In addition to PRP8, a number of other crosslinked proteins were specifically precipitated with anti-8.6 antibodies (lanes 3 and 6). Anti-8.6 antibodies have been shown to precipitate intact spliceosomes, and these bands most likely represent other spliceosomal proteins cross-linked to pre-mRNA that were coprecipitated with PRP8, as denaturation of the samples prior to immunoprecipitation resulted in the loss of these additional proteins (data not presented). Thus, immunoprecipitation of radiolabelled protein from UVirradiated in vitro splicing reactions with anti-8.4 and anti-8.6 antibodies demonstrated that PRP8 protein contacts pre-mRNA during the splicing reaction. The interaction of PRP8 protein with pre-mRNA is splicing-dependent; the contact event occurs only with pre-mRNA molecules that participate in the splicing reaction and only in the presence of ATP, which is required for spliceosome assembly as well as for the splicing reaction.
Association of PRP8 protein with splicing complexes and binding of PRP8 protein to pre-mRNA are two separable events To investigate the substrate requirements for interaction between PRP8 protein and pre-mRNA, modified substrates were tested. Truncated RNA substrates (28/1 RNA and 28/2 RNA) were generated by oligodeoxynucleotide-directed RNase H cleavage of rp28 pre-mRNA with oligos 1 or 2, complementary to the 3' splice site and to the sequence immediately downstream of the 3' splice site in exon 2 (Figure 2a). A
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Figure 1. Crosslinking of PRP8 protein to pre-mRNA. A. In vitro splicing reactions (10 i1), containing wild-type (WT) rp5lA pre-mRNA with (lane 1) or without (lane 2) ATP, or reaction mixtures containing mutant rp5 lA pre-mRNA with a deletion of the 5' splice site (5'-0; lane 3) or a deletion of the UACUAAC box (A3B; lane 4) were UV-irradiated, treated with RNase TI, and the proteins were fractionated by SDS-PAGE. Splicing reaction mixtures (50 /1) containing wild-type pre-mRNA (lanes 5, 6, 9 and 10) or mutant pre-mRNA (lanes 7 and 8) were UV-irradiated, treated with RNase T I and subjected to immunoprecipitation with anti-8.4 antibodies (lanes 5 to 8) or with anti-8.4 antibodies pre-saturated with PRP8 fusion protein 8.4 (FP; lane 9) or with ,B-galactosidase (,B-g; lane 10). The immunoprecipitated proteins were resolved by SDS-PAGE. Protein-RNA adducts were visualised by autoradiography. The positions of marker proteins are indicated (sizes in kD). B. In vitro splicing reactions were assembled with rp28 pre-mRNA with (lane 1) or without (lane 2) added ATP, exposed to shortwave UV light, treated with RNase TI, and the RNA-protein adducts resolved by SDS-PAGE. UV-irradiated, RNase Ti-treated splicing reaction mixtures with (lanes 3, 5 and 6) or without added ATP (lane 4) were subjected to immunoprecipitation with anti-8.6 antibodies (lanes 3 to 6), in the presence of the aminoterminal PRP8 peptide against which the anti-8.6 antibodies were raised (lane 5) or in the presence of control (C) peptide (lane 6). Asterisks (*) indicate radiolabelled proteins present in the anti-8.6 precipitates which do not correspond to PRP8 protein (lanes 3 and 6). Sizes of protein molecular weight standards are given in kD.
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