INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Nuclear pre-mRNA splicing is a dynamic process in which intervening sequences (introns) are excised from pre-mRNAs in a two-step mechanism that is catalyzed by the spliceosome (for review, see references 15 and 28). The spliceosome assembles on the pre-mRNA by the stepwise integration of the small nuclear ribonucleoproteins (snRNP), U1, U2, U4/U6, and U5, and an as-yet-undefined number of non-snRNP proteins. In the early phase of spliceosome formation, U1 snRNA base pairs with the 5' splice site, and U2 snRNA interacts with the branch site to form the prespliceosomal E and A complexes, respectively. Spliceosomal assembly is completed by addition of the 25S U4/U6.U5 tri-snRNP complex, which is formed under splicing conditions from the U4/U6 and U5 snRNP particles (reviewed in reference 28). After assembly, the spliceosome undergoes several RNA and protein conformational rearrangements. For example, the U4/U6 duplex, which is present in the tri-snRNP, is unwound, and the U6 snRNA interacts instead with the 5' end of the U2 snRNA (7, 26, 47, 58) and with intron sequences at the 5' splice site (14, 21, 40, 41, 44, 56). The rearranged U2/U6 snRNA network is thought to be involved in the catalytic step of splicing (25), and U5 snRNA is involved in aligning the two exons for ligation (32). After the splicing reaction, the spliceosome is dissolved, and the tri-snRNP is generally believed to be assembled on the U5 snRNP again. The U5 snRNP therefore plays an important role in both spliceosome assembly and splicing.

The protein composition of the U5 snRNP complex has been most thoroughly investigated for HeLa cells; this complex contains at least 17 proteins, many of which have been characterized by cDNA cloning. These proteins can be classified into two groups. Like all other snRNPs of the spliceosome, U5 snRNP contains a set of the so-called Sm proteins: B/B', D1, D2, D3, E, F, and G. These proteins bind to a conserved site on the snRNA, thus forming the "core complex." It has been shown that the Sm proteins play an essential part in the biogenesis of the snRNPs (33) and that they also have an important task in the stabilization of the snRNPs and their integration into the spliceosome. In addition to the Sm proteins, the U5 snRNPs contain a number of particle-specific proteins with molecular masses of 15, 40, 52, 100, 102, 110, 116, 200, and 220 kDa. In accordance with the conservation of the splicing mechanism, the yeast Saccharomyces cerevisiae possesses a very similar set of proteins (10): not only are the Sm proteins all strongly conserved (42), but clear homology could also be established for many of the U5-specific proteins.

Interestingly, many of the U5-specific proteins are essential for splicing and have important tasks in the dynamics of the spliceosome (reviewed in references 45 and 57). Some examples of these are as follows. (i) The 200-kDa protein (200kD) and its yeast homologue, Snu246p (18)also termed Brr2p, Slt22p, or Rss1p (22, 34, 60)contain both DEXH-box RNA helicase motifs, and the isolated human 200kD protein possesses RNA unwindase activity (16). (ii) The 100-kDa protein of U5 (U5-100kD) also possesses an RNA helicase domain, in this case of the DEAD box type (50). Its yeast orthologue, Prp28p, is needed for the maturation of the spliceosome before the first step of the splicing reaction; however, the target of its action is not known (46). (iii) The U5-116kD protein probably catalyzes rearrangements during the spliceosomal cycle. Both this protein and its orthologue in yeast, Snu114p, show extensive homology with the translational elongation factor EF-2, a GTPase that is needed for the translocation of mRNA on the ribosome (11). The human U5-116kD protein has been shown to bind GTP in vitro, and a point mutation in the GTP-binding motif of Snu114p yields a lethal phenotype. (iv) The 220kD protein and its yeast orthologue, Prp8p, have also been associated with rearrangements on the spliceosome. In both systems, this protein has been shown to contact pre-mRNA. Interestingly, the 220kD protein (or Prp8p) contacts the pre-mRNA at the 5' splicing site before the first catalytic step of splicing (38, 52, 59) and then moves over and contacts the 3' splicing site after the first step (6, 51, 53). Furthermore, this protein has been shown to be close to the branching point (24, 52). Thus, the 220kD protein (or Prp8p) is in contact with all three elements of the pre-mRNA involved in splicing, and it clearly plays a key role in the spliceosome. This central role is underlined by the strikingly high degree of conservation of this protein (12, 18, 23).

As the 220kD protein binds to different parts of the pre-mRNA, there arises the interesting possibility that it plays a central role in the communication between the pre-mRNA and the enzymes in the U5 snRNPfor example, by triggering their activity. This would require these proteins to interact with one another, directly or indirectly. However, nothing is known at present about the interactions between the U5-specific proteins. In the work described here, we studied protein-protein interactions in the U5 snRNP. In one approach, U5-specific proteins were dissociated from the U5 core particle by using the chaotropic salt sodium thiocyanate, allowing the investigation of snRNA-free subcomplexes of these proteins. As described below, we have been able to show that the 220kD protein, the 200kD RNA unwindase, the 116kD G protein, and the U5-40kD protein (here shown to be a new member of the WD-40 repeat family) together make up an snRNA-free complex that comprises more than one-half of the total mass of the U5 snRNPs. The core of this complex is the 220kD protein, which binds directly to the 40- and 116kD proteins and probably also to the 200kD RNA unwindase.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Preparation of 20S U5 snRNPs from HeLa nuclear extracts. Nuclear extracts were prepared from HeLa cells (Computer Cell Culture Company, Mons, Belgium) according to the method of Dignam et al. (8). Total snRNPs containing U5 and U4/U6 snRNPs were purified from nuclear extract by affinity chromatography at 420 mM NaCl with the monoclonal antibody H20 as described by Laggerbauer et al. (17). U5 snRNPs and U4/U6.U5 tri-snRNPs were obtained by the same procedure at 250 mM NaCl. 20S U5 snRNPs were obtained by subsequent fractionation of the snRNP particles on a 10-to-30% (wt/wt) glycerol gradient containing 150 mM KCl according to Laggerbauer et al. (17).

Fractionation of U5 snRNP components in thiocyanate-containing glycerol gradients. In a typical experiment, 750 µg of purified U5 snRNP was concentrated by centrifugation in a Beckman TLA 100.3 rotor at 70,000 rpm for 17 h at 4°C. The supernatant was discarded, and the U5 snRNP-containing pellet was resuspended in 800 µl of buffer R400 (20 mM HEPES-KOH, [pH 7.9], 400 mM sodium thiocyanate [NaSCN], 1.5 mM MgCl₂). For fractionation, the U5 snRNPs were layered onto a linear 12-ml, 5-to-20% (wt/wt) glycerol gradient prepared with buffer R400. The gradients were centrifuged in a Beckman SW40 Ti rotor at 33,000 rpm for 21 h at 4°C, and 30 420-µl fractions were harvested manually from top to bottom. Proteins were recovered from 50-µl aliquots by acetone precipitation, separated by sodium dodecyl sulfate (SDS)-13% polyacrylamide gel electrophoresis (PAGE) with a high concentration of TEMED (N,N,N',N'-tetramethylethylenediamine) (20), and visualized by Coomassie blue staining. RNA was recovered from 30-µl aliquots by phenol extraction and ethanol precipitation, separated by urea-10% PAGE, and visualized by silver staining. Alternatively, the U5 snRNPs in 800 µl of R400 buffer were layered onto a 10-to-30% (wt/wt) glycerol gradient in buffer R200 (20 mM HEPES-KOH [pH 7.9], 200 mM NaSCN, 1.5 mM MgCl₂) and centrifuged in an SW40 Ti rotor at 30,500 rpm for 18.5 h at 4°C. The gradient was harvested as before. In reassociation experiments, fractions from glycerol gradients in buffer R400 were mixed and dialyzed against one change of 200 volumes of R200 buffer without glycerol. Four hundred microliters of the dialysate was layered onto a 4.2-ml, 5-to-20% (wt/wt) glycerol gradient prepared in buffer R200 and centrifuged in a Beckman SW60 Ti rotor for 12 h at 40,000 rpm at 4°C. Twenty-eight 140-µl fractions were collected from the top.

Microsequencing and database search. snRNP proteins were extracted from 20 mg of affinity-purified snRNPs with 1 volume of phenol, and proteins were precipitated from the phenolic phase with 5 volumes of acetone. The precipitated proteins were separated by preparative SDS-PAGE. Coomassie-stained bands of individual snRNP proteins were excised from the acrylamide gel, concentrated by funnel-well SDS-PAGE, and electroblotted onto a sequencing-grade polyvinylidene difluoride membrane (Bio-Rad). Partial amino acid sequences of tryptic peptides of the snRNP proteins were obtained by microsequencing on an ABI 477A protein sequencer. Sequence analysis was performed at the microchemical facility of Harvard University, Cambridge, Mass.

Expression of His-tagged 40kD protein and immunization of rabbits. To clone the 40kD cDNA into the pET 28b vector (Novagen), NcoI and XhoI restriction sites were added via PCR amplification to the N and C termini, respectively, of the 40kD EST. The PCR fragment was cloned into the pET expression vector, thus encoding the full-length 40kD protein fused to a C-terminal His tag (six histidines). Correct orientation and the presence of an ORF were verified by DNA sequencing. The fusion protein was expressed in Escherichia coli BL21 cells for 3 h at 30°C after induction with 1 mM IPTG (isopropyl--D-thiogalactopyranoside). The protein was found to accumulate in inclusion bodies, from which it was extracted and purified with Ni-agarose under denaturing conditions essentially as described by the supplier (Novagen). This fusion protein was used for the immunization of rabbits.

Translation and radioimmunoprecipitation in vitro. For translation in vitro, the StuI-NotI fragment containing the entire ORF of the 40kD protein was subcloned into the pBluescript SK() vector. Coupled transcription and translation reactions in vitro were performed by using the Promega TNT (T7) system, applying procedures recommended by the manufacturer.

Far-Western analysis. Protein fractions were separated on an SDS-9% polyacrylamide gel and transferred to nitrocellulose. Proteins were denatured and subsequently renatured by washing (for 15 min at 4°C) the blot in NET₁₅₀ (50 mM Tris-HCl [pH 7.9], 150 mM NaCl, 0.1% Triton X-100) containing 6 M guanidine-HCl, then in NET₁₅₀ containing 2 M guanidine-HCl, and finally in NET₁₅₀ containing 0.66 M guanidine-HCl. The nitrocellulose was then blocked for 3 h with NET₁₅₀ containing 5% fat-free dried milk, washed with NET₁₅₀, and incubated overnight at 4°C with 10⁶ cpm of [³⁵S]Met-labelled protein prepared by in vitro translation (40kD or 116kD) in NET₁₅₀. The blot was washed three times for 15 min with NET₁₅₀ and autoradiographed with X-ray film.

Nucleotide sequence accession number. The nucleotide and amino acid sequences for the 40kD protein have been deposited in the GenBank database under accession no. AF090988.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Dissociation of 20S U5 snRNPs with NaSCN. To study possible protein-protein interactions, we first attempted to dissociate purified U5 snRNPs by treatment with various salts, with the aim of isolating stable protein heteromers. Interpretation of the results obtained by this approach relies on the assumption that strong protein-protein interactions, once established in RNA-free assembly intermediates, may persist in the intact particle and may also survive RNP dissociation under mild conditions. This method has been successfully employed to elucidate protein-protein interactions in the ribosome (9) and the Sm core (36) and to characterize the complex of the 20kD, 60kD, and 90kD proteins in the U4/U6 snRNP (49). With the U5 snRNP, this approach is especially useful because the high molecular weight of several of the U5-specific proteins renders recombinant expression of full-length proteins difficult (our unpublished results). Treatment of U5 snRNPs with a high concentration of salt, such as KCl, causes only a few proteins, including the 100kD putative RNA helicase, to dissociate almost quantitatively (16). The remaining, salt-resistant U5 snRNP particle contains, in addition to the Sm core proteins, the U5-specific 40kD, 102kD, 116kD, 200kD, and 220kD proteins, which are therefore good candidates for studying stable protein-protein interactions.

View larger version (87K): [in this window] [in a new window]   FIG. 1.   Dissociation of U5 snRNP particles by using sodium thiocyanate. U5 snRNPs were disrupted in 0.4 M NaSCN and separated on a 5-to-20% glycerol gradient containing the same buffer as described in Materials and Methods. Proteins were recovered from aliquots of each fraction by acetone precipitation, separated on an SDS-13% polyacrylamide gel and visualized by staining with Coomassie blue. The top 24 fractions are shown (no protein was seen in the bottom 6 fractions). The positions of the U5 proteins are indicated on the right, and those of molecular mass markers are indicated on the left. The positions to which ovalbumin (3.5S), albumin (4.6S), and immunoglobin G antibodies (7.0S) sedimented on an identical gradient are shown at the top. The bottom panel shows the RNA that was recovered by phenol extraction and ethanol precipitation, separated by urea-10% PAGE, and visualized by silver staining.

The 220kD protein forms an RNA-free, heteromeric complex with the 40kD, 116kD, and 200kD proteins. Figure 1 shows that the 116kD protein directly and stably associates with the 220kD protein and provides initial evidence that the 40kD protein associates with this dimer. To investigate whether further U5-specific proteins also interact directly with the 220kD protein, we lowered the stringency of the dissociation conditions under which the U5 components were separated. The proteins and complexes obtained by resuspending U5 RNP particles in 0.4 M NaSCN were separated on a glycerol gradient containing only 0.2 M NaSCN, so that protein-protein interactions that are stable under these conditions could be reestablished upon entry into the glycerol gradient. As shown in Fig. 2, under these conditions the bulk of the 40kD protein cosedimented with the 220kD and 116kD proteins (fractions 13 to 17), while a minor part of the 40kD protein stayed on top (fractions 1 to 3), as expected for a monomeric protein of this size. This result substantiates the hypothesis, posited above, that the 40kD protein forms a specific complex with the 116kD and 220kD proteins and that this complex is semistable in 0.4 M NaSCN.

View larger version (77K): [in this window] [in a new window]   FIG. 2.   Dissociated U5 snRNP particles fractionated at reduced sodium thiocyanate concentration. U5 snRNPs were disrupted in 0.4 M NaSCN as in Fig. 1 and then layered onto a 10-to-30% glycerol gradient containing 0.2 M NaSCN in the same buffer. Proteins (top panel) and RNA (bottom panel) were recovered and visualized as described for Fig. 1. The positions of the U5 proteins are indicated on the right, and those of molecular mass markers are shown on the left.

View larger version (123K): [in this window] [in a new window]   FIG. 3.   The U5-200kD RNA helicase associates at 0.2 M sodium thiocyanate with the 40/116/200 complex. Fractions enriched in the 40kD, 116kD, and 220kD proteins (similar to fractions 16 and 17 in Fig. 1) or enriched in the 200kD protein (corresponding to fractions 9 to 11) were pooled from a gradient similar to that shown in Fig. 1, dialyzed separately (top and middle panels) or together (bottom panel) against buffer containing 0.2 M NaSCN, and then run on separate glycerol gradients prepared with the dialysis buffer as described in Materials and Methods. Proteins from 40 µl of 140 µl of each fraction, or from 20 µl of the sample loaded onto the gradients (first lane), were separated by SDS-10% PAGE and visualized by staining with Coomassie blue. The positions of the relevant proteins are indicated on the left.

The U5-40kD protein is a novel WD-40 repeat protein. The experiments discussed above demonstrate that the 40-, 116-, and 200kD proteins form a stable, snRNA-free complex with the 220kD protein. While the 116kD and 200kD proteins have been characterized by cDNA cloning and are implicated in the dynamics of the spliceosome (see the introduction), the 40kD protein has not yet been characterized at a molecular level. To obtain further details of the protein-protein interactions in this interesting heteromer, it was first necessary to characterize the 40kD protein by cDNA cloning.

View larger version (47K): [in this window] [in a new window]   FIG. 4.   The U5-40kD protein is a novel WD-40 repeat protein. (A) Predicted amino acid sequence of the 40kD protein. Peptide sequences obtained by microsequencing are underlined; amino acids highly conserved in WD-40 repeats are shown in bold. (B) Alignment of the seven WD-40 repeats. Amino acids that agree with the consensus at the five highly conserved positions are boxed in black, and those meeting the requirements in the less conserved positions are shaded in grey. The bottom lines list all amino acids that may occur in a given position of the consensus sequence. Lowercase letters indicate groups of amino acids: h, ACMFWYVIL; t, DGNP; s, GSTACY. The brackets indicate how many nonconserved residues can be found in that region. These data are adapted from reference 31.

View larger version (50K): [in this window] [in a new window]   FIG. 5.   Characterization of the cDNA-encoded 40kD protein. Antibodies raised against the recombinant 40kD protein recognize the native U5 snRNP protein. Proteins present in 20 µl of nuclear extract (lanes 3 and 5) or in 5 µg of purified snRNP (lanes 4 and 6) were separated by SDS-PAGE and blotted onto nitrocellulose. The membrane was immunostained with antibodies directed against the 40kD protein (lanes 5 and 6) or preimmune serum derived from the same rabbit (lanes 3 and 4). Lanes 1 and 2 show marker proteins and snRNP proteins, respectively, which were separated on the same gel and visualized by Coomassie blue staining. Molecular mass markers are shown on the left, and the position of the 40kD protein is shown on the right.

View larger version (74K): [in this window] [in a new window]   FIG. 6.   Alignment of the U5-40kD protein with highly homologous WD proteins. The U5-40kD protein was aligned with a hypothetical 38kD protein from A. thaliana (accession no. AC002333), and hypothetical proteins of 38.8 and 38.7 kDa from C. elegans (accession no. AF000265 and Z66561, respectively). The position of the WD domains is indicated by the numbered double arrows. Amino acids that are identical in at least three sequences are boxed in black, and those that are conserved in at least three sequences are shaded grey. Conserved amino acids are grouped as follows: DE, HKR, CILMVFYW, ST, and AG.

The 220kD protein interacts directly with the 40kD protein and presumably also with the 200kD protein. Our sedimentation studies demonstrate that the 40kD, 116kD, and 200kD proteins form a specific, snRNA-free complex with the 220kD protein. However, only the 116kD protein has been shown so far to interact directly with the 220kD protein, because this is the only protein stably and stoichiometrically associated with it at 0.4 M NaSCN. Since the 220kD protein plays a key role in the spliceosome, we were interested to see whether the other two proteins also interact directly with the 220kD protein. For this purpose, we first tested which protein 40kD interacts with. Fractions enriched in the 100kD and 200kD proteins as well as those containing the 116/220 complex were taken from an NaSCN gradient similar to the one shown in Fig. 1 and used to coprecipitate ³⁵S-labelled 40kD protein prepared by translation in vitro. In the presence of the U5-specific 100kD protein (Fig. 7, lanes 5 to 8) or the 200kD protein (lanes 9 to 12), none of the sera directed against the 100kD, 116kD, 200kD, or 220kD protein coprecipitated the 40kD protein, whereas all four sera coprecipitated the 40kD protein when it was incorporated into U5 snRNPs (lanes 1 to 4). This indicates that the 40kD protein interacts neither with the 200kD protein (at least not directly) nor with the 100kD protein. In contrast, radioactively labelled 40kD protein coprecipitated in significant amounts with the 116/220 dimer (Fig. 7, lanes 14 and 16). This interaction is specific, since no precipitation was observed in the absence of the 116/220 complex (lanes 6, 8, 10, and 12).

View larger version (71K): [in this window] [in a new window]   FIG. 7.   35S-labelled 40kD protein prepared by translation in vitro associates specifically with the 116/220 dimer. The translated 40kD protein was incubated in the presence of U5 snRNP particles (lanes 1 to 4) or with fractions enriched in proteins in the 1000-kDa range (lanes 5 to 8), the 200kD protein (lanes 9 to 12), or the 40kD, 116kD, and 220kD proteins (lanes 13 to 16). The protein composition of these fractions is shown in Fig. 8, left panel. Proteins precipitated with protein A-Sepharose-bound antibodies specific for the 100kD, 116kD, 200kD, or 220kD protein, as indicated above each lane, were separated by SDS-PAGE, and the 40kD protein was visualized by fluorography.

View larger version (88K): [in this window] [in a new window]   FIG. 8.   The 40kD and 116kD proteins both interact with the 220kD protein on far-Western blots. U5 snRNP proteins and gradient fractions containing proteins in the 100-kDa range, the 200kD protein, or the 40kD, 116kD, and 220kD proteins were separated on three identical SDS-9% polyacrylamide gels. The proteins were either visualized by staining with Coomassie blue (left panel) or blotted onto nitrocellulose and probed with the 40kD protein prepared by translation in vitro (middle panel) or the 116kD protein (right panel) as described in Materials and Methods. The autoradiograms of the membranes are shown.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We have shown here that four of the U5-specific proteins, together comprising more than one-half of the mass of the U5 snRNP particle, form a complex which is stable in the absence of snRNA or the Sm proteins. Two of these proteins, the U5- 116kD G protein and the U5-200kD RNA unwindase possess domain structures which suggest that they participate in rearrangement steps during spliceosome assembly or the splicing process itself. The third protein in the complex is the 220kD human orthologue of the yeast protein Prp8p, which binds on both cutting sites of the pre-mRNA and thus plays a central part in the functioning of the spliceosome. Finally, the protein complex contains the U5-40kD protein, which we here show to be a novel WD-40 repeat protein. WD-40 repeat proteins are frequently involved in the regulated association and dissociation of protein complexes; it can therefore be assumed that the 40kD protein also plays a part in the assembly of the spliceosome.

As an initial approach to investigating protein-protein interactions in the U5 snRNP particle, we dissociated purified U5 snRNP particles in the presence of 0.4 M NaSCN. Under these conditions, the U5-specific proteins are completely released while the snRNP core particle is left intact. The resulting pool of U5-specific proteins yields a reconstituted complex of the 40kD, 116kD, 200kD, and 220kD proteins when the NaSCN concentration is reduced to 0.2 M (Fig. 2). Even though all other U5 components were present during this step, no interaction between these four proteins and the other U5-specific proteins or the core complex was observed. This shows that the other proteins do not compete for the interactions that keep the tetrameric complex intact, and it can therefore be inferred that these four proteins also interact with one another in the intact U5 snRNP. Further evidence for this is found in the fact that none of these four proteins can be made to dissociate from the U5 snRNP by treatment with high concentrations of potassium chloride whereas other U5-specific proteins do dissociate under these conditions (16). Finally, our reconstitution method allows the preparation of the complex in pure form (Fig. 2 and 3) so that the participation of other proteins can be ruled out.

The detailed investigation showed that the 220kD protein must be centrally placed in the complex because it interacts with at least two of the three other proteins. Its interaction with the 116kD protein is strong enough that the 116/220 dimer remains completely intact even in the presence of 0.4 M NaSCN and can be isolated on a glycerol density gradient (Fig. 1). The 40kD protein is found by radioimmunoprecipitation to associate specifically with the 116/220 dimer (Fig. 7), and far-Western blots show that it interacts directly with the 220kD protein (Fig. 8). Finally, the 200kD protein also interacts with the 220/116 complex (Fig. 3); an interaction with the isolated 40kD or 116kD protein could not be shown, either by radioimmunoprecipitation (Fig. 7 and unpublished data) or by far-Western blotting. We conclude, therefore, that the 200kD RNA unwindase also binds directly to the 220kD protein, although the possibility remains that a composite binding site for the 200kD protein is created only upon formation of the 116/220 protein complex. Clearly, further experiments are required to provide us with a more comprehensive map of all interactions occurring among the four proteins in the 40/116/200/220 heteromer. In any case, the large number of specific interactions of the 220kD protein with other proteins of the U5 snRNP and also with other components of the spliceosome (see below) could also explain why this protein is so highly conserved among all eucaryotic organisms (12, 18, 23).

Apart from its obvious importance for the structural organization of the U5 snRNP particle, the four-protein complex may also play a major part in the biogenesis of the U5 snRNP, by first assembling on its own and then becoming integrated into the nascent particle. The remarkably strong association among the four proteins raises the possibility that at least some of them may already associate with one another in the cytoplasm, directly after synthesis, and are then cotransported into the nucleus. From this idea, it would be predicted that the proteins of the complex (i.e., half of the total snRNP mass) cannot be integrated into the snRNP particle in the absence of the 220kD protein. In support of this, it has been observed that genetic depletion of the Prp8p protein in yeast results in a dramatic decrease in mass of the U5 snRNP particle (shown by sedimentation) (5).

The 220kD protein not only interacts with the components of the U5 snRNP; like its yeast homologue Prp8p, it also contacts both splicing sites of the pre-mRNA (38, 52, 59) and thus has not only a central location but also probably a central role in the spliceosome. This appears to reflect the dynamics of the spliceosome: before the first step of splicing it is in contact with the 5' splicing site, and after this step it contacts the 3' splice site (6, 51, 53). Furthermore, the PRP8 gene in yeast shows genetic interaction with a group of splicing factors that interact with the spliceosome only after the first step of the splicing reaction, which may indicate the existence of further, splicing-step-dependent contacts involving the Prp8p protein (54). At the same time, the 220kD (or Prp8p) protein interacts strongly, as we have shown here, with the 200kD and 116kD proteins, which are probably involved in the dynamic processes of the spliceosome. The former contains, as does its homologue in yeast, two DEXH RNA-helicase motifs (18, 22, 34, 60), and mutations in the yeast gene show that at least one of these helicase domains is essential. Moreover, it was shown that the highly purified 200kD protein is able to unwind the duplex of U4 and U6 RNAs (16). The 116kD protein is a putative GTPase with strong homology to the elongation factor EF-2, which catalyzes the rearrangement of the ribosome during the translocation step (11). Both of these proteins belong to families that generally require other proteins as coactivatorsfor example, the GTPase-activating proteins and the guanyl exchange factors in the case of the GTPases, or the protein eIF-4B in the case of the best-studied RNA unwindase, eIF-4A (1, 37, 39). There is thus the interesting possibility that the 220kD protein may be an activating factor for the 200kD RNA unwindase and/or for the 116kD EF-2 homologue. Considering that the 220kD protein also contacts in a sequential manner the 5' and 3' splice sites, its potential activation of the 200kD RNA unwindase or the 116kD protein might be triggered by certain functional states of the spliceosome. It will be interesting to see which, if any, of the protein-protein contacts described in this paper change in the course of the splicing reaction and how this affects the activity of the RNA unwindase and the GTPase.

In this connection, the domain structure of the 40kD protein also present in the heteromeric complex raises some interesting questions. As we have shown here, the 40kD protein is a new member of the family of WD-40 repeat proteins. The principal function of the WD-40 proteins is the mediation of reversible protein-protein interactions. For example, the  subunits of the heterotrimeric G proteins bind to the  subunits to which GTP is bound and dissociate after hydrolysis of the GTP. In fact, there is also a G protein in the U5 snRNP, the 116kD protein; however, it is not very likely that these two proteins form a functional pair, both because no interaction between them has been detected and because the 116kD protein does not belong to the same subfamily of GTPases as the G- proteins do. WD-40 proteins are not only found in connection with the heterotrimeric GTPases; they are also present among the transcription factors (reviewed in reference 31). Furthermore, these proteins have also recently been identified as common constituents of large RNA-protein complexesfor example, in the spliceosome (4, 6a, 13, 19, 35, 43, 55, 61) (also this work), in the polyadenylation complex (48), in the initiation complex of translation (3, 30), and in telomerase (29). Common to all these complexes is the fact that they are continually built up de novo on an appropriate substrate and then dismantled in a later reaction step. It is possible that the WD-40 proteins in the multifactoral RNP complexes, as in the trimeric G proteins, may contribute to the assembly of the relevant complex. Since these interactions are reversible, the components can be reused after the dissolution of the complex. A similar scenario may be envisioned for the 40kD WD-40 protein as part of the U4/U6.U5 tri-snRNP. During the splicing of pre-mRNA, the U4/U6.U5 tri-snRNP proceeds through a cycle in which it enters into various transient interactions: first of all, the U5 and U4/U6 snRNPs bind to one another; then the tri-snRNP thus formed becomes part of the prespliceosome; then the U4 and U6 RNA duplexes are unwound, and the U4 RNA is released along with an as-yet-undefined protein complement. After the splicing reaction, the spliceosome dissociates, and it is generally believed that the tri-snRNP must then be reassembled out of its components. Interestingly, the human U4/U6.U5 tri-snRNP contains, in addition to the 40kD protein, a second WD-40 repeat protein, the U4/U6 60kD protein (Prp4p in yeast) (4, 13, 19, 35, 55). Thus, the U4/U6.U5 tri-snRNP contains at least two proteins capable of participating in changing protein-protein contacts. It will be of great interest to see which of the steps described involve the U5-40kD protein and which other proteins it interacts with during these steps.

It is clear that the protein-protein interactions described here reveal the existence of a large number of functional interactions between proteins in the spliceosome that may have a decisive influence upon the timing of the assembly of the spliceosome and the rearrangements that occur during its maturation.