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Departments of Biology,1 Chemistry, University of Kentucky, Lexington, Kentucky 40506-02252
Received 15 June 2005/ Returned for modification 12 July 2005/ Accepted 21 September 2005
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ABSTRACT |
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INTRODUCTION |
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Early on it was shown that U2 snRNP addition in mammals is stimulated by U2AF65, a protein bound to a pyrimidine-rich sequence near the 3' end of the intron (40). The UAP56 DExD/H-box protein interacts with U2AF65 to further enhance U2 snRNP recruitment (17). UAP56 likely stimulates a reorganization step within the U1 snRNP-bearing commitment complex that results in the displacement of U2AF65 and the branchpoint binding protein (also called SF1 or BBP) from the targeted site of U2 snRNA/pre-mRNA interaction. The budding yeast homolog of U2AF65 is encoded by the nonessential MUD2 gene (1). Mud2p and yeast BBP (also called Msl5p) are released from (or less stably bound to) the splicing apparatus after U2 snRNP addition, consistent with a conserved mRNP reorganization step during yeast prespliceosome formation (41). The yeast UAP56 homolog, Sub2p, is essential for splicing unless MUD2 is first deleted (28), supporting its involvement in early spliceosome assembly. The ATPase activity of a second DExD/H-box protein, Prp5p, is needed to configure the U2 snRNP into a state competent for prespliceosome assembly (37). Prp5p also serves a separate, ill-defined ATPase-independent function in prespliceosome assembly (36). Recent evidence from the fission yeast system suggests that Prp5p physically tethers the U1 and U2 snRNP particles, although the protein contacts mediating this association are unknown (55).
In 1993, Kramer and coworkers reported the resolution of mammalian splicing factor SF3 into subfractions SF3a and SF3b (11). Binding of SF3a and SF3b to the 12S U2 snRNP core converts this structure into the mature 17S particle recruited during spliceosome assembly (5, 10). Photo-cross-linking experiments show that multiple SF3 subunits, including at least the p14a, SAP49, SAP145, and SAP155 subunits of SF3b, are in close contact with the splicing substrate (22, 23). Intriguingly, SAP155 (22, 23) and possibly the yeast counterpart, Hsh155p (31), straddle the pre-mRNA branchpoint consensus sequence in the splicing complex. The p14a subunit, although buried within a deep cleft of the SF3b particle (20), can bind the bulged branchpoint nucleotide (51) during spliceosome assembly. The contribution of individual SF3b proteins to mammalian SF3b assembly or splicing remains largely untested.
We biochemically purified yeast SF3b and documented the presence of the conserved Hsh49p/SAP49, Hsh155p/SAP155, Cus1p/SAP145, Rse1p/SAP130, and Rds3p/SF3b14b proteins (50). Two small proteins, possibly counterparts to the mammalian p10 and p14a proteins, were observed but could not be identified by mass spectroscopy. No strong match to p10 was reported in the yeast genome (52), and deletion of the gene encoding the proposed p14a homolog, Ist3p (also called Snu17p [21]), reduced but did not eliminate the SF3b p17 band, indicating the presence of a second protein. More recently Dziembowski et al. (16) reported a p10 counterpart in yeast SF3b, although this study did not address its function in splicing nor provide evidence for the presence or identity of p17 in SF3b.
Here we show that the yeast SF3b p10 protein, Rcp10p, is essential for splicing and required for stable SF3b assembly. Yeasts that lack Rcp10p fail to support U2 snRNP recruitment in vitro and have much-reduced levels of the essential SF3b subunit, Cus1p. Genetic and biochemical evidence reveals that two proteins, Ist3p and Bud31p, interact with SF3b as p17. Ist3p independently binds a complex implicated in nuclear pre-mRNA retention called RES (16). Finally a directed yeast two-hybrid analysis reveals an intriguing network of putative interactions suggesting, for instance, that Rcp10p and the Prp5 DExD/H-box factor function in part through contact with the proposed branchpoint straddling protein Hsh155p. These and related observations offer a new perspective on the branchpoint-associated proteins acting from the earliest steps of pre-mRNP assembly through late stages of splicing.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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his3
1/his31 leu20/leu20 ura30/ura30 met150/MET15 lys20/LYS2). The structure of the disrupted allele in this strain (QW201) was confirmed by Southern blotting.
GAL1::RCP10 was prepared by inserting the RCP10 ORF prepared by PCR (primers Ynl138wa-3 and Ynl138wa-4) into the BamHI site of pBM150. Ycplac111-RCP10-TAP was prepared by first inserting the tandem affinity purification (TAP) fragment as previously described (49) into the SmaI site of YCplac111 and then adding RCP10 as a PCR fragment prepared with primers Ynl138wa-1 and Ynl138wa-2. This construct contains approximately 400 bp of RCP10 5' flanking sequence. The deletions of codons 2 to 19 and 2 to 32 were constructed by inverse PCR on this template using primer RCP10-delta-UP with RCP1019 or RCP1032. The related rcp10fs frameshift allele was fortuitously isolated as a PCR artifact. To compensate for the rcp10fs frameshift (see Results), a 5-bp insertion was placed in the intron by inverse PCR with primers RCP10-intron-1 and RCP10-intron-2 followed by BamHI digestion and ligation. The structures of all gene constructs were confirmed by DNA sequencing and, where appropriate, Western blotting for the TAP fusion. QW201 was transformed with RCP10 derivatives and haploid isolates that uniquely expressed the modified alleles isolated by standard yeast techniques. TAP-tagged versions of all other genes were obtained from Open Biosystems.
The C-terminal V5 epitope fusion alleles were prepared by inserting PCR fragments of BUD31 (primers Bud31-V5-1 and Bud31-V5-2) and IST3 (primers Ist3-3 and Ist3-5) into pYC6/CT (Invitrogen) cleaved at the BamHI site. The resulting constructs were transformed into the corresponding KAN disruption mutants in the BY4742 background (Open Biosystems).
Yeast two-hybrid analysis was conducted by insertion of full-length ORF PCR fragments (primer sequences available on request) into the GAL4 activation domain plasmid pACT and the GAL4 DNA binding plasmid pAS2. Reporter gene transactivation in strain PJ69-4A (26) was scored after 4 days of growth at 30°C on medium lacking adenine and on histidine-deficient medium supplemented with 3-aminotriazole as described previously (26).
DNA oligonucleotides were as shown in Table 1.
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Metabolic depletion of Rcp10fs was done by harvesting the GAL1::rcp10fs culture grown in yeast extract-peptone broth with 2% galactose, washing the recovered cells with sterile water, and then resuspending the pellet in 20x to 200x original culture volume with yeast extract-peptone broth containing 2% glucose. After 2 to 16 h, the cells were harvested for the preparation of splicing extracts (described above) or for RNA analysis (7). Conditions for the resolution of snRNP complexes by glycerol gradient centrifugation were previously described (8) as were in vitro splicing assays (50) and the conditions for affinity selection of biotin-substituted splicing complexes (54). Northern hybridization and the 35S-labeled protein images were acquired with a Typhoon 9600 phosphoimager (Amersham/Pharmacia). The anti-Rds3p polyclonal antibody was prepared by Proteintech Group Inc., using a glutathione S-transferase fusion protein expressed from pGEX-2T in the BL21 Escherichia coli strain as recommended by the plasmid distributor (Amersham Biosciences). A polyclonal antibody against the TAP segment present after TEV cleavage was obtained from Open Biosystems. The anti-V5 monoclonal antibody and appropriate secondary antibodies were purchased from Invitrogen.
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RESULTS |
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RCP10 encodes the yeast SF3b p10 protein. Given the limited sequence similarity of the yeast and human p10 homologs (i.e., roughly 26%), it was critical to show that Rcp10p resides within SF3b. Toward this end, haploid yeast strains that express TAP-tagged (39) versions of Rcp10p, the previously verified SF3b subunits Rds3p and Cus1p, or no TAP tag were metabolically labeled and the tagged proteins were recovered by tandem affinity selection (Fig. 1B). To enhance specificity, the purifications were performed at 0.5 M NaCl rather than at the lower salt concentration originally described (39). Only trace amounts of U2 snRNA copurify under these conditions due to the dissociation of the SF3b structure from the U2 snRNP particle (data not shown but see reference 35). With the exception of the tagged protein, which retains approximately 5 kDa of residual TAP sequence (Fig. 1B, asterisk), equivalent protein banding patterns are observed for the Rds3-TAP, Cus1-TAP, and Rcp10-TAP (lanes 2 to 4) selections, confirming the presence of Rcp10p in the SF3b particle. The identities of five SF3b proteins (i.e., Rse1, Hsh155p, Cus1, Hsh49p, and Rds3p) were verified by mass spectroscopy (see Materials and Methods). While this work was in progress, Dziembowski et al. (16) independently identified Rcp10p (also known as Ysf3) as SF3b associated.
In addition to the proteins mentioned above, an unidentified 17-kDa band is consistently found in the SF3b preparations (Fig. 1B, lanes 2 to 5; note that this protein comigrates with Rcp10-TAP*; also see reference 50). Mass analysis identified a single protein in this size range, the 18-kDa Bud31p. Mass analysis also detected the Prp8p and Brr2p splicing factors as copurifying with SF3b at 150 mM NaCl, and both proteins can be seen in an equivalent preparation stained with Coomassie blue (Fig. 1B, lane 5). Unlike the SF3b subunits, the yields of Prp8p, Brr2p, and the RNA export factor Yra1 (50) (see below) varied from preparation to preparation (for example, compare the region above Rse1p in Fig. 1B, 6C, and 7A). In addition, the Prp9p, Prp21p, Snu114p, and Smd2p splicing factors were occasionally observed by mass spectroscopy (see Materials and Methods). The lower mass analysis confidence scores and the absence of clearly corresponding protein bands when assayed by polyacrylamide gel electrophoresis (PAGE) suggest that these are minor components in the preparation. Contaminants in the preparation include a number of abundant cellular proteins common with TAP selections (see Materials and Methods).
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32 construct being especially temperature sensitive. Based on these observations, we conclude that all essential features of Rcp10p reside 33 to 85 amino acids downstream of the initiating methionine. Prespliceosome assembly requires Rcp10p. Rcp10p-depleted extracts do not splice pre-mRNA and assemble little or no stable prespliceosome (complex A) or spliceosome (complex B) when assayed by native gel electrophoresis (Fig. 4A). To further investigate the impact of Rcp10p depletion on early events in spliceosome assembly, we used biotin-substituted pre-mRNA to affinity purify in vitro-assembled complexes prepared with or without Rcp10p. Spliceosomes affinity selected from wild-type extract show an accumulation of snRNAs consistent with the assembly profile noted by native gel electrophoresis (Fig. 4B). Background binding of snRNAs to the matrix is negligible when a nonbiotinylated pre-mRNA is used (lanes 6 and 12). As shown previously (38), mature splicing complexes selectively shed U4 snRNA, here indicated by a fivefold reduction in the U4/U6 signal ratio when assembly progresses from 1.5 min (U4/U6, 0.7) to 45 min (U4/U6, 0.15). Although the yeast U1 snRNP association is weakened after prespliceosome formation, causing its dissociation from standard native gels (38), this particle can still be specifically recovered with the splicing apparatus when assayed by gentler selection procedures such as gel filtration or affinity selection (compare lanes 2 to 5 with background in lane 6) (also see references 32 and 54 and review in reference 33).
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Rcp10p depletion influences SF3b integrity. Cellular lysates prepared before and after transcriptional repression of a TAP-tagged GAL1::rcp10fs allele were resolved on a 15 to 40% glycerol gradient and assayed by Western blotting for two SF3b-associated proteins (Fig. 5A). In galactose, the coexpressed Cus1-TAP and Hsh49-TAP proteins colocalize and are most enriched in the SF3b region of the gradient (fractions 9 to 15 between the 7S and 19S markers). Note that, in the galactose-grown culture, the ratio of Cus1-TAP to Hsh49-TAP is constant throughout the first half of the gradient, even where the overall levels are low (e.g., fractions 5 and 7). The overexpressed Rcp10fs-TAP is less tightly focused but is enriched in the same fractions as Cus1-TAP and Hsh49-TAP. Extracts prepared from cells that express Rcp10-TAP from its natural promoter show an equivalent pattern except that the Rcp10-TAP is restricted to the same fractions as Hsh49-TAP and Cus1-TAP (data not shown). After 4 h of GAL1::rcp10fs-TAP repression, the Rcp10fs-TAP levels are reduced approximately 10-fold (compare lanes labeled Total). Gradient fractions 13 and 15 retain equivalent amounts of Cus1-TAP and Hsh49-TAP but lack Rcp10fs-TAP, showing that at least these two proteins and perhaps all of SF3b can assemble in the absence of its smallest subunit. Equivalent results are observed if TAP selection is used to recover these proteins (data not shown). Fraction 11 is similar but shows a 40% reduction in Cus1-TAP compared with the denser fractions. In fractions 7 and 9 the level of Cus1p is further reduced relative to Hsh49p, an indication of incomplete SF3b particles not observed when RCP10 is expressed.
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Ist3p is associated with SF3b proteins. Ist3p has been alternatively described as a component of SF3b (21, 50) and as an RES complex subunit that does not stably associate with the U2 snRNP (16). To further investigate the proposed Ist3p-SF3b association, we coexpressed a V5-tagged Ist3 protein and Rcp10-TAP and assayed for protein recovery at increasing NaCl concentrations (Fig. 6A). Sequential IgG agarose and calmodulin agarose chromatography was performed, and the eluted samples were probed with a monoclonal anti-V5 antibody and with a polyclonal antibody specific for a second (and untagged) SF3b subunit, Rds3p. The two proteins show similar levels of recovery at 150, 300, and 450 mM NaCl, indicating a stable association. In extracts that lack the epitope-tagged Ist3 protein, only the Rds3p band is observed and only background proteins are recovered if the TAP tag is missing (Fig. 6A). Equivalent Ist3-V5 recovery is observed when Cus1-TAP is used to select SF3b (data not shown). The salt-stable association of Ist3p with SF3b contrasts with the Yra1p mRNA export factor, which we find bound only under low-salt conditions (Fig. 6B) (see reference 50).
To learn what proteins stably associated with Ist3p, we purified the Ist3-TAP complex and compared the recovered protein pattern with that observed with Cus1-TAP and Rds3-TAP under high-salt conditions (Fig. 6C). As previously reported (16) the 30-kDa Bud13p and the 24-kDa Plm1p proteins copurify with Ist3-TAP. Although we do not know the stoichiometry of the associated proteins, the low sulfur content of Ist3p (one methionine and no cysteine residues) likely contributes to its comparatively weak 35S signal. In addition, we consistently observe the Rse1p, Hsh155p, and Cus1p proteins of SF3b in the Ist3-TAP preparation (Fig. 6C). The Rse1p band appears underrepresented relative to Hsh155p and Cus1p, possibly due to the presence of the TAP tag (see Discussion). Bands corresponding to the smaller SF3b subunits are more difficult to evaluate, although a band comigrating with the Rds3 protein can be seen with longer image exposure (lower panel, Fig. 6C). Western analysis confirms this assignment (data not shown). The overlapping migration of Ist3-TAP with Hsh49p makes Hsh49p assignment problematic, and the status of Rcp10p is unknown. Bud13-TAP results in the recovery of a protein set containing SF3b subunits (data not shown). In addition, we note that a minor band the size of Bud13p is observed in the Cus1-TAP preparation in Fig. 6C. A Pml1p-sized band has not been observed, and its association with SF3b is uncertain. This overlapping pattern of recovered proteins is most consistent with Ist3p existing in two states, with and without associated SF3b.
Bud31p contributes to the p17 signal. Deletion of the nonessential IST3 gene reduces but does not eliminate the 17-kDa signal of SF3b (Fig. 7A, lanes 4 and 5) (see reference 50), showing that at least one other protein exists in this band. Consistent with its detection in this complex by mass spectroscopy, deletion of BUD31 also reduces the p17 signal. Rse1p recovery also drops in the bud31::KAN mutant and can be restored to normal levels by the coexpression of an N-terminal Gal4-Bud31 fusion protein (compare lane 1 with lanes 3 and 5). While weak signals make visualization of the smaller bands difficult, labeled Rds3p and Rcp10p are recovered with the ist3::KAN and bud31::KAN single mutants, although at reduced levels with bud31::KAN (Fig. 7A, lower panel, lanes 3 to 5). Unexpectedly, the ist3::KAN and bud31::KAN double mutant proved to be inviable. To circumvent this synthetic lethality, an Ist3-V5 fusion protein was coexpressed in the double-knockout background. In this case, the p17 band is fully and reproducibly lost and, similar to the original bud31::KAN mutant, reduced recovery of labeled Rse1p, Rds3p, and Rc10p is observed (Fig. 7A, lane 2).
As an alternative means to investigate Bud31p-SF3b association, a Western blot of SF3b recovered at 450 mM NaCl was probed for the presence of an epitope-tagged Bud31-V5 protein. Bud31-V5 copurified with Cus1-TAP (Fig. 7B, lanes 2 and 3) but not from an extract that lacks a TAP-tagged protein (lane 1) or when a nonspecific resin matrix was substituted in TAP selection (lane 4). Together the combined biochemical, immunological, and genetic data make a strong case that Ist3p and Bud31p both contribute to the p17 signal of SF3b.
Two-hybrid interaction map of SF3b. Although it was first identified as a stable complex over a decade ago (11), surprisingly little is known about the subunit organization of SF3b. Since only a few of the subunits were recovered as interacting partners in the published global two-hybrid screens (18, 25, 45), we constructed full-length (i.e., entire-ORF) two-hybrid clones for each subunit and assayed all pairwise combinations for reporter gene stimulation. Several other genes implicated by genetic or biochemical association with the U2 snRNP function (CLF1, YRA1, BUD13, PRP5, MUD2, and BBP/MSL5) were included as well (Table 2; see Fig. S3 in the supplemental material for photographic images of all positive interactions).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gottschalk and coworkers (21) reported yeast Ist3p/Snu17p as a possible SF3b p14a homolog based on its structural similarity to the mammalian p14a protein and on the specific recovery of U2 snRNA with this RRM-bearing protein (52). Importantly, they also showed splicing inhibition and altered U2 snRNP electrophoretic mobility in an ist3 mutant background, thereby linking Ist3p with U2 snRNP biogenesis and function. More recently, Ist3p was shown to be necessary for Mer1p activation of splicing in strains with mutated branchpoint sequences (43), reinforcing a role for this protein in U2-associated events of intron selection. Here we show that a V5-tagged Ist3p protein specifically copurifies with the SF3b subcomplex of the U2 snRNP. In addition, we show that the purified Ist3-TAP complex(es) contains SF3b subunits. Given the preponderance of evidence for Ist3p-U2 snRNP association, it is surprising that Dziembowski et al. (16) failed to detect Ist3p in SF3b or SF3b subunits with Ist3p. The reason for this discrepancy is unclear but presumably reflects differences in the protein isolation or detection techniques.
The identification of Bud31p as the second p17 protein of SF3b is supported by mass spectroscopy of this complex, Western analysis results showing its salt-stable association with Cus1-TAP, and the reduction in p17 signal in the bud31::KAN background. Bud31p has been found in multiple other splicing-related protein complexes including the endogenous spliceosome (34, 43a, 49). Deletion of this nonessential gene clearly impairs yeast splicing (30), supporting a physiologically relevant basis for these interactions. The partial recovery of Bud31p and other splicing factors such as Brr2p and Prp8p among multiple complexes (e.g., intact snRNP particles, SF3b, and endogenous spliceosome) or intermediates in the extract presumably reflects partitioning based on relative affinity. While little is known about Bud31p function, the synthetic lethality of the bud31::KAN ist3::KAN double knockout suggests a related role in splicing. Conceivably, the lower levels of Rse1p recovered with SF3b from the bud31::KAN mutant reflect impaired SF3b biogenesis or stability. We note with caution, however, that Rse1p interactions appear particularly sensitive to perturbation, as this protein can be released from the endogenous spliceosome with temperature inactivation of the Clf12p splicing factor (49), from SF3b after Rds3-1p inactivation (50), or seemingly when the TAP tag is introduced into Ist3p (this study). Consequently, the lower level of Rse1p recovered with SF3b from the bud31::KAN mutant may reflect a less direct consequence of splicing inhibition.
Our directed two-hybrid study with full-length bait and prey constructs identified a number of interactions missed in earlier screens of peptide fusion libraries (e.g., references 18, 25, and 45), not a surprising result given the number of unique interactions reported for each of these earlier studies. While not proving direct or functional association, positive two-hybrid results often define interactions worthy of additional consideration, especially when supported by other relevant observations. For instance, the mammalian SAP155 protein interacts with the SF3b p14a subunit that cross-links to the catalytic branchpoint adenosine (21). While we do not observe an Hsh155p-Ist3p interaction, we do detect an interaction between Hsh155p and the Ist3p binding partner, Bud13p (25, 45), that might serve an equivalent role in organizing the pre-mRNP in yeast. Similarly, a reciprocal two-hybrid interaction was found between Hsh155p and the DExD/H-box protein Prp5p. This is particularly interesting since recent results from the fission yeast system suggest that the ATP-independent function of Prp5p may include tethering the U1 and U2 snRNP particles (55); our results suggest that Hsh155p may serve as a U2 snRNP contact. Novel interactions were also observed between the NTC subunit Clf1p and the Cus1p and Hsh155p components of SF3b. Independent support for a Clf1p-U2 snRNP association is provided by the observation that temperature inactivation of Clf12p in vitro (or in vivo) causes the release of at least two SF3b proteins, Hsh155p and Rse1p, from endogenous spliceosomes (49), suggesting a stabilizing effect of Clf1p on U2 snRNP components of the splicing complex. Indeed, enhanced U2 snRNA expression partially alleviates the growth defect of certain clf1 mutants (6), indicating a mechanism of splicing compensation dependent on the U2 snRNP. Mutations in RDS3 and the RES-associated BUD13 genes were identified in a synthetic lethal screen with clf12 and shown to independently impair splicing (47). Taken in this context, the Hsh155p-Clf1p-Cus1p two-hybrid interactions reported here suggest a means of physically linking the U2 snRNP with the NTC structure essential for late events in spliceosome assembly and splicing (13, 44). Finally, the previously reported Rse1p-Prp9p and Cus1p-Brr2p two-hybrid interactions (18) are consistent with the SF3b association of Prp9p and Brr2p indicated by our mass analysis of SF3b (see Materials and Methods).
The relationship between the RES and SF3b complexes is an interesting unresolved issue. The results of reporter gene assays suggest that RES components may help retain the unprocessed pre-mRNA in the nucleus independently of U2 snRNP function (16). While Ist3p is structurally similar to mammalian SF3b 14a, an argument has been made that higher eukaryotes possess two 14a-like RNA binding proteins, with the SF3b-specific subunit being absent in budding yeast (16). Since results presented here establish physical association of Ist3p with SF3b, we propose resolution of this conundrum by suggesting that, in yeast, the putative intron retention and splicing functions reside within a single protein. If they are functionally conserved, extrapolation from the yeast system predicts that the second SF3b 14a-like protein in mammals may serve some role in pre-mRNA retention. A speculative model consistent with current data is that RES binds the splicing substrate, possibly near the branchpoint in a pre-mRNA retention capacity prior to prespliceosome formation. With U2 snRNP recruitment, Ist3p and Bud13p (and possibly Pml1p) become SF3b associated and function in splicing. Afterwards, the RES factors may recycle free of U2 snRNP components. Alternatively, the RES proteins may simply serve as accessory splicing factors, intimately associated with the SF3b branchpoint binding subcomplex of the U2 snRNP.
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ACKNOWLEDGMENTS |
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Support for this work was provided by NIH GM42476 to B.C.R. and infrastructure funds provided by NSF EPS-0132295.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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