spliceosome assembly through stable U4/U6.U5 ...

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Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition. S Chung, M R McLean and B C Rymond RNA 1999 5: 1042-1054

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© 1999 RNA Society


RNA (1999), 5:1042–1054+ Cambridge University Press+ Printed in the USA+ Copyright © 1999 RNA Society+

Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition

SEYUNG CHUNG, MITCH R. McLEAN, and BRIAN C. RYMOND T+H+ Morgan School of Biological Sciences and the Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40506-0225, USA

ABSTRACT Mutants in the Drosophila crooked neck (crn ) gene show an embryonic lethal phenotype with severe developmental defects. The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain. Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development. We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor. CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame. Clf1p is required in vivo and in vitro for pre-mRNA 59 splice site cleavage. Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association. Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p. Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1). Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition. Keywords: pre-mRNA splicing; Saccharomyces cerevisiae ; TPR motif; U1 snRNA

INTRODUCTION Spliceosome assembly occurs through a temporally defined set of interactions between the pre-mRNA and the trans -acting components of the splicing apparatus (Nilsen, 1998; Staley & Guthrie, 1998; Burge et al+, 1999)+ The U1 snRNP particle initiates spliceosome assembly through ATP-independent contacts with the 59 splice site and the branchpoint regions of the premRNA+ The 59 splice site interaction includes ill-defined protein contacts and base pairing between the 59 end of the U1 snRNA and 4– 6 nt at the upstream intron border+ In contrast, branchpoint recognition by the U1 snRNP particle may be mediated exclusively by protein contacts+ A phylogenetically conserved protein, BBP/SF1, binds to the branchpoint sequence and interacts with the U1 snRNP Prp40p protein and with the yeast U2AF65 homolog, Mud2p (Arning et al+, 1996; Abovich & Rosbash, 1997; Berglund et al+, 1997, 1998; Reprint requests to: Brian C+ Rymond, T+H+ Morgan School of Biological Sciences and the Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40506-0225, USA; e-mail: rymond@pop+ uky+edu+

Rain et al+, 1998)+ The BBP/SF1-Prp40p-Mud2p interactions suggest that a “cross-intron bridge” forms in the commitment complex to juxtapose the 59 splice site and the branchpoint regions of the intron+ With the addition of U2 snRNP, BBP/SF1 is likely displaced from the pre-mRNA to permit U2 snRNA to base pair with the branchpoint sequence (Berglund et al+, 1998)+ Although it is clear that the chemically reactive nucleotides of the pre-mRNA must be juxtaposed prior to splicing, it is not known how the proposed cross-intron bridge is maintained in the U2 bearing prespliceosome or, later, in the U4/U6+U5 snRNP-enriched spliceosome+ The Drosophila crooked neck (crn ) gene resides in region 2E3 of the X chromosome and encodes a 702amino-acid protein composed almost exclusively of sixteen direct copies of a variant tetratricopeptide repeat (TPR) element (Zhang et al+, 1991)+ Perrimon and colleagues (Zhang et al+, 1991) have shown that the crn gene is expressed throughout the embryo and is present in the larval, pupal, and adult stages+ Null allele mutants of crn die in late embryogenesis with impaired neurological and muscle development (Zhang et al+, 1991; Drysdale et al+, 1993)+ For instance, the horizon-

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clf1p acts as a protein scaffold in splicing tal commissures of the ventral nerve cord are much thinner than normal and the corresponding longitudinal connectives are reduced or absent+ Likewise, some muscle groups fail to develop and the yolk remains as a fixed plug within the crn mutant embryo+ The ubiquitous gene expression pattern of the crn gene and the embryonic lethal phenotype of null mutants indicate that crn supports a fundamental, essential cellular process+ The gross similarity of the crn TPR elements with similar elements in the fungal cdc16, cdc23, nuc21, and BimA cell cycle proteins contributed to an early suggestion that crn might be a component of the Drosophila cell cycle machinery (Zhang et al+, 1991)+ However, the crn TPR motif contains distinctive sequence features not found in the cell cycle protein set (Sikorski et al+, 1991)+ It remains uncertain whether crn activity directly contributes to cell cycle progression+ TPR-bearing proteins are present in the three genetic kingdoms and function in support of many cellular processes, such as transcription, peroxisome biogenesis, cell cycle progression and PKR protein kinase inhibition and pre-mRNA splicing (Legrain & Choulika, 1990; Goebl & Yanagida, 1991; Sikorski et al+, 1991; Gindhart & Goldstein, 1996; Urushiyama et al+, 1997; Kyrpides & Woese, 1998)+ Generally, TPR elements are clustered in three or more repeats and act to promote site-specific protein/protein contact+ Although the crn-like TPR motif is not common, the yeast genome codes for several proteins with multiple copies of this element (McLean & Rymond, 1998)+ Among the proteins with the best matches are three RNA processing proteins, two U1 snRNP proteins (Prp39p and Prp42) and a phylogenetically conserved mRNA 39 end processing factor (Rna14p)+ This correlation between the crn-like TPR repeat and RNA processing proteins led us to ask whether the apparent yeast crn counterpart, CLF1, also contributed to RNA metabolism+ Here we present evidence to show that the Clf1p protein is an essential and conserved pre-mRNA splicing factor+ The data support a model in which Clf1p/crn functions as a scaffold to organize the intron and advance spliceosome assembly through its distinctive TPR repeats+ In addition, this study provides evidence that the crn-like TPR motif is restricted to RNA processing proteins and thus a valuable predictor of protein function+ RESULTS Identification of CLF1 A hypothetical protein with approximately 40% sequence identity and 65% sequence similarity to the Drosophila crooked neck (crn) protein was identified through a Blastp search (Altschul et al+, 1990) of the nonredundant yeast protein database (Fig+ 1A)+ This 687-amino-acid protein is encoded by the uncharacterized open reading frame (orf), YLR117c, which we have

1043 renamed CLF1 (for crooked neck like factor)+ Similar to the Drosophila protein, Clf1p consists of direct iterations of the crn TPR motif flanked by short N-terminal and C-terminal extensions of 30 and 109 amino acids, respectively+ The somewhat smaller yeast protein appears to lack the equivalent of the first fly crn TPR repeat and is missing a portion of the non-TPR carboxyl terminus+ As expected for homologous proteins, alignment of the yeast and fly sequences showed the greatest similarity when the degenerate TPR elements are aligned in register (Clf1p TPR1 with crn TPR2, Clf1p TRP2 with crn TRP3, etc+)+ The fifteen Clf1p TPR elements form a consensus that is an excellent match to the Drosophila crn consensus (Fig+ 1B)+ The crn-like TPR variant is distinguished from the more general TPR repeat most notably by the presence of an aromatic residue in position 7, the lack of the conserved glycine at position 8, substitution of a glutamic acid for a hydrophobic residue at position 11, and the prevalence of basic amino acid at position 21 and a leucine or an isoleucine at positions 23 and 28 (Fig+ 1B and McLean & Rymond, 1998)+ The Drosophila crn protein contains a putative nuclear localization sequence (NLS) in its carboxyl terminus (PRRIKER, amino acids 620– 626) (Zhang et al+, 1991)+ Clf1p has a similar sequence (PKARKIL) located between amino acids 421 and 427 (Fig+ 1A)+ In addition to these C-terminal structures, NLS-like sequences are present in the N terminus of Crn and Clf1p at positions 60–63 (RKRK) and 48–51 (RRKR), respectively+ CLF1 is essential and encodes the Saccharomyces cerevisiae crooked neck ortholog A null allele was created by substitution of all the CLF1 protein coding sequence downstream of the translation initiation codon with the HIS3 gene+ Chromosomal replacement of the CLF1 locus by this clf1::HIS3 allele proved lethal in the haploid state (Fig+ 2A)+ In 20 dissected tetrads of a CLF1/clf1::HIS3 heterozygous diploid, no His1 colonies were recovered, whereas the unlinked mating type marker, MAT a, segregated with the expected 2:2 frequency+ The chromosomal disruption of CLF1 does not inhibit the expression of adjacent essential genes because clf1::HIS3 is complemented in trans by the CLF1 orf expressed from the galactosedependent GAL1 promoter on plasmid pBM150 (Johnston & Davis, 1984) (Fig+ 2B)+ As expected, transcriptional repression of GAL1::CLF1 on glucose-based medium inhibits colony formation+ Tiny colonies do form after extended incubation but these appear to result from residual GAL1::CLF1 expression and not from Clf1p-independent growth+ This conclusion is based on three observations+ First, as stated above, no clf1::HIS3 colonies were isolated by meiotic segregation in the absence of GAL1::CLF1. Second, GAL1::CLF1 cul-


S. Chung et al.

1044 tures fail to form colonies on medium that selects for the loss of plasmid-linked URA3 gene (i+e+, 59FOA plates; data not shown)+ Finally, a mutation of GAL1::CLF1 that introduces a C-terminal insertion after amino acid 679 [ GAL1::clf(679) ] efficiently supports growth on galactose medium but completely blocks colony formation on the glucose-based medium (Fig+ 2B)+ Presumably, this Clf1p-derivative has somewhat diminished activity or stability and thus is unable to support cellular growth when synthesized in trace amounts on glucose medium+ The extensive sequence similarity between crn and Clf1p suggested that CLF1 might be the functional homolog of crn+ To directly address this point, the ability of Drosophila crn to complement the yeast clf1::HIS3

allele was determined+ A cDNA copy of crn was placed downstream of the yeast translation elongation factor 1a (TEF ) promoter on plasmid TEF424 (Mumberg et al+, 1995) and the resulting construct assayed for function in the GAL1::CLF1 -dependent yeast strain described above+ The TEF::crn gene is sufficient for cell viability, as yeast that spontaneously lost GAL1::CLF1 could now be recovered by 59FOA selection and the resultant strains grow well (Fig+ 2C)+ This positive complementation result provides strong evidence that CLF1 is the yeast ortholog of Drosophila crn+ TEF::crn complementation of clf1::HIS3 is efficient at room temperature and at 30 8C but somewhat impaired at 37 8C+ Thus, the crn function is modestly temperature sensitive in yeast+

FIGURE 1. ( Figure continues on facing page.)


clf1p acts as a protein scaffold in splicing Spliceosome assembly is impeded in the absence of Clf1p The presence of crn-like TPR elements in several RNAprocessing proteins (McLean & Rymond, 1998) suggested that Clf1p might also contribute to pre-mRNA splicing+ To test this, the efficiency of yeast pre-mRNA splicing was monitored as a function of time after transcriptional repression of GAL1::CLF1+ Metabolic depletion of Clf1p clearly inhibited pre-mRNA splicing as RP51A (and ACT1, see below) mRNA levels dropped and pre-mRNA levels increased with incubation in the glucose-based medium (Fig+ 3A)+ The time course of splicing impairment and the subsequent growth arrest were indistinguishable from what has been previously reported for other GAL fusions, such as PRP8 (Brown & Beggs, 1992) and the genes for TPR proteins Prp39p (Lockhart & Rymond, 1994) and Prp42p (McLean & Rymond, 1998)+ No Clf1p-dependent changes in RNA mobility were observed with several intron-free pol II transcripts, including the U2 snRNA (Fig+ 3B)+ Primer extension analysis confirmed that the more slowly migrating RNAs observed with RP51A and actin (ACT1 ) hybridization probes were largely due to increased levels of pre-mRNA and not the similarly sized lariat intermediate (Fig+ 4 and data not shown)+ From these re-

1045 sults we conclude that the growth arrest observed after GAL1::CLF1 repression results from a defect in cellular pre-mRNA splicing+ Extracts prepared from yeast cultures depleted of Clf1p were unable to process exogenously added premRNA (Fig+ 5A)+ This splicing deficiency was associated with a specific defect in spliceosome assembly (Fig+ 5B)+ When splicing reactions were resolved by native polyacrylamide gel electrophoresis, pre-mRNA from the Clf1p-complete extract was rapidly assembled into the U1, U2-containing prespliceosome band (complex A, Fig+ 5B)+ As expected based on previous studies (Pikielny et al+, 1986; Cheng & Abelson, 1987), the prespliceosome was converted with time into the more slowly migrating, snRNP-complete spliceosome band (complex B, Fig+ 5B)+ In contrast to the wildtype extract, a single splicing complex band formed in the Clf1p-depleted extract that comigrated with the well-characterized prespliceosome complex+ Prespliceosome arrest is clearly not a de facto consequence of inhibited growth or pre-mRNA splicing, as depletion of other essential splicing factors block assembly at earlier or later times in assembly (e+g+, see Lockhart & Rymond, 1994; McLean & Rymond, 1998; Xie et al+, 1998)+ The time of appearance and level of abundance of the putative Clf1-defective presplice-

FIGURE 1. Protein sequence analysis of the yeast crooked neck-like factor, Clf1p+ A: The protein coding sequences S. cerevisiae Clf1p and Drosophila melanogaster crn are aligned to show maximum sequence similarity+ Similar amino acid residues are indicated by a single dot, identical residues by double dots+ The putative NLS sequences are shown in bold and underlined+ B: Alignment of the Clf1p TPRs+ The fifteen Clf1p TPR repeats are highlighted to show positions with 40–69% similarity (light boxes) and sequences .70% similarity (dark boxes)+ The residues were classified as polar charged (D,E,H,K, and R), polar uncharged (N,Q,S, and T), nonpolar hydrophobic (L,I,V,M,F,Y, and W), small (A and G), or other (C and P)+ The Clf1p and crn consensus sequences were created using the stadenpep comparison matrix for the Pretty program within Genetics Computer Group (GCG) Wisconsin Package Version 9+0+ The crn consensus is nearly identical to what was previously published (Zhang et al+, 1991)+


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S. Chung et al.

FIGURE 3. Analysis of cellular pre-mRNA splicing after Clf1pdepletion+ A: Wild-type yeast (CLF1 ) and the GAL1:CLF1 yeast culture were grown continuously on galactose (T 5 0) or shifted to glucose-based medium for the indicated times+ Total cellular RNA was extracted at each time point and then resolved on a denaturing agarose-formaldehyde gel+ A: The results of hybridization with the intron-containing RP51A probe+ The positions of the pre-mRNA and mRNA are schematically represented at the left of the image+ B: The same filter after hybridization with the SNR20 U2 snRNA gene+

FIGURE 2. Disruption and complementation of the CLF1 gene+ A: PCR analysis of the clf1::HIS3 disruptant+ Total yeast DNA was extracted from a wild-type diploid (lane 1) or a heterozygous clf1::HIS3 disruptant (lane 2) and amplified by PCR with primers flanking the CLF1 gene+ The positions of the wild-type and disruptant alleles are noted by the diagram on the right+ M is a DNA molecular weight ladder; the sizes of representative fragments are given on the left+ B: Phenotypic assay of growth in the presence or absence of Clf1p+ Yeast cultures containing the wild-type CLF1 gene (CLF1 ) and the clf1::HIS3 disruptant transformed with GAL1::CLF1 or its viable mutant derivative GAL1::clf1(679) were streaked on galactose-based rich medium (GAL) or glucose-based medium (GLU) and incubated at 30 8C+ C: Complementation by the Drosophila crn gene+ Wild-type yeast (CLF1 ) and the clf1::HIS3 disruptant transformed with TEF::crn were streaked on YPD medium and incubated at 30 8C+

osome were similar to that observed for the wild-type prespliceosome, indicating that Clf1p-depletion did not significantly impair earlier steps in splicing complex assembly+

The native gel assay results suggested that tri-snRNP addition was impaired in the Clf1p-depleted extract+ However, because the electrophoretic mobility of a Clf1p-deficient spliceosome cannot be predicted with certainty, it remained possible that the U4, U5, or U6 snRNA bound in the absence of Clf1p+ If so, then the Clf1p-defective complex would need to be viewed as more elaborate than the normal prespliceosome+ To address this issue, splicing complexes were affinity purified on biotin-substituted pre-mRNA and then assayed for snRNA content by Northern blot (Fig+ 6)+ When a Clf1HAp-complete control extract was used, each of the spliceosomal snRNAs was recovered with the biotinylated RP51A pre-mRNA (Fig+ 6, lane 4)+ As previously observed (Pikielny et al+, 1986; Cheng & Abelson, 1987; Konarska & Sharp, 1987; Xie et al+, 1998), the U4 snRNA was underrepresented in the mature splicing complexes (Fig+ 6, lane 4) relative to the unfractionated extract (Fig+ 6, lane 1) due to U4 release prior to 59 splice site cleavage+ The U1 and U2 snRNAs, but few U4, U5, or U6 snRNAs, were recovered from splicing complexes assembled in the Clf1p-depleted extract (Fig+ 6, lane 3)+ Equivalent snRNA profiles were observed with earlier (5 and 15 min) time points of assembly in the Clf1p-depleted extract (data not shown)+ In all cases, however, the U1 and U2 snRNA recovery was substrate dependent, as virtually no snRNA copurified with a nonbiotinylated control substrate RNA (Fig+ 6, lane 5) or on RNAs lacking splice sites (Rymond et al+, 1987 and data not shown)+ The low level of tri-snRNP-derived snRNA in the Clf1p-defective complexes was not due to general degradation in the extract, as the U4, U5, and U6 snRNAs were abundantly present in the unbound fraction (Fig+ 6, lane 2)+ Together with the data presented above these results show


clf1p acts as a protein scaffold in splicing

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FIGURE 5. Pre-mRNA splicing and spliceosome assembly in Clf1pdepleted yeast extracts+ A: Analysis of pre-mRNA splicing+ Splicing extracts were prepared from GAL1:CLF1 cultures grown continuously in galactose (1Clf1p) or after 14 h in glucose-based medium (2Clf1p)+ The splicing reactions were initiated by the addition of RP51A pre-mRNA+ At the indicated time points aliquots of each reaction were terminated and the reaction products resolved on a denaturing 5% polyacrylamide 7 M urea gel+ The positions of the lariat intermediate (LI), excised intron (I), pre-mRNA (P), and mRNA (M) are noted with arrowheads+ B: Assay of splicing complex formation+ One half of each reaction in A was resolved in parallel on a 3% polyacrylamide/0+5% agarose gel to assay for splicing complex formation+ The positions of the prespliceosome (complex B) and spliceosome (complex A) are noted+ The dark band below these complexes represents the unassembled pre-mRNA+

Rymond, unpubl+)+ In contrast, in vitro-translated Prp40p (or an unprogramed reticulocyte lysate) failed to restore splicing (Fig+ 7, lane 4, and data not shown)+ Thus, Clf1p is essential for the assembly of catalytically active spliceosomes and this activity is specifically lost after transcriptional repression of GAL1::CLF1+

FIGURE 4. Impact of Clf1p-depletion on the two RNA cleavage/ ligation steps of pre-mRNA splicing+ Total yeast RNA was isolated from the GAL1:CLF1 yeast culture grown continuously in galactose (lane 5) or after a 12-h shift to glucose (lane 6)+ For the convenience of assay, a high-copy ACT1 plasmid (see Materials and Methods) was cotransformed into this strain+ Primer extension was performed with an oligonucleotide specific to ACT1 exon II+ The positions of the doublet ACT1 pre-mRNA and mRNA cDNA bands as well as the lariat intermediate are indicated on the right+ The lengths of each cDNA product were confirmed by DNA sequence analysis performed with the same oligonucleotide used for reverse transcription (lanes 1– 4)+

that the U4/U6+U5 tri-snRNP particle does not bind productively to the prespliceosome in the absence of Clf1p+ Weak or transient interactions between the trisnRNP and prespliceosome might occur without Clf1p, but such interactions are insufficient to support premRNA splicing in vivo or in vitro+ Splicing in the Clf1p-depleted extracts was partially reconstituted with a hemagglutinin-tagged Clf1p protein (ClfHAp) synthesized in a rabbit reticulocyte lysate (Fig+ 7, lanes 1–3 and see Fig+ 8) or with Clf1p protein present in a micrococcal nuclease-treated (i+e+, snRNP-free) wild-type yeast extract (M+ McLean & B+C+

Clf1p interactions with the Prp40p and Mud2p commitment complex proteins and the role of the TPR motif The results presented above suggest that Clf1p likely promotes spliceosome assembly through TPR-based interactions that help organize the U4/U6+U5 tri-snRNP particle or help tether this particle to the prespliceosome+ Consistent with the latter suggestion, twohybrid studies revealed that Clf1p interacts with at least two components of the yeast commitment complex and prespliceosome, the U1 snRNP protein Prp40p and the likely yeast U2AF65 homolog, Mud2p (Table 1)+ Phylogenetic support for the specificity of the Mud2p and Prp40p associations is provided by the fact that Drosophila crn protein also interacts with the yeast Mud2p and Prp40p proteins+ No two-hybrid interactions were observed when Clf1p was paired with itself, with several other spliceosomal proteins (BBP, Prp39p, Prp42p, U1-C, and Mud1p), or with approximately 200,000 activation domain fusions screened from a yeast genomic DNA library (James et al+, 1996)+ Thus, although it is possible that the large Clf1p protein interacts specifically with additional yeast proteins, Clf1p is not inherently “sticky” in the two-hybrid system+


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S. Chung et al. The Clf1p binding site is within the amino terminus of Prp40p+ Two WW domain elements are present in the amino terminus of Prp40p (Kao & Siliciano, 1996) and have been suggested as possible sites of BBP/SF1 interaction (Abovich & Rosbash, 1997)+ This region of Prp40p is unnecessary for Clf1p interaction, however (Table 1)+ Clf1p does not interact with the isolated WW domain (Prp40p-WW)+ In addition, TPR elements are believed to bind alpha helical surfaces in their target proteins (e+g+, see Tzamarias & Struhl, 1995; Das et al+, 1998), three of which are predicted in the N-terminal half of Prp40p downstream of the WW region+ Removal of the WW domain from the amino half of Prp40p only slightly reduces the level of lacZ reporter gene transactivation when these three helical segments are present (Prp40p-HI, II, III)+ However, deletion of the first of these helices (Prp40p-HII,III), significantly reduces the level of transactivation, suggesting that helix I may form a portion of the Clf1p recognition domain+ Both halves of Clf1p interact with Prp40p, although not as well as the full-length Clf1p+ Presumably Prp40p binds Clf1p through contacts that fall on either side of (and perhaps

FIGURE 6. Affinity purification of Clf1p-defective splicing complexes+ Biotin-substituted RP51A (lanes 1– 4) or RP51A without biotin (lane 5) was incubated for 45 min under standard splicing conditions in the Clf1p-complete extract (lane 4) or in an extract depleted of Clf1p prior to preparation by transcriptional repression of GAL1::CLF1 (lanes 1–3, 5)+ Splicing complexes (Bound) were recovered by streptavidin chromatography and then assayed by Northern blot for their snRNA contents+ In addition, the total unfractionated snRNA (Total) and the snRNAs remaining unselected after streptavidin chromatography (Unbound) were assayed for the Clf1p-depleted samples+ The location of the spliceosomal snRNAs are indicated by arrow heads on the left+

As a first approach to characterize the domain organization of the Clf1p-dependent interactions, Clf1p, Mud2p, and Prp40p were bisected into their amino (N) and carboxyl (C) terminal halves and each assayed by the two-hybrid system+ The results show that the Prp40p and Mud2p interactions with Clf1p are complex yet distinct from one another+ For example, the amino terminus of Clf1p (through TPR 9) interacts preferentially with Mud2p, and this interaction includes both amino and carboxyl regions of Mud2p+ For unknown reasons, the amino half of Clf1p interacts better with the Mud2p half molecules than the full-length Clf1p+ Deletion of the second Clf1p TPR (DTPR2) blocks the Mud2p/Clf1p interaction+ Western blot analyses of the Clf1p fusion proteins show roughly equivalent protein levels for the full-length and half molecules, indicating that differential protein turnover is not likely a factor in these transactivation results (S+ Chung & B+C+ Rymond, unpubl+)+ Based on these observations, we propose that the Mud2p binding domain resides in the N-terminal half of Clf1p and that its site of association straddles both halves of Mud2p+

FIGURE 7. In vitro complementation of Clf1p activity+ Wild-type extracts (lane 1) or extracts metabolically depleted of Clf1p (lanes 2– 4) were incubated under standard splicing conditions in the presence of in vitro-translated Cfl1HAp protein (lane 3), with in vitro-translated Prp40p (lane 4) or water (lane 2)+ The positions of the RP51A lariat intermediate (LI), excised intron (I), pre-mRNA (P), and mRNA (M) are noted with arrowheads+


clf1p acts as a protein scaffold in splicing

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FIGURE 8. In vitro association of Clf1HAp with Prp40p and Mud2p+ A: Gene-specific DNA was used in a coupled in vitro transcription/ translation reaction to synthesize Clf1HAp (lanes 1–3), Prp40p (lanes 4– 6), Mud2p (lanes 7–9), and the negative control, luciferase (lanes 10–12)+ Lanes 13–16 contain roughly equal amounts of each protein+ The total in vitro translation products (T) were assayed directly after translation (lanes 1, 4, 7, 10, 13) or as immune pellets (B) after immune precipitation with the HA-specific antibody, HA+11 (lanes 2, 5, 8, 11, 14–16)+ The unbound supernatants following immune precipitation (U) were assayed in parallel (lanes 3, 6, 9, 12)+ The immune precipitation and washing steps were performed with 100 mM NaCl except where indicated+ The electrophoretic mobility of each translation product is noted at the right of the figure+ B: Immune precipitation of in vitro-translated proteins as described in A+ Lane 1, total, nonprecipitated Clf1HAp, Prp40p, Mud2p, and Xef-1 (indicated by arrows)+ Bound (B) and unbound (U) samples of Clf1p with Prp40p and Xef-1 (lanes 2, 3), ClfHAp with Mud2p and Xef-1 (lanes 4, 5), and Prp40p, Mud2p, and Xef-1 (lanes 6–7)+ 100 mM NaCl was used for the precipitation and wash steps+ To reduce visual distortion of the protein bands, the total and unbound samples were exposed for one half the time of the bound samples+

straddle) the TPR 9-TPR10 boundary used in the twohybrid constructs+ The high level of reporter gene transactivation suggests, but does not prove, that the interactions of Clf1p with Mud2p and Prp40p are direct+ Additional support for this interpretation is provided by the observation that in vitro-synthesized Mud2p and Prp40p bind a hemagglutinin-tagged version of Clf1p (i+e+, Clf1HAp)+

An antihemagglutinin specific antibody, HA+11, precipitates CLF1HAp but not Mud2p, Prp40p, or a luciferase control protein (Fig+ 8A)+ However, when the in vitro translated proteins are mixed prior to immune precipitation, Mud2p and Prp40, but not the luciferase control coimmune precipitate with the Clf1HAp protein+ The Mud2p/Prp40p/Clf1HAp coprecipitation was maximal at 100 mM NaCl; at 200 mM NaCl, lowered levels of all

TABLE 1+ Summary of the Clf1p two-hybrid interactions+

Clf1p Clf1p-N Clf1p-C Clf1p DTPR Dros. crn

Mud2

Mud2p-N

Mud2p-C

Prp40p

Prp40p-N

Prp40p-C

Prp40p WW

Prp40p HI,II,III

Prp40p HII,III

UlC, others a

87 31 5 2 23

36 84 8 3 nd

33 49 3 nd nd

100 31 23 84 36

72 28 13 nd nd

5 3 3 nd nd

3 nd b nd nd nd

64 nd nd nd nd

15 nd nd nd nd

,5 ,5 ,5 nd ,5

The values are arbitrarily normalized to 100% based on the 39 b-galactosidase units measured for the Clf1p/Prp40p interaction+ The numbers are the average of three or more trials and have standard deviations of 610%+ The amino acid boundaries in the incomplete construct are: Clf1p-N (1–355), Clf1p-C (356– 687), Clf1D2 (1– 64; 99– 687), Mud2p-N (1–264), Mud2p-C (265–527), Prp40p-N (1–292), Prp40p-C (293–583), Prp40p WW (1–76), Prp40p HI, II, III (77–292), Prp40p HII, III (175–292)+ a Includes Clf1p placed in the DNA activation domain, a random activation library isolate, BBP, Mud1p, Prp39p, Prp42p, and reverse-orientation activation domain constructs for Mud2p and Prp40p+ b Interactions not determined+


S. Chung et al.

1050 three proteins are observed+ As this level of salt is unlikely to disrupt the antibody/antigen interaction, the reduced level of Mud2p/Prp40p/Clf1HAp precipitation at 200 mM NaCl might result from salt-induced (interor intramolecular) conformational changes that obscure the Clf1HAp hemagglutinin epitope+ Prp40p and Mud2p also bound Clf1HAp individually under conditions where luciferase or another negative control, the Xenopus translation elongation factor 1 (Xef-1), failed to interact (Fig+ 8B and data not shown)+ Although not quantitative, the level of Prp40p and Mud2p recovered in the Clf1HAp-immune pellet was reproducibly much higher than the background observed in the absence of the epitope-tagged protein (Fig+ 8B)+ Thus, although it is possible one factor (i+e+, Prp40p or Mud2p) changes the affinity of Clf1p for the other, a three-factor complex is not an absolute requirement for coprecipitation+ A preliminary mutational analysis revealed that not all TPR elements act equivalently in Cfl1p function+ For instance, removal of the second TPR repeat of Clf1p (i+e+, DTPR2) inhibits splicing and results in a temperature-sensitive growth defect (Table 2)+ In contrast, deletion of another TPR (DTPR11) has no apparent impact on splicing or growth+ Interestingly, TPR11 contains the strong C-terminal NLS sequence motif+ The lack of mutant phenotype in the DTPR11 construct shows that this sequence is not essential and may be redundant with other elements of the Clf1p protein (e+g+, the N-terminal NLS-like motif)+ A frameshift mutation placed directly upstream of TPR4 proved lethal whereas a similar frameshift mutation placed upstream of TPR11 produced a partially functional but not temperaturesensitive allele+ Finally, similar to the GAL1::clf(679) allele described earlier, removal of the C-terminal one third of Clf1p by a frameshift mutation placed upstream of TPR13 had little or no impact on cellular splicing or growth+ This last result is hardly surprising, as the C-terminal region of Clf1p has the lowest level of sequence conservation with Drosophila crn, a protein that can functionally substitute for Clf1p+

DISCUSSION We have identified the S. cerevisiae ortholog of the Drosophila crooked neck protein, Clf1p, and have shown that it is needed for the formation of catalytically active splicing complexes+ Based on this characterization, we propose a model for Clf1p function in splicing (Fig+ 9)+ Early branchpoint recognition is promoted in part through an interaction between BBP/SF1 bound to the UAC UAAC sequence and the U1 snRNP protein Prp40p associated with the 59 end of the intron+ Other proteins, including the yeast U2AF65 counterpart, Mud2p (Abovich et al+, 1994), and possibly Prp39 (Lockhart & Rymond, 1994; Fromont-Racine et al+, 1997) support this putative “cross-intron bridge” formed in the commitment complex+ U2 snRNA is incorporated into the complex at the prespliceosome stage and likely displaces BBP/SF1 from the pre-mRNA branchpoint sequence (Berglund et al+, 1998)+ A recently identified protein associated with the U2 snRNP, FBP21, interacts with the branchpoint binding protein and the U1 snRNP (Bedford et al+, 1998) and may participate in this reorganization+ We propose that Clf1p supports the juxtaposition

TABLE 2+ Growth and splicing phenotypes of CLF1 mutants+

CLF1 (WT) clf1(679) DTPR2 DTPR11 DTPR13-end DTPR11-end DTPR4-end

Growth a

Temperature sensitivity b

Pre-mRNA splicing c

111 111 1 111 111 11 2

No No Yes No No No No

111 111 1 111 111 11/2 2

a Growth assayed as colony formation after 3 days at 30 8C on YPD (glucose) medium to repress expression of the endogenous GAL1::CLF11 gene+ The clf1(679) construct was assayed similarly on YPGal (galactose) in the clf1::HIS3 deletion background+ b Assayed as colony formation after 3 days 37 8C on YPD medium+ c Measured after 24 h growth on YPD at 30 8C+

FIGURE 9. Model of Clf1p function in spliceosome assembly+ The results presented in this article show that Clf1p interacts with two proteins, Mud2p and Prp40p, that also bind the yeast branchpoint binding protein (BBP)+ The in vitro assembly data suggest that Clf1p acts after BBP+ Here these interactions are diagramed as a “hand-off” of Mud2p and Prp40p contacts from BBP to Clf1p+ Such a transfer might maintain the cross-intron bridge after BBP displacement by the U2 snRNP+ The stable addition of the U4/U6+U5 tri-snRNP is facilitated by Clf1p+ This observation suggests that contacts may exist between Clf1p and components of the U4/U6+U5 tri-snRNP particle+


clf1p acts as a protein scaffold in splicing of the intron ends after BBP/SF1 displacement by maintaining the contacts between U1-Prp40p and Mud2p+ The prespliceosome arrest observed in the absence of Clf1p shows that productive U4/U5+U6 recruitment also requires Clf1p function+ It is possible that Clf1p tethers the tri-snRNP to the prespliceosome through direct contact between the two complexes+ An attractive interpretation of the two-hybrid, immune precipitation, and spliceosome assembly data is that Clf1p interacts with Prp40p and Mud2p in the prespliceosome and that these contacts together with a tri-snRNP interaction are critical for prespliceosome-to-spliceosome transition+ It remains possible, however, that the Clf1p-Prp40p (or -Mud2p) contacts are indirect and that other (unidentified) Clf1p-dependent interactions with the prespliceosome actually mediate tri-snRNP recruitment+ In addition, the thumbnail sketch of Clf1p function presented (Fig+ 9) does not rule out other Clf1p-dependent steps in spliceosome assembly or splicing+ The work presented here suggests that Clf1p may associate with the U1 snRNP or the U4/U6+U5 snRNP particle+ However, no snRNAs coprecipitate with Clf1HAp under a range of salt concentrations (50– 200 mM NaCl) (M+ McLean & B+C+ Rymond, unpubl+)+ In addition, Clf1p is not found in purified preparations of the yeast U1 snRNP (Gottschalk et al+, 1998)+ Although several unidentified proteins are present in the U4/ U6+U5 tri-snRNP preparation (Fabrizio et al+, 1994), none show an electrophoretic mobility comparable to Clf1p (M+ McLean & B+C+ Rymond, unpubl+)+ Clearly, these negative results do not rule out the possibility that Clf1p is transiently (or weakly) bound to a snRNP particle or stably bound but inaccessible to the available antibody+ Indeed, some evidence suggests that Clf1p may be part of a larger complex+ Only 50% of the epitopetagged Clf1HAp can be precipitated from yeast extracts (M+ McLean & B+C+ Rymond, unpubl+), and the remaining Clf1HAp is completely sufficient for splicing+ Clf1p may be similar to Prp19p and found in a large non-snRNP protein complex (Tarn et al+, 1994)+ The need to bind other splicing factors could explain the limited complementation of the Clf1-depleted extracts by in vitro translated Clf1HAp+ Little pre-mRNA, splicing intermediates, or products precipitate with the anti-HA antibody, suggesting that the HA-epitope of Clf1HAp likely remains obscured in the spliceosomal complex+ The basic unit of TPR-ligand association remains poorly understood+ A single TPR repeat or a set of clustered repeats may mediate specific protein interactions+ For proteins with directly iterated TPR elements, individual cassettes may bind the same target (cooperatively or independently) or associate with different surfaces on one or more ligands+ In the case of Clf1p, at least five of the C-terminal TPR elements are dispensable and possibly redundant in function+ In contrast, TPR2 is critical for Mud2p association yet unnec-

1051 essary for the Prp40p interaction+ Of the few proteins characterized that interact with TPR repeats the only common feature appears to be the presence of alpha helical sequence (e+g+, see Lamb et al+, 1995; Smith et al+, 1995; Tzamarias & Struhl, 1995; Shpungin et al+, 1996)+ This observation is consistent with the interaction of Clf1p with the Prp40p helix I, II, III regions located directly downstream of its WW repeats+ The Mud2p binding domains are less clearly defined+ However, secondary structure analysis (Smith et al+, 1996) suggests that significant alpha helical structure exists in both the amino and carboxyl halves of Mud2p and these sequences may support the Clf1p interaction+ This study confirms that four yeast proteins with multiple copies of the crn-like TPR motif (i+e+, Clf1p, Prp39p, Prp42p, and Rna14p) contribute to cellular pre-mRNA processing+ A fifth protein, Rrp5p, with related repeats was shown necessary for proper 18S and 5+8S rRNA biosynthesis (Torchet et al+, 1998)+ Although a human counterpart to Clf1p has yet to be reported, likely Clf1p/ crn orthologs are present in Schizosaccharomyces pombe (accession number P87312) and Caenorhabditis elegans (accession number O16376)+ Presumably, sequences within the crn-like TPR motif have evolved to accommodate interactions with RNA or RNA-bound proteins+ Precedent exists for a nonprotein ligand of the TPR motif+ Activation of the PP5 phosphatase can be achieved by the specific association of polyunsaturated fatty acids with the PP5 TPR domain, thus inducing a favorable conformation change within this enzyme (Chen & Cohen, 1997)+ Although several TPR proteins associate with RNA or DNA complexes (e+g+, Ssn6p, Prp6p, Prp39p, Prp42p, TFIIIC, and Rrp5p), none have been shown to directly bind nucleic acids+ Intriguingly, however, in addition to its TPR elements, the Rrp5p protein contains 12 copies of the S1 RNA binding motif+ Independent of whether Clf1p directly binds RNA, we believe that the crn TPR motif serves as a good predictor of RNP association for eukaryotic proteins+ Spliceosomes form only where the cis- and trans acting factors are present and compatible, that is, found in the correct location, orientation, and stoichiometry+ The step-wise assembly process both reduces the likelihood of mRNA cleavage at improper sites and provides the means for cellular control of gene expression through regulated splice site selection (Lopez, 1998)+ Clf1p is clearly established as an essential factor in spliceosome assembly+ Yet, several intriguing questions remain regarding its function+ For instance, our model shows a “swap” of Clf1p for BBP/SF1 in early spliceosome assembly+ This order agrees with the commitment complex arrest observed in the absence of BBP/SF1 and the prespliceosome arrest noted in the absence of Clf1p+ However, it remains to be shown that the Mud2p/Prp40p contacts of Clf1p and BBP/SF1 are temporally distinct or mutually exclusive+ The model of


S. Chung et al.

1052 Clf1p function assumes that intron alignment throughout spliceosome assembly is critical+ If Clf1p plays an essential role in this alignment, then Cfl1p interaction with the 39 end of the intron must include other unidentified contacts, as Mud2p is not essential for splicing or cell viability (Abovich et al+, 1994)+ Based on our analysis of Clf1p, we predict that crn will be a component of the pre-mRNA splicing machinery of Drosophila+ This prediction is supported by recent evidence implicating a role for crn function in exon inclusion within the alternatively spliced Ultrabithorax mRNA (Burnette et al+, 1999)+ The impaired neurological and muscle development of the Drosophila crn mutants might reflect a greater sensitivity of these tissues to a general reduction in splicing efficiency or tissue-specific crn contributions+ Finally, even the relationship between CLF1 and BBP (MSL5 ) genes remains enigmatic+ In addition to interacting with the same two proteins, Clf1p and BBP are encoded by adjacent convergent transcription units on the right arm of chromosome XII+ It remains to be seen if this arrangement is simply fortuitous or in someway influences the expression of one or both of these genes+ The first crystal structure of a TPR protein, a three repeat segment of the PP5 protein phosphatase, was recently solved (Das et al+, 1998)+ The authors of this study (Das et al+, 1998) predict that clustered TPR elements interact to form a right-handed super-helix well suited to act as a platform for the assembly of protein complexes+ Based on its highly repetitive TPR structure and the experimental data presented here, we propose that Clf1p serves as such a multifaceted platform and drives splicing complex assembly by binding multiple spliceosomal proteins+ While unique in its repetitive simplicity, Clf1p serves as a valuable model for how other TPR proteins might punctuate spliceosome assembly through the parallel recruitment of trans acting factors to their multiple TPR surfaces+ MATERIALS AND METHODS Yeast strains and plasmid constructions: Cloning of GAL1::CLF1, CLF1, and TEF::crn The CLF1 orf was amplified by the polymerase chain reaction (PCR) from yeast genomic DNA with upstream primer 59-GGA TCC TAA AAT GGA CAC TTT A-39 and downstream primer 59-GGA TCC GTA TGT AGT CTC TAT TTT GCA-39 (Bam HI site underlined)+ The primer ends were cleaved within Bam HI and the resultant DNA fragment cloned downstream of the GAL1 promoter by insertion into the Bam HI site of plasmid pBM150 (Johnston & Davis, 1984)+ Alternatively, the CLF1 promoter was included in the amplification by substitution of 59-GGA TCC TGA GCG CTA TCG AAT ATG AAT GGA CTT AG-39 for the upstream primer+ The resulting PCR fragment was then cloned directly into the Sma I site of plasmid YCplac22 (Gietz & Sugino, 1988)+ The clf1::HIS3 null allele was prepared by PCR using the HIS3 gene of plasmid

YDp-H (Berben et al+, 1991) as a template and primers consisting of 40 bases of CLF1 sequence followed by 25–27 nt of HIS complementarity (59-GAC AAA CTT GAG CTA AAA GTA CTC TAC TAT TGC CAG CAT G GA GTC ACT GCC AGG TAT CGT TTG AA-39 and 59-TGA CAA AAA TAT TAT TTC ACG ACG ATA CCC TTA CCT TTA TTC ACA AAA AGA AAA AAG GAA AGC GCG CCT C-39; CLF1 sequence underlined)+ The amplification product was then used for targeted gene disruption through homologous recombination in the diploid yeast strain YPH274 (MATa/a ura3-52, lys2-801a, ade2-101, trp1D1, his3-D200, leu2-D)+ Tetrads were dissected as described (Kaiser et al+, 1994) from the heterozygous disruptant or from the same strain after transformation with GAL1::CLF1 or the mutant derivative GAL1::clf1(679)+ GAL1::clf1(679) was identified as an aberrant PCR cloning product+ This construct substitutes sequences found downstream of CLF1 codon 679 with an in-frame fusion of codons for the peptide, KNGNRNRHFILMMFLIMLEDP, followed by sequences from the Escherichia coli tetracycline resistance gene (codons 60–396)+ The other deletions and frameshift mutations used in this study were introduced by PCR (Hemsley et al+, 1989) with primer pairs (59-ATC CAG TCG GTT TCT CTT-39 and 59-ATC CCC CTT TGG ATA CGA-39 (D2); 59-AAA AGT GAA ATG TTT ATG-39 and 59-AAA ACT TTC AAA GGA TAT-39 (D11); 59-ATA CTC TGG GCA ACG TAC TAA-39 and 59-AAT GCA TGG AAT TCT TTC GT-39 (TPR 4 frameshift); 59-AAA AAG TGA AAT GTT TAT G-39 and 59-AAA ACT TTC AAA GGA TAT-39 (TPR 11 frameshift); 59-ATC AGA GGG CTG AAA TTC AA-39 and 59-TTA ACA AAG GAG GCT AAAAT-39 (TPR 13 frameshift)+ The frameshift mutations were introduced into the YCplac22 plasmid+ Phenotypic characterizations of the mutant phenotypes were performed in the clf1::HIS3 null mutant on agar medium containing 1% yeast extract, 2% Bactopeptone, and 2% glucose (YPD) (Kaiser et al+, 1994)+ The Drosophila crn orf was amplified from crn -containing cDNA (gift of N+ Perrimon) by PCR with upstream primer 59-GGA TCC TAA AAA TGG AGC GGC CAC AGA AGA TGC CC-39 and downstream primer 59-GGA TCC TCA GTC ACC GCT ATC CGT CGT ATC-39+ The crn PCR product was gelpurified, phosphorylated, and inserted into the Sma I site of plasmid vector TEF424 (Mumberg et al+, 1995) to create TEF::crn+ The correct orientation of the insert was confirmed by Pst I restriction enzyme digestion and direct DNA sequence analysis+

Pre-mRNA splicing analysis, immune precipitations Splicing extracts were prepared by the method of Umen and Guthrie (1995) in cultures grown continuously in YP media (Kaiser et al+, 1994) with 2% galactose or after shift to 2% glucose for 14 h as previously described (Lockhart & Rymond, 1994)+ Splicing reactions were assembled on the RP51A pre-mRNA synthesized by SP6 transcription of the Eco RI-cleaved plasmid, pSPrp51A (Pikielny & Rosbash, 1986)+ Spliceosome assembly was monitored on 3% polyacrylamide (59:1 ratio of acrylamide to bis-acrylamide), 0+5% agarose gel run in 0+53 TBE buffer (Pikielny et al+, 1986)+ In vitro-translated Clf1HAp was prepared by coupled in vitro transcription-translation with the Single Tube Protein SystemTM according to the manufacturer’s instructions (Novagen)+ For


clf1p acts as a protein scaffold in splicing complementation of the Clf1p-depleted extract, 2+5 mL of the in vitro translation reaction was substituted for water in a 10 mL splicing reaction+ The Clf1HAp or control translations were incubated with the yeast extract for 30 min+ at room temperature prior to adding the labeled substrate+ Immune precipitations of Clf1HAp, Prp40p, Mud2p, luciferase, and Xenopus elongation factor 1 (Xef-1) were conducted by preincubating equivalent amounts of each translation product (approximately 3 mL each) at room temperature for 3 h+ For each 4 mL of protein sample, 6 mL of assembly mix (1 mL 25 mM MgCl2 , 1 mL 30% PEG 8000, 1+5 mL 100 mM HEPES, pH 7+0, 2+5 mL water) were added and the samples adjusted for NaCl concentration as needed+ Next, 1 mL of HA+11 antibody (Babco, Inc+) was added and the incubation continued for an additional 15 min+ To recover the antibody-bound complexes, 20 mL of a 50% protein A-agarose bead slurry (Gibco/BRL) equilibrated in HNT (20 mM HEPES, pH 7+5, 100 mM NaCl, 0+05% Triton-X 100) were added+ Nonspecific protein adsorption to the beads was suppressed by incubating the protein A-agarose for 15 min with 5% nonfat dry milk prior to equilibration+ The immune precipitation samples were adjusted to 100 mL with HNT and incubated with continuous rotation at 4 8C for 30 min+ Subsequently, the samples were spun at 4,000 3 g for 1 min to pellet the beads+ The supernatant was removed for assay as the unbound fraction+ To reduce background, the pellets were transferred to a fresh microfuge tube and then washed 4 3 1 mL with HNT+ Proteins were eluted from the beads with gel sample buffer and heated to 100 8C for 10 min prior to electrophoresis+ For the analysis of spliceosomal snRNAs, in vitro transcripts of pSPrp51A were prepared by incorporation of biotin16-UTP (Boehringer Mannheim)+ Each 40-mL splicing reaction received 7 ng of biotin-substituted pre-mRNA+ After 5, 15 or 45 min, spliceosome assembly was stopped with 40 mL of buffer Q (400 mM KCl, 2 mM Mg(OAC)2 , 100 mM HEPES, pH 7+5) and 20 mL of immobilized streptavidin (Boehringer Mannheim)+ The bead mixtures were incubated for 45 min at 4 8C and then washed four times with 800 mL of NET-2 buffer (50 mM Tris-HCl, pH 8+0, 50 mM KCl, 1 mM dithiothreitol (DTT), and 0+5% NP-40)+ An equal volume of 23 PK buffer (200 mM Tris-HCl, pH 7+5, 25 mM ethylene diamine tetraacetic acid (EDTA), 300 mM NaCl, 2% sodium dodecyl sulfate (SDS), 2 mg/mL proteinase K) was then added for 10 min at 37 8C to release the bound snRNAs+ The samples were phenol extracted and the associated RNAs ethanol precipitated+ The subsequent RNA blotting and snRNA detection steps were previously described in detail (Blanton et al+, 1992)+ The analysis of cellular pre-mRNA splicing by primer extension was performed as described in Teem and Rosbash (1983), using yeast transformed with ACT1 -bearing plasmid pBA3 (Domdey et al+, 1984) and the exon II primer, 59-GAA CCG TTA TCA ATA ACC AAA G-39+

Two-hybrid analysis Primers complementary to the CLF1, MUD2, PRP40, BBP/ SF1, U1-C, SNP 1, PRP3 9, PRP42, and crn orfs were used to amplify yeast genomic DNA or the existing DNA clones+ Plasmids pACT and pAS2 were used for two-hybrid analysis (Matsuoka et al+, 1995)+ All subcloned DNAs were sequenced to confirm their identities and structures+ The clones were

1053 assayed in yeast strain PJ69-4A (MATa trp1-901, leu2-3, 112, ura3-52, his3-200 gal4D gal80D LYS::GAL1-HIS3, GAL2ADE2, met2::GAL7-lacZ ) for transactivation of the resident HIS3 and ADE2 reporter genes by growth on -histidine, -adenine media and for the levels of b-galactosidase (Kaiser et al+, 1994)+ In addition to the directed assays, a yeast twohybrid library (James et al+, 1996) was screened for transactivation through selection of his1, ade1 transformants+

ACKNOWLEDGMENTS We thank Martha Peterson and our lab colleagues, Neetu Dembla, and Rebecca Seipelt for their helpful comments and suggestions throughout the preparation of this manuscript+ In addition, we thank Norbert Perrimon for his generous gift of the crn cDNA, Phil James for the yeast two-hybrid library, and John Abelson for the ACT1 plasmid, pBA3+ This work was supported by the National Institutes of Health Grant GM42476 to BCR+

Received March 19, 1999; returned for revision April 19, 1999; revised manuscript received May 26, 1999

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