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© 1996 Oxford University Press 1849-1855

Footnote

The small subunit of the splicing factor U2AF is conserved in fission yeast

The small subunit of the splicing factor U2AF is conserved in fission yeast Kelly Wentz-Hunter and Judith Potashkin*

Department of Pharmacology and Molecular Biology, Finch University of Health Sciences/The Chicago Medical School, 3333 Green Bay Road, North Chicago , IL 60064, USA

Received February 15, 1996; Revised and Accepted March 26, 1996 GenBank accession no. U48234

ABSTRACT

The human splicing factor U2 auxiliary factor (hsU2AF) is comprised of two interacting subunits of 65 and 35 kDa. Previously we identified the Schizosaccharomyces pombe homolog, spU2AF 59 , of the human large subunit. We have screened a fission yeast cDNA library in search of proteins that interact with spU2AF 59 using the yeast two-hybrid system and have identified a homolog of the hsU2AF 35 subunit. The S.pombe U2AF small subunit is a single copy gene that encodes a protein which shares 55% amino acid identity and 17% similarity with the human small subunit. Unlike the human protein, the yeast protein lacks an arginine/serine-rich region. The predicted molecular mass of the spU2AF small subunit is 23 kDa. The region of spU2AF 59 that interacts with spU2AF 23 is similar to the region in which the human small and large subunits interact.

INTRODUCTION

Splicing proceeds in a two-step pathway ( 1 - 3 ). The first step involves cleavage of the pre-mRNA at the highly conserved 5' splice site (GU). The result of this step is a linear first exon and a lariat intron-second exon intermediate. The second step of splicing involves cleavage of the 3' splice site (AG) and ligation of the 5' phosphate of the second exon to the 3' hydroxyl of the first exon, producing the mRNA and the lariat intron.

Splicing takes place in a dynamic complex termed the spliceosome ( 4 - 6 ). The spliceosome is thought to be comprised of at least 50 components, many which still need to be identified and characterized ( 7 ). The components that have been previously identified include small nuclear ribonucleoproteins (snRNPs) and non-snRNP splicing factors. These factors interact with the pre-mRNA as well as each other to drive splicing.

The first ATP-dependent step in spliceosome assembly involves binding of the U2 snRNP particle to the branch point sequence ( 8 - 10 ). In mammals this step is facilitated by the non-RNP auxiliary factor U2AF, which recognizes and binds to the 3' splice site prior to U2 snRNP binding ( 11 ). Human U2AF consists of two subunits of 65 and 35 kDa ( 12 ). The specific interaction of the U2 snRNP with the branch point sequence requires the U2AF large subunit ( 11 ). hsU2AF 65 binds in vitro to the characteristic polypyrimidine-rich region found in mammalian introns between the branch point sequence and the 3' splice site ( 13 ). hsU2AF 65 is essential for splicing in vitro and the fission yeast homolog, spU2AF 59 , and the fruit fly homolog, dmU2AF 50 , are essential splicing factors in vivo ( 14 - 16 ).

The small subunit of hsU2AF was identified through its co-purification with the large subunit ( 12 , 13 ). The gene for the small hsU2AF subunit has been cloned and sequenced ( 17 ). hsU2AF 35 contains two serine/arginine (SR) motifs in its C-terminus separated by a stretch of 12 glycines. Deletion studies using in vitro translated proteins have determined that the central region of hsU2AF 35 is necessary for binding to hsU2AF 65 . This region does not include either of the S/R domains. In contrast to hsU2AF 65 , hsU2AF 35 has not been shown to be essential for splicing in vitro . The function of hsU2AF 35 remains unknown.

Recently, Wu and Maniatis have identified protein-protein interactions that take place between hsU2AF and other known splicing factors ( 18 ). Specifically, they have shown that hsU2AF 65 interacts with hsU2AF 35 in vivo using the yeast two-hybrid system. hsU2AF 35 in addition to binding with the large subunit can also interact with itself and the SR splicing factors SC35 and SF2/ASF. SC35 and SF2/ASF in turn bind through protein-protein interactions to the U1 70 kDa protein, which is part of the U1 snRNP complex that binds to the 5' splice site of introns. The yeast two-hybrid system is a sensitive assay that allows rapid identification of protein interactions that are weak and transient.

Here we report the cloning of the fission yeast U2AF small subunit. The spU2AF small subunit was identified by its protein-protein interaction with spU2AF large subunit through screening of a fission yeast cDNA library using the yeast two-hybrid system. The predicted amino acid sequence shows a high degree of similarity with the hsU2AF small subunit. Through deletion studies, we have identified the region of spU2AF 59 that interacts with the spU2AF small subunit.

MATERIALS AND METHODS

Yeast two-hybrid screen

The yeast two-hybrid screen for protein interactions with spU2AF 59 was done using a Schizosaccharomyces pombe cDNA library subcloned into the GAL4 activation domain (AD) of the plasmid pGADGH which carries the LEU2 gene of Saccharomyces cerevisiae (Clonetech, Palo Alto, CA). Plasmid from the library was prepared as described in the Clonetech Matchmaker library protocol. spU2AF 59 was subcloned in-frame using the Bam HI site downstream of the GAL4 DNA binding domain in the plasmid pGBT9, which carries the TRP1 gene. Yeast transformations were carried out sequentially as described in Schiestl et al . ( 20 ). Y190 (MAT a ade2 gal4- [Delta] gal80- [Delta] his3 leu2-3 , 112 trp1- [Delta] 901 ura3-52 LYS2 :: GAL1-HIS3 URA3 :: GAL1-lacZ ) was used as the host yeast strain for screening the library ( 21 ). HF7c [MAT a , ura3-52 his3-200 lys2-801 ade2-101 trp1-901 leu2-3 , 112 gal4-542 gal80-538 LYS2 :: GAL1-HIS3 URA3 ::( GAL4 17mers) 3 - CYC1-lacZ ] was used as the host yeast strain for the promoter control experiments ( 22 ). Transformants carrying pGBT9/spU2AF 59 were selected on minimal medium lacking tryptophan. Transformants containing pGBT9/spU2AF 59 and the S.pombe cDNA (spcDNA) library were selected on minimal medium lacking leucine, tryptophan, uracil and histidine and containing 3-aminotriazole. [beta]-Galactosidase filter assays were performed on colony lifts from the yeast transformation plates (Clonetech Matchmaker Protocol). spcDNA library plasmids that gave positive results in the [beta]-galactosidase filter assay were recovered by growing the strain for several generations in minimal medium without leucine and plating the cells on minimal medium either without leucine or without leucine and tryptophan. This allowed the identification of strains that were missing the pGBT9/spU2AF 59 plasmid but carrying the pGADGH/spcDNA library plasmid. The strains carrying only the pGADGH/spcDNA library plasmids were grown in minimal medium without leucine and plasmids were prepared after glass bead breakage according to the Clonetech Matchmaker protocol.

Construction of spU2AF 59 and spU2AF 23 deletion series

Full-length or shorter fragments of spU2AF 59 and spU2AF 23 were amplified by polymerase chain reaction (PCR) ( 23 ). Refer to Table 1 for oligonucleotides PRP2- Bam HI 5', PRP2-[Delta]111 5', PRP2-[Delta]284 3' and PRP2- Bam HI 3' used to subclone fragments of the large subunit gene and Clone A- Bam HI 5', Clone A- Bam HI 3', Clone A-[Delta]1-49 5' and Clone A-[Delta]169-200 3' for the small subunit gene. The PCR buffer contained 10 ng pIRT3/spU2AF 59 plasmid, 20 pmol 5' and 3' oligonucleotides, 1 mM MgCl 2 , 20 [mu]mol dNTPs, 1* Taq polymerase buffer and 2.5 U Taq polymerase (Promega, Madison, WI) in a 100 [mu]l reaction. Full-length or shorter fragments of spU2AF 23 were amplified by PCR from wild-type S.pombe 972 genomic DNA. The PCR conditions were the same as above except 30 ng genomic DNA was used as template. The purified fragments were digested with Bam HI (New England Biolabs, Beverly, MA). pGBT9 and pGAD424 were digested with Bam HI and treated with 10 U calf alkaline phosphatase (New England Biolabs) according to the conditions suggested by the manufacturer. The PCR fragments were then subcloned in-frame into either pGBT9 or pGAD424. Correct orientation of the inserts was determined either through restriction enzyme analysis or dideoxy sequencing. All pairwise interactions were conducted with each construct in both the pGBT9 and pGAD424 plasmids.

Table 1 . The nucleotide sequences of the deoxyoligonucleotides used in this study are listed
Oligonucleotides

Sequence

Experiments

Clone A-1 sense

5'-TTTAGTCAAACGATC-3'

Sequencing

Clone A-1 antisense

5'-GATCGTTTGACTAAA-3'

Sequencing

Clone A-2 sense

5'-CGGTGAAGTTGAACA-3'

Sequencing

Clone A-2 antisense

5'-TGTTCAACTTCACCG-3'

Sequencing

Clone A-5 sense

5'-GCATGCCAAAAAACC-3'

Sequencing

Clone A-5 antisense

5'-GGTTTTTTGGCATGC-3'

Sequencing

Clone A-6 sense

5'-GCCTTTGTAATTTTA-3'

Sequenicng

Clone A-11 antisense

5'-CGCGTAAAAGACTGAGGACTTGG-3'

PCR, 5'-end cloning

Adaptor primer 1

5'-CCATCCTAATACGACTCACTATAGGGC-3'

PCR, 5'-end cloning

M13 universal primer

5'-GTAAAACGACGGCCAT-3'

Sequencing

M13 reverse -20

5'-TTCACACAGGAAACAG-3'

Sequenincg

PRP2- Bam HI 5'

5'-TTGGATCCCAATGGATTTGTCTTCCAGA-3'

PCR, full-length, deletion series

PRP2- Bam HI 3'

5'-TTGGATCCCTTTTCATTGAAACTG-3'

PCR, full-length

PRP2-[Delta]111 5'

5'-TTGGATCCCAATCGAACAGGAACTC-3'

PCR, deletion series

PRP2-[Delta]284-518 3'

5'-TTGGATCCGATGTAATTTTGAATTCTT-3'

PCR, deletion series

Clone A- Bam HI 5'

5'-TTGGATCCCAATGGCAAGTCATTTGGCAAGTATTT-3'

PCR, full-length

Clone A- Bam HI 3'

5'-TTGGATCCTCGTGGAATCACTATTGGG-3'

PCR, full-length

Clone A-[Delta]1-49

5'-TTGGATCCCAAACATGTATAAAAACCCAATTCAT-3'

PCR, deletion series

Clone A-[Delta]169-200

5'-TTGGATCCTGAAAACTCACAAAACATGTCCT-3'

PCR, deletion series

The experiment(s) in which the oligonucleotides were used are listed in the third column.

Oligonucleotides

Oligonucleotides were synthesized by Gibco BRL (New York, NY). The oligonucleotides used for sequencing and PCR experiments are listed in Table 1 .

5 ' -End cloning

The 5'-end of spU2AF 23 was obtained using the Clonetech Marathon cDNA Amplification Kit. Total RNA was prepared from S.pombe 972 (h - ) as described ( 24 ). Poly(A) + RNA was isolated using the Poly(A) + Quick kit (Stratagene, La Jolla, CA). Reverse transcription of mRNA, second strand synthesis, adaptor ligation and PCR were according to the Marathon cDNA protocol using oligonucleotides Clone A-11 antisense and adaptor primer (Table 1 ). PCR products were ligated into pCR II cloning vector (Invitrogen San Diego,CA) using T4 DNA ligase (New England Biolabs). Ligations were transformed into Escherichia coli XL Blue and transformants with plasmids containing PCR inserts were selected by blue/white screening on LB-amp plates containing 5-bromo-4-chloro-3-indoyl-[beta]-D-galactopyranoside (X-gal) ( 25 ). The 5'-end of spU2AF 23 was determined by dideoxy sequencing using oligonucleotides M13 reverse and Clone A1 antisense (Table 1 ).

Sequencing and sequence analysis

Sequencing of full-length spU2AF 23 and the spU2AF 59 deletions was completed using the dideoxy sequencing method ( 26 ). [[alpha]- 35 S]dATP (1000 Ci/mmol; Amersham Arlington Heights, IL) and Sequenase version 2.0 DNA polymerase (US Biochemicals, Cleveland, OH) were used for sequencing according to the suppliers kit and protocol.

Sequence data for spU2AF 23 was assembled using Assemblyalign version 1.0.7 (Eastman Kodak Co., New Haven, CT). The nucleic acid sequence was translated and analyzed with MacVector version 4.0.1 (Eastman Kodak Co.). GenBank was searched using the BLASTP program version 1.4.8. The Profile Gap program (GCG Sequence Analysis Software Package version 8.0.1; GCG, Madison, WI) was used to align the hsU2AF 35 and spU2AF 23 peptide sequences.

Southern blot analysis

Genomic DNA was prepared from S.pombe 972 (h - ) as described ( 27 ) and 20 [mu]g DNA were digested with either Bam HI (New England Biolabs), Eco RI (New England Biolabs), Pst I (New England Biolabs), Hin dIII (Promega) or Xba I (Promega) according to the conditions suggested by the manufacturer. Digests were run on a 0.8% agarose gel and transferred overnight onto a Gene Screen Plus (New England Nuclear, Boston, MA) filter. The filter was prehybridized in 4* SSCP (60 mM sodium citrate, 0.48 M sodium chloride, 32 mM sodium phosphate and 18 mM sodium phosphate monobasic), 1* Denhardts, 1% SDS and 0.5 [mu]g/ml denatured salmon sperm DNA at 48oC and hybridized as above without salmon sperm DNA. spU2AF 23 cDNA was random primer-labeled with [[alpha]- 32 P]dCTP (3000 Ci/mmol; New England Nuclear) using the Random Primer Plus Extension Labeling System kit (New England Nuclear). Aliquots of 4.5 * 10 6 c.p.m. of labeled cDNA was used to probe the Southern blot. The filter was washed at room temperature in 2* SSC ( 25 ), 1% SDS and 0.1* SSC, 1% SDS for 30 min each with a final wash in 0.1* SSC, 1% SDS at 65oC for 1 h. The filter was exposed to X-Omat AR film (Eastman Kodak Company) at -70oC overnight.

RESULTS

Identification of protein-protein interactions

To identify interactions between spU2AF 59 and other proteins, we used the yeast two-hybrid system. The cDNA encoding spU2AF 59 was subcloned into the yeast expression vector pGBT9 in-frame with the sequence encoding the DNA binding domain of the GAL4 protein. The pGBT9/spU2AF 59 construct was the bait for our library screen. pGBT9/spU2AF 59 was transformed into the yeast strain Y190 ( 21 ), which contains two reporter genes, HIS3 and LacZ , under the control of a promoter containing the GAL1 binding site. The pGBT9/spU2AF 59 fusion protein can bind the promoter of these reporter genes but lacks a transcription activation domain and thus cannot activate transcription. An S.pombe cDNA library containing cDNAs subcloned into a yeast expression vector in-frame with a sequence encoding the transcription activation domain of the GAL4 protein was transformed into the Y190 strain carrying pGBT9/spU2AF 59 . Interaction between the spU2AF 59 protein and a protein encoded by the cDNA library vector was identified by growth on medium lacking histidine and the expression of [beta]-galactosidase in a X-gal filter assay.

Approximately 6 * 10 6 independent transformants of the S.pombe library were screened. Sixty two transformants that showed both growth on medium lacking histidine and production of [beta]-galactosidase in a filter assay were identified. A series of controls were run to determine whether positives identified in the initial library screen were false positives. cDNA library clones were isolated as described in Materials and Methods. The cDNA plasmids were transformed into Y190 alone and assayed for [beta]-galactosidase activity using X-gal filter assays to determine if they contained any intrinsic DNA binding ability. The cDNA plasmids were also transformed into strain Y190 with pGBT9 containing no inserts or with pVA3, the murine p53 gene (amino acids 72-390), subcloned into pGBT9, to rule out the possibility of non-specific protein interactions ( 28 ). cDNA plasmids that have [beta]-galactosidase activity only in the presence of pGBT9/spU2AF 59 fusion protein were considered true positives. These cDNA plasmids were then transformed into strain HF7c carrying pGBT9/spU2AF 59 ( 22 ). HF7c contains a LacZ reporter gene under the control of a promoter containing the GAL4 binding site. The transformants were assayed for [beta]-galactosidase activity. Positives indicated that the activation of the reporter gene by the interaction of pGBT9/spU2AF 59 and the cDNA plasmid was promoter independent. Any clones that produced a positive [beta]-galactosidase assay in the presence of pVA3 or without pGBT9/spU2AF 59 were not pursued further. Thirty of the cDNA clones were true positives according to these criteria. These clones were sequenced from their 3'-end and this preliminary sequence analysis was used to determine that the 30 clones encoded seven distinct cDNA clones. The cDNAs were designated A-G, with clone A represented five times, clone B represented 19 times, clone F represented twice and all others represented once each. Preliminary sequence analysis of clones B and F indicates that these are novel proteins that are not represented in GenBank.

Sequence analysis

The 5'-end of clone A was isolated using the Marathon cDNA amplification system from Clonetech. Both strands of clone A were sequenced using the dideoxy sequencing method ( 26 ). The open reading frame of clone A encodes a putative protein of 200 amino acids if translation begins at the first ATG codon. The predicted molecular mass of the protein encoded by clone A is 23 kDa. The sequence of clone A was compared with those in the GenBank database and found to be similar to the human splicing factor U2AF small subunit (Fig. 2 ; 17 ). An alignment of spU2AF 23 and hsU2AF 35 indicates 55% of the amino acids are identical and 17% differ by conserved changes. The putative protein encoded by spU2AF 23 is smaller than hsU2AF 35 and does not contain the arginine/serine (R/S)-rich motifs or glycine run found at the C-terminus of the human protein. There are three regions of the small subunit that have been highly conserved throughout evolution, amino acids 14-35, 100-109 and 136-151.


Figure 1 . Comparison between the predicted amino acid sequence of the spU2AF 23 gene product and the 35 kDa subunit of hsU2AF. Solid bars indicate identical amino acids and colons indicate amino acids that have similar physicochemical properties. The asterisk indicates the stop codon. The GenBank accession no. for spU2AF 23 is U48234.

We have cloned a genomic copy of spU2AF 23 by PCR amplification. Sequence analysis of the genomic clone indicates that there are no introns present within the coding region. Southern blot analysis of wild-type fission yeast genomic DNA digested with several restriction enzymes and probed with 32 P-labeled spU2AF 23 cDNA shows a single band in each digest, suggesting that spU2AF 23 is represented by a single copy gene (Fig. 3 ).

Interaction between spU2AF 59 and spU2AF 23

The identification of spU2AF 23 from the library screen which used spU2AF 59 as bait suggests that the small and large subunits interact. A polyclonal antibody that recognizes spU2AF 23 co-immunoprecipitates the large subunit in a wild-type strain of fission yeast (data not shown). To determine the region of interaction between the spU2AF 59 and spU2AF 23 proteins, a series of deletions was designed. Two deletions of spU2AF 59 were constructed and subcloned in-frame into the DNA binding domain vector. One deletion ([Delta]284-518) removed the second and third RNA recognition motifs (RRM) at the C-terminus of spU2AF 59 (spU2AF 59 [Delta]RRM2,3) and the other removed both RRM 2 and 3 and the N-terminus R/S-rich region ([Delta]1-111 and [Delta]284-518, spU2AF 59 [Delta]R/S, [Delta]RRM 2,3). Each deletion was transformed into strain Y190 carrying pGBT9/spU2AF 59 . The interaction of the spU2AF large subunit deletions with the spU2AF small subunit was tested using [beta]-galactosidase filter assays. The results of the filter assays are shown in Figure 4 . The results show that spU2AF 59 [Delta]RRM 2,3 interacts with spU2AF 23 , but spU2AF 59 [Delta]R/S, [Delta]RRM 2,3 does not.


Figure 2 . Southern blot analysis of wild-type fission yeast genomic DNA. A DNA blot of S.pombe wild-type DNA digested with Bam HI, Eco RI, Hin dIII, Pst I and Xba I was probed as described in Materials and Methods. The DNA blot was exposed to film overnight at -70oC with an intensifying screen.


Figure 3 . Heterodimer formation between the large and small subunits of fission yeast U2AF. Interaction between spU2AF 59 and spU2AF 23 was assayed by the [beta]-galactosidase filter assay. A + denotes blue color production, indicating an interaction between the subunits. A - indicates there was no color production and therefore the two subunits do not interact. The spU2AF 59 [Delta]RRM 2,3 deletion has amino acids 284-518 removed and spU2AF 59 [Delta]R/S, [Delta]RRM 2,3 has amino acids 1-111 (R/S) and 284-518 (RRM 2,3) removed. The spU2AF 23 deletion has amino acids 1-49 and 169-200 removed.

A deletion of spU2AF 23 was constructed and subcloned in-frame into the transcription activation domain vector that contained only amino acids 49-169 (spU2AF 23 [Delta]1-49, [Delta]169-200). The [Delta]spU2AF 23 construct was transformed into strain Y190 carrying spU2AF59, spU2AF 59 [Delta]RRM 2,3 or spU2AF 59 [Delta]R/S, [Delta]RRM 2,3. Interaction between spU2AF 23 [Delta]1-49, [Delta]169-200 and spU2AF 59 or its deletions was determined using the [beta]-galactosidase filter assay. spU2AF 23 [Delta]1-49, [Delta]169-200 interacts with full-length spU2AF 59 and the spU2AF 59 [Delta]RRM 2,3 deletion, but not with the spU2AF 59 [Delta]R/S, [Delta]RRM 2,3 deletion (Fig. 4 ).

spU2AF 59 and spU2AF 23 were also assayed for the ability to form dimers (Fig. 5 ). spU2AF 59 does not display [beta]-galactosidase activity when assayed with itself. In contrast, spU2AF 23 does produce a positive [beta]-galactosidase assay, indicating that it is capable of forming a homodimer. This interaction is lost, however, when spU2AF 23 is assayed for interaction with the [Delta]spU2AF 23 construct.

DISCUSSION

We have screened a fission yeast cDNA library using the yeast two-hybrid system to identify proteins that interact with spU2AF 59 . Through this screen we found a fission yeast homolog of the small subunit of the human splicing factor U2AF. The region of interaction between spU2AF 59 and spU2AF 23 was also determined using the yeast two-hybrid system by testing deletions within both the large and small subunits for their interaction with either full-length or shortened versions of the other subunit. Our results show that this region has been conserved over the billion years of evolution that separate yeast and man ( 29 ).

spU2AF 23 and hsU2AF 35 share 55% amino acid identity and 17% conserved amino acid changes. There are three regions of the two proteins that are highly conserved, corresponding to spU2AF 23 amino acids 14-35, 100-109 and 136-151. By contrast, the two proteins diverge at their C-terminus. It is interesting that spU2AF 23 lacks both R/S motifs and the glycine run that are present in the C-terminus of hsU2AF 35 . The large hsU2AF subunit has an R/S domain that is located in the N-terminus. Both hsU2AF subunits contain the type of R/S domains in which arginine and serine are enriched but dispersed among other amino acids. This arrangement differs from SR proteins, in which the serines and arginines alternate in dinucleotide repeats, share a common epitope and co-purify in an ammonium sulfate/magnesium chloride two-step precipitation ( 30 , 31 ). R/S motifs similar to that found in hsU2AF are found in a number of other splicing factors, including U1 snRNP-specific 70 kDa protein ( 32 ), Drosophila tra ( 33 ), tra-2 ( 34 ), dmU2AF 50 ( 16 ) and spU2AF 59 ( 15 ).


Figure 4 . Homodimer formation between the large and small subunits of fission yeast U2AF. Protein-protein interaction was assayed by the [beta]-galactosidase filter assay. A + denotes blue color production, indicating an interaction. A - indicates there was no color production and therefore no interaction. The spU2AF 23 deletion has amino acids 1-49 and 169-200 removed.

The results from several experiments suggest possible roles for R/S domains in splicing factors. Deletion and substitution experiments with the Drosophila su(w a ) and tra protein R/S domains indicate that this motif is necessary for the localization of these splicing factors to the subnuclear region which is enriched with splicing factors ( 35 ). The R/S domain is also required for some protein-protein interactions. A number of splicing factors containing R/S domains are present in the bridge of proteins that link the 5' splice site to the 3' splice site and the exon bridge that extends from the 3' splice site to the downstream 5' splice site ( 18 , 36 , 37 ). In the light of these studies, it is interesting that neither of the R/S regions of the small subunit of hsU2AF is required for its interaction with the large subunit ( 17 ). The function of the R/S regions of hsU2AF 35 remains unknown. The absence of R/S motifs in spU2AF 23 may indicate that some of the protein-protein interactions that have been identified in the mammalian spliceosome are not present in fission yeast. It is also possible that the same protein-protein interactions do exist in fission yeast but these interactions might be independent of the R/S domain.

We have identified the region of interaction between spU2AF 59 and spU2AF 23 . This interaction does not require the second and third RRMs of spU2AF 59 but is dependent on the presence of its R/S motif. hsU2AF 65 and hsU2AF 35 interaction is similar in that it is autonomous of the second and third RRM. However, the human subunit interaction is only dependent on the existence of the linker region and the first RRM of the large subunit and does not require the R/S motif ( 17 ). This difference in the region of interaction between the small and large subunits may be either a true difference between fission yeast and human U2AF interaction or the result of the deletions that were designed. The R/S domain of the large subunits of U2AF in fission yeast and humans are scattered and ill defined. As a result, the amino acids that we have chosen to remove in the R/S deletion may include amino acids that are actually part of the linker region as defined through mammalian studies. This arbitrary distinction between the R/S motif and the linker region could account for the difference that we observe in regions of spU2AF 59 required for the interaction with spU2AF 23 .

The region of the small subunit that is required for interaction with the large subunit is contained within amino acids 49-169. This fragment of the spU2AF small subunit is capable of interacting with both the full-length large subunit and the deletion lacking RRMs 2 and 3. This region is similar to amino acids 47-172 of the human small subunit that are required for interaction with hsU2AF 65 ( 17 ). The existence of similar regions of interaction between the fission yeast and human U2AF subunits suggests that other protein-protein interactions involving these subunits may also be evolutionarily conserved in fission yeast. In addition, we have also shown that the ability of the small subunit of U2AF to form a dimer with itself has been conserved in fission yeast. This suggests the possibility that the bridge between the 5' and 3' splice sites of mammalian introns proposed by Wu and Maniatis ( 18 ) may also exist in fission yeast. In this regard it will be interesting to determine whether homologs of the U1 snRNP 70 kDa protein, SC35 and SF2/ASF also exist in fission yeast.

ACKNOWLEDGEMENTS

We would like to thank R.Daniel Gietz for helpful advice with S.cerevisiae transformations and the yeast two-hybrid system; Claudia Wing and Yelina Noskina for technical assistance in the screening of the yeast two-hybrid system; John Callaci for assistance in the 5'-end cloning and helpful discussions and Joel Saban for assistance in computer analysis. We are grateful to David Mueller for helpful discussions. This work was supported by a Grant in Aid of Research from Sigma Xi to KW-H and National Institutes of Health grant R01GM47487 and American Cancer Society grant JFRA-545 to JP.

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