Mol Gen Genet (1991) 226:277-282 002689259100102E

OIGG

© Springer-Verlag 1991

Genetic studies of the P R P l l gene of Saccharomyces cerevisiae Keith Schappert t' 2 and James D. Friesen 1, 2

Department of Genetics, The Hospital for Sick Children, 555 UniversityAvenue, Toronto, Ontario M5G IX8, Canada 2 Department of Medical Genetics,Universityof Toronto, Toronto, Ontario M5S I A8, Canada Received September 28, 1990 / November 19, 1990

PRPll is a gene t h a t encodes an essential function for pre-messenger RNA (mRNA) processing in Saecharomyces cerevisiae. We have carried out a mutational study to locate essential and non-essential regions of the PRP11 protein. The existing temperaturesensitive (ts) mutation (prpll-1) was isolated from the chromosome of the original mutant and its position in the gene was determined. When the prpll-1 gene was transcribed from the GALl promoter, the overproduced protein was able to reverse the ts prpll-1 phenotype; this is compatible with the possibility that the defect in the prpll-1 gene product affects its binding to the spliceosome. Thirteen linker-insertion mutations were constructed. Only five (prpl l-4, 11-6, 11-10, -13 and -14) resulted in a null phenotype. One of these became temperature-sensitive when the insertion was, reduced in size from four (prpll-lO) to two (prpll-15) amino acids. A sequence of ten amino acids of which also occurs in the human U1 small nuclear ribonucleoprotein particle (snRNP) A protein and the U2 snRNP B" protein, when deleted from P R P l l , had no phenotype and thus appears to be nonessential for PRP11 function. However, a linker-insertion mutation (prpll-lO) immediately adjacent to this region resulted in a null phenotype. Summary.

Key words: mRNA processing - Linker-insertion muta-

genesis

Introduction

The precise removal of introns from nuclear precursor messenger RNA (pre-mRNA) is an essential process in gene expression. Splicing takes place in a protein-RNA complex called the spliceosome, which is composed of proteins and small nuclear RNAs (snRNAs) in addition to the pre-mRNA. The splicing process in yeast and higher eukaryotes is remarkably similar. However, the

Offprint requeststo: K. Schappert

yeast Saccharomyces cerevisiae has an advantage as an experimental system in that the mechanism of splicing can be studied using both biochemistry and the welldeveloped genetic system of that organism. The genetics of pre-mRNA processing in yeast centres on a set of genes, identified as temperature-sensitive (ts) mutants, called PRP for Pre-RNA Processing (Hartwell et al. 1970; Teem and Rosbash 1983; Yijayraghavan etal. 1989). Most PRP mutants (prp2-11 and prp16-27) are thought to be involved in the processing of pre-mRNA in yeast because they are defective in one step or other of splicing in vivo and in some cases also in vitro (Teem and Rosbash 1983; Lustig etal. 1986; Vijayraghavan and Abelson 1990). A number of the PRP genes have been cloned and their characterization has begun. PRP2,3,4,5,6,8,9,11 and 16 have been shown to encode proteins of Mr 100 (PRP2; Lee et al. 1984), 56 (PRP3; Last and Woolford 1986), 52 (PRP4; Petersen-Bjorn et al. 1989; Banroques and Abelson 1989), 96 (PRP5; Balbadie-McFarland and Abelson 1990), 140 (PRP6; Legrain and Choulika 1990) 260 (PRP8; Jackson et al. 1988), 63 (PRP9; Legrain and Choulika 1990), 30 (PRPll; Soltyk et al. 1984; Chang et al. 1988) and 121 kDa (PRP16; Burgess et al. t990). PRP2 protein functions in splicing at a step following formation of the 40S splicing complex (Lin et al. 1987). PRPI1 protein is associated with the yeast spliceosome (40S), as well as with a 30S complex (Chang et al. 1988). PRP4 protein shows amino acid sequence similarity to /?-transducin, STE4 protein and CDC4 protein, although the significance of this observation as it relates to PRP4 function is not yet clear (Dalrymple et al. 1989). PRP5 and PRP16 proteins show similarity to RNA helicase proteins (Balbadie-McFarland and Abeslon 1990; Burgess et al. 1990). PRP18 protein is required for the second reaction in splicing (Vijayraghavan and Abelson 1990). Some of the PRP gene products are associated with specific snRNPs: antibodies to PRP8 protein have been used in immunoprecipitation experiments to show association with the U5 small nuclear ribonueleoprotein par-

278 ticle (snRNP) (Lossky et al. 1987). Similarly, immunoprecipitation studies have shown that PRP4 protein is part of the U5/U4/U6 snRNPs (Petersen-Bjorn et al. 1989; Banroques and Abelson 1989); further experiments indicate that PRP4 protein is associated preferentially with the 5' portion of the U4 snRNA (Xu et al. 1990; Bordonn6 et al. 1990). Genetic evidence indicates an interaction of PRP3 and PRP4, suggesting that the former might also be associated with the U4/U6 snRNP (Last et al. 1987). In this communication our aim is to determine some of the essential and nonessential regions of the P R P l l protein through a mutational study of PRPll. To do this we cloned and sequenced the prpll-1 ts allele. In addition we performed linker-insertion and site-directed mutagenesis in regions of the gene which might encode functionally important protein segments. Materials and methods

Plasmids, strains and growth media. P R P l l is carried on a 4.5 kb HindIII fragment (Soltyk et al. 1984). Fortuitously, Shore et al. (1984) cloned and sequenced a 4.5 kb HindIII fragment containing SIR2 and a second open reading-frame (ORF) (Genbank accession number X01419). The latter proved to be P R P l l (Chang et al. 1988). The 2.1 kb EcoRV-HindIII fragment containing P R P l l with flanking BamHI linkers (Soltyk et al. 1984) was used for the mutagenesis in the present study. Yeast integration plasmid pFL34 is a URA3-marked pUC derivative; plasmid pFL39 is a CEN-ARS-TRP1 vehicle. Both plasmids were gifts from Frangois Lacroute. Plasmid pYF1089 is pFL39 carrying PRPll. Plasmid pYF1088 has PRPI1 carried on pAPS2 (CEN-ARSURA3) (Percival-Smith and Segall 1986). The transfer of the prpll-1 mutant gene to plasmid pYF372 (also known as AL14RV in Soltyk et al. 1984) was carried out by gap-repair (Orr-Weaver et al. 1983) as follows: pYF432 was digested with BstXI and SnaBI to excise the P R P l l ORF, which was discarded leaving only PRP11 flanking regions on the plasmid; this DNA fragment was gel-purified and was used to transform strain YF413 (see below), selecting for Ura + transformants. Chromosomal D N A from two independent transformants was digested with BstXI, ligated under dilute conditions, and used to transform Escherichia coli strain JF1754 (Soltyk et al. 1984). The resulting prpll-1 clones were sequenced with synthetic oligonucleotide primers (see below). One of the prpll-1 clones was chosen for further studies (pYFI090). Plasmid pYF1091 was constructed by digesting pYF1090 with EcoRV and HindIII and ligating the prpll-i fragment into the SmaI to HindIII site of pFL34. Plasmid pYF1092 has prpll-1 under the control of the GALl promoter, pGAL1. It was constructed by creating a BamHI site eleven nucleotides upstream of the translational initiation codon of prpti-1 by using site-directed mutagenesis (see below). A 1.6 kb BamHIHindIII fragment carrying the prpli-1 gene was then ligated in the BamHI and HindIII sites of pFL34 which had a pGAL1 fragment inserted into it.

Saccharomyces cerevisiae strain W303-1A (MATa, canl-lO0, his3-11,15, leu2-3, 112, trpl-1, ura3-1, ade2-1) was given by Rodney Rothstein. The original prpll-1 strain, called ts382 (Hartwell et al. 1970), is carried in the A364A background (MATa, adel, ade2, his7, lys2, ural, gall). This strain was crossed with strain SR25-1A (MATa his4-912, ura3-52) to yield a strain with the appropriate genetic markers for further studies (YF413: MATe ura3-52, prp11-1), Strain YF1629 (W303-1A background) has a chromosomal null mutation of the P R P l l gene (prpll: :HIS3); wild-type P R P l l function in this strain is provided by plasmid pYF1088. This strain was used to assay the phenotypes of the linkerinsertion and site-directed mutants of P R P l l (see Fig. 2). The chromosomal null mutation of PRP11 in strain YF1629 was constructed as follows: mutant gene prp114 (see Table 1) carried on pFL39 was digested at its unique site with XhoI; the HIS3 gene was inserted by ligation. Then the P R P l l gene on the chromosome was replaced with a BamHI fragment (carrying the prp11::HIS3 insertion) from this plasmid, by transformation followed by recombination (Rothstein 1983). The final construction was verified by DNA blot hybridization. Strain YF1630, a W303-1A derivative in which P R P l l is transplaced with prp11-1 (Scherer and Davis 1979), was constructed as follows: plasmid pYFI091 (see above) was digested with BstXI (unique to the P R P l l fragment thus targeting the fragment to the chromosomal P R P l l gene) and was used to transform W303-1A, selecting for Ura + transformants. These cells were grown on 5-fluoro-orotic acid (5-FOA) to select for those cells that had lost the URA3 gene plus a copy of either P R P l l or prp11-1. These were then screened at two temperatures to identify those that had retained the prp11-1 gene. The final construction was varified by DNA blot hybridization. Yeast strain YF1630 was transformed with pYF1092 previously digested with EcoRV (unique site in the URA3 gene), selecting for U R A + transformants. The resulting genotype of this yeast strain is prp11-1 with pGAL-prpll-1 at the URA3 locus. The final construction was verified by DNA blot hybridization. Growth media were prepared as described by Sherman et al. (1986). Plasmid shuffling was carried out on 5-fluoro-orotic acid-containing solid medium as described by Boeke et al. (1987). Mutagenesis. In-frame, linker-insertion mutagenesis was essentially as described by Stone et al. (1984). Plasmid pYF1089 was linearized by incomplete digestion in the presence of ethidium bromide using one of the following restriction endonucleases: AluI, DraI, RsaI, HaeIII, or SspI. Unphosphorylated J(hoI dimer linkers (5'CTCGAGCTCGAG-3') were ligated to the plasmid DNA. Linear, linker-tailed molecules were purified on a 0.8% agarose gel, allowed to recircularize and were transformed into E. coli without further ligation. This resulted in an insertion of four amino acids, the sequence of which is determined by the frame of the insertion. In some cases the XhoI dimer was digested with restriction endonuclease XhoI to reduce a four-amino-acid in-

279 sertion to a two-amino-acid insertion at the same site. The exact location of each linker insertion was determined by nucleotide sequencing. Site-directed mutagenesis was carried out as described by Zoller and Smith (1983). The sequence of the oligonucleotide used to delete the region of similarity is 5'-GCTATTGAGCTAAGCGAAAAC-3'. The sequence of the oligonucleotide used to create the BamHI site upstream ofprpll-1 is 5'-CATTTAACGGATCCATACATT-3'.

DNA manipulations and analysis. The relevant DNA fragments were sequenced by the chain-termination method (Sanger et al. 1987) using three oligonucleotide primers that spanned the gene. The sequences of the primers were 5'-GTAAGAAGGGCAGGTGG-3', 5'GAAAGGCATTTGGGTGGT-Y and 5'-GGTTCAGTAGGTTTGGC-3'. Other DNA manipulations were essentially as described by Maniatis et al. (1982). Results and discussion

In the original characterization of the PRP ts alleles, prplO-1 and prpll-1 were placed in separate complementation groups (Hartwell et al. 1970). However, recent genetic evidence shows that these two mutations in fact are in the same complementation group (Lustig et al. 1986; our own unpublished observations). Therefore, we have designated prplO-1 as prpii-2.

Location of the prpl 1-1 temperature-sensitive mutation The prpll-1 allele was cloned using a gap-repair and excision method. Two independent clones were isolated. The nucleotide sequence of prpll-1 showed a single base-pair change, a C to T transition at position two of codon 178. The mutation results in a proline (CCT) to leucine (CTT) substitution. The mutation is consistent with the mechanism of nitrosoguanidine mutagenesis (Burns et al. 1987), which was the mutagen used in the original isolation of the prp mutants (Hartwell et al. 1970).

Over-expression of p r p l l - i suppresses its temperaturesensitive phenotype The ts characteristic of the prpll-1 gene product could either be due to its failure to function in the spliceosorne, or to its inability to enter the spliceosome because of an increased binding constant for some other spliceosome components. We sought to distinguish between these two possibilities by overproducing the PRPll-1 protein, reasoning that an elevated intracellular concentration of a protein that had a binding constant higher than normal should restore activity by driving the equilibrium of its association. The prptl-I gene was placed under the control of the strong GALl promoter (pGAL1) carried on an integrating vector (pYF1092). Strain YF1630, which carries the prpll-1 allele on the chromo-

Fig. 1. Phenotypicreversion of the prpll-1 temperature sensitivity by over-expression of the ptTli-1 gene. Three independent transformants of strain YF1630 (prpll-1/pGAL-prpll-1) (1, 2, 3) and strain YF1630 containing vector along (4, 5, 6) were spread on minimal-agar medium with galactose as the sole carbon source and incubatedfor 3 days at 37° C

some, was then transformed with pYF1092, selecting and screening for integrants. When grown with glucose as the sole carbon source, the only source of PRPll-1 protein is from the prpll-1 promoter, However, in medium containing galactose as sole carbon source, there will be additional transcription of prpll-1 from the pGAL-prpil-1 gene and thus, one assumes, increased levels of the PRP11-1 protein. The effects of overproducing prpIl-1 were tested at 37° C. The results, shown in Fig. 1, indicate that extra copies of the prpll-1 gene product phenotypically revert the ts phenotype of the prpli-1 single-copy allele. This is consistent with the possibility that the defect in the PRP11-1 protein affects its binding to the splicesome, rather than its ability to carry out some other function while in the spliceosome. We cannot exclude the alternative explanation that the prpli-1 gene product might be relatively unstable, resulting in insufficient protein to support normal splicing and growth. Overproducing of such a protein could result in a normal pool size and thus normal function.

Linker-insertion mutagenesis ofPRP11 Linker-insertion mutagenesis was chosen because it has the advantage of easy mutation localization and results in relatively large local structural changes in the protein. A XhoI dimer linker (5'-CTCGAGCTCGAG-Y) was inserted at various blunt-end restriction sites. This results in an insertion of four amino acids, or of three amino acids plus an alteration of one or both flanking residues, depending on the sequence and the frame of the insertion (Table 1). In some cases, the 32hoi dimer was digested with restriction endonuclease XhoI to reduce insertion of an four amino acids to a two-amino-acid insertion

280 Table 1. Location and phenotype of the linker insertion mutations in PRP11 Allele

(prpll-) - 3 4 - 5 - 6 '- 7 - 8 - 9 - 10 11 - 12 - 13 -

-

14

- 15

Location of linker insertiona

Residuechange

57 92 110 129 133 135 187 208 212 218 92 u

SRARD FSSSR GSSSS FSSSR LELE TRARV LELE LELE LELE ISSSR FSR

129 b

FS R

208 b

LE

ll-:H

Phenotype wildtype null c wildtype null wildtype wildtype wildtype null wildtype wildtype null null ts

a The mutation created by the addition of a linker depends upon whether the restriction enzyme used cut between codons or within a codon. If the cut is between codons then the mutations are due solely to the addition of the linker. However, if the cut is within a codon then, in addition to the linker mutations, and additional mutation is created by the interruption of the codon. In the latter case, up to five amino acid changes can occur. If the cut occurs between codons the codon location immediately 5' to the the cut site is indicated in the table b These mutants were created by the digestion of the XhoI dimer linker with XhoI, followed by religation. Thus prpll-13, -14 and -15 derive from prpl l-4 , -6 and -10, respectively c Null means nonfunctional allele

at the same site. The phenotype of the insertion mutants was determined by a plasmid-shuffling technique (Fig. 2) in strain YF1629, which carries a chromosomal null mutation of the PRPI1 gene. Thirteen linker-insertion mutants were isolated and their phenotyes were determined on the single-copy plasmid, pFL39. The results of this analysis are shown in Table 1 and summarized in Fig. 4. Only three of the ten four-amino-acid insertions had a phenotype distinguishable from the wild type. The location of these three insertions (called prpll-4, prpll-6 and prpll-lO) is shown in Table 1. The insertion at codon 92 (prpll-4) is adjacent to a possible zinc-binding region which was first described by Chang et al. (1988). The three insertion mutations which displayed a null phenotype were digested with XhoI and the resulting two-amino-acid insertions were assayed for phenotype. Linker-insertion mutations at codons 92 (prpll-4, prpll-13) and 129 (prpll6, prpll-14) retained the null (nonfunctional) phenotype. However, the two-amino-acid insertion at codon 208 (prpll-iO, prpll-15) acquired a temperature-sensitive phenotype.

Mutagenesis of a region of the PRP11 gene product which is similar to two proteins found in snRNPs of HeLa cells We compared the amino acid sequence of P R P I 1 to the amino-acid sequences of two recently cloned proteins that are known to be associated with the U1 s n R N P

, ~

TRANSFORMYF1629 WITH MUTANT ALLELES OF PRP11 CARRIED ON pFL39. SELECT FOR TRP+ "TRANSFORMANTS

PATCH TRP+ TRANSFORMANTS ONTO MEDIUM CONTAINING FOA TO SELECT FOR CELLS WHICH HAVE LOST THE URA3 PLASMID. INCUBATE PLATES AT 23°C FOR 3 DAYS

A

B

FOA SENSITIVE (NONFUNCTIONALA L L E L E )

FOA RESISTANT (FUNCTIONAL ALLELE AT 23°C) TEST FOR GROWTH AT 37°C

Fig. 2. Plasmid-shuffle method used to analyze linker-insertion mutations (A protein; Sillikens et al. 1987) or U2 s n R N P (B" antigen; Habets et al. 1987) of human cells. These two proteins are approximately 80% homologous to each other and share antigenic epitopes (Habets et al. 1985). As is shown in Fig. 3A, P R P l l , A-antigen and B"-antigen (266, 283, and 225 amino acids, respectively) are all similar to one another in a region of ten amino acids, which in all three proteins is located approximately 65% to 70% from the amino-terminus of the protein. We searched a protein and nucleic acid data base (Genbank release 63) with the ten-amino-acid region of similarity and did not find any other proteins with this motif. We reasoned that the similarity between yeast and human proteins in these regions might indicate an essential sequence of amino acids that is necessary for the function of these proteins in R N A processing. To our surprise, deletion of the seven amino acids that included residues 21d~220 in P R P l l (prpll-16, see Fig. 3B) had no effect on the ability of the deletion protein to support cell growth when assayed in the plasmid-shuffle system described above. This result indicates that the ten-aminoacid region is not essential for P R P I I function. It is not known whether it is essential in proteins A and B". During the mutagenesis of the ten-amino-acid region described above we fortuitously isolated a single basepair deletion mutant in codon 212 (prplI-17). The conse-

281

and nonessential regions in the PRP11 protein emerges. The three null (prpll-4, prpll-6 and prpll-lO) and the two ts mutations (prpll-t and prpll-15) are scattered throughout the protein and lie with in no obvious motifs. The null insertion mutation, prpll-4, lies very near a possible zinc-binding region (Chang et al. 1988), suggesting that this region is essential for function. The mutations in prpll-lO and prpll-15 are adjacent to a region of ten amino acids with high similarity to regions in two snRNP-associated human proteins; this region itself is not essential for P R P l l function, yet the insertion of four amino acids immediately adjacent to it results in an inactive protein, and a two-amino-acid insertion results in a ts protein. We cannot exclude, at this time, the possible explanation that one or more of the mutant phenotypes found in this study result from an unstable gene product. In these cases, the mutations could lie in a region essential to the stability of the protein. Seven of the linker-insertion mutations had no phenotype, thus the native configuration of the protein is not essential in these regions. Some of these position (all of which now have a XhoI restriction site) might be suitable for the insertion of protein motifs or epitopes in further experiments. The PRPI 1 protein does not appear to be associated with any of the five snRNPs that are found in spliceosomes; rather it is bound elsewhere in the spliceosome (Chang et al. 1988). Our results from the overproduction of the prpll-I protein suggest that the binding is weakened by this particular mutational change, apparently leading to a block in m R N A splicing at an early stage (Lustig et al. 1986; Chang et al. 1988).

A NH2~

A-protein

B"antigen

NH21

PRP1 1

NH21

tCOO H

I

. ,.,", ,.,

•

tcoo.

• . j-"

...

Foo. ,

.-,'" ,..,,"'""

"',, "N x,

A-protein

PRO PRO ASN His ILE LEU PHE Leu Thr ASN 205

B"antigen

PRO PRO ASN Tyr ILE LEU PHE Leu Asn ASN 148

PRP11

PRO PRO ASN Glu ILE LEU PHE Ser Glu ASN

214

B PRP11

Ala lie Glu Leu Pro Pro Asn Glu lie Leu Phe Ser Glu Asn ASh 214

prpl 1-16

Ala lie Glu Leu Ser Glu Ash Asn

Fig. 3. A Diagram of the regions of similarity among HeLa UJ snRNP A protein, HeLa U2 snRNP B" protein and yeast P R P l l protein, with details of the amino acid similarity among the three proteins. B Details of deletion mutation, prpll-16

quence of this deletion is the mutation and deletion of 55 amino acids at the carboxy-terminus of PRP11. When this mutant was tested in the plasmid-shuffle system it did not support growth of the cell, indicating that the 55 carboxy-terminal amino acids of PRPI 1 are essential for its function and/or stability. The results of the mutational analysis presented here are summarized in Fig. 4. No clear pattern of essential

0-13

Acknowledgements. K.S. held a Medical Research Council of Canada Postdoctoral Fellowship. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.

A-15

0"14

NH2"[

~ COOH

1-16

txxxxxxx;K;4~l~ ?

PRP11 prp11-4 prpl 1-15

C KL C NTMHMSWSSVER H LGGKK H GLN H GRSSSRN H GRSRN PRP11 prp11-10 prp11-11 prp11-12 prp11-15

N[AIELPPNEILFSENN

I NLEI

ISSSRL

Fig. 4. Summary of the location of PRPll mutations. Nonfunctional (e) and temperature-sensitive (~,) alleles are indicated. The dotted region indicates the possible zinc-binding motif; the filled rectangle the region of similarity to HeLa U I snRNP A protein and HeLa U2 snRNP B" protein; and the crosshatched rectangle the region of deletion of 55 carboxy-terminal amino acids (prp11-17). The detailed locations of some of the selected mutations are also shown

282

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C o m m u n i c a t e d by D.Y. T h o m a s