prpl' gene required for pre-mrna splicing in ...the prpl' gene encodes a protein of 906...

Copyright 0 1997 by the Genetics Society of America

The Prpl' Gene Required for Pre-mRNA Splicing in Schizosaccharomyces pornbe Encodes a Protein That Contains TPR Motifs and Is Similar

to Prp6p of Budding Yeast

Seiichi Urushiyama, Tokio Tani and Yasumi Ohshima Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-81, Japan

Manuscript received January 2, 1997 Accepted for publication June 13, 1997

ABSTRACT The prp (Ere-m-RNA processing) mutants of the fission yeast Schizosaccharomyces pombe have a defect in

pre-mRNA splicing and accumulate mRNA precursors at a restrictive temperature. One of the prp mutants, prpl-4, also has a defect in poly(A)+ RNA transport. The prpl' gene encodes a protein of 906 amino acid residues that contains 19 repeats of 34 amino acids termed tetratrico peptide repeat (TPR) motifs, which were proposed to mediate protein-protein interactions. The amino acid sequence of Prplp shares 29.6% identity and 50.6% similarity with that of the PRP6 protein of Saccharomyces cerevisiae, which is a component of the U4/U6 snRNP required for spliceosome assembly. No functional complementation was observed between S. pombe prpl' and S. cermisiae PRP6. We examined synthetic lethality of prpl-4 with the other known prp mutations in S. pornbe. The results suggest that Prplp interacts either physically or functionally with Prp4p, Prp6p and PrplSp. Interestingly, the ppl' gene was found to be identical with the zerl+ gene that functions in cell cycle control. These results suggest that Prplp/Zerlp is either directly or indirectly involved in cell cycle progression and/or poly(A)+ RNA nuclear export, in addition to pre-mRNA splicing.

T HE removal of intervening sequences from nuclear pre-mRNA is an essential step in the expression

of many eukaryotic genes. Pre-mRNA splicing occurs via two transesterification reactions in a large complex called the spliceosome (reviewed by GREEN 1991; MOORE et al. 1993).. The spliceosome consists of U1, U2, U4/U6 and U5 small nuclear ribonucleoprotein particles (snRNPs) and a number of non-snRNP protein factors, which are either integral components of, or transiently interact with, the spliceosome. Recent studies suggest that both the accurate recognition and exci- sion of an intron require many complex interactions, such as RNA-RNA interactions through base pairing, RNA-protein interactions mediated by RNA binding domains (RBD) in the proteins, and protein-protein interactions mediated by such domains as arginine/serine- rich (RS) domains (reviewed by MOORE et al. 1993; WHANI and GUTHRIE 1994; FU 1995).

To elucidate the mechanism of pre-mRNA splicing, many protein factors have been identified either by biochemical approaches, using an in vitro splicing system from mammalian cells, or by genetic approaches, using conditional mutants defective in pre-mRNA splicing in yeast. In budding yeast S. cerevisiae, more than 40 heat- sensitive (ts) or cold-sensitive (cs) mutants were isolated including prp (Ere-mRNA processing) mutants (reviewed by GUTHRIE 1991; RUBY and ABELSON 1991;

Corresponding uuthrmYasumi Ohshima, Department of Biology, Fac- ulty of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-81, Japan. E-mail: [email protected]

Genetics 147: 101-115 (September, 1997)

MOORE et al. 1993) and brr (bad response to refrigera- tion) mutants (NOBLE and GUTHRIE 1996). Many genes defined by these mutations have been cloned and the gene products were then characterized by in vitro splicing systems. Furthermore, suppressor genes of some prp mutations were also identified (JAMIESON et al. 1991; CHAPON and LECRAIN 1992; MADDOCK et al. 1994). In S. pornbe, 14 prp mutants defective in pre-mRNA splicing were isolated as ts or cs mutants: prpl, prp2 and Pg3 by POTASHKIN et al. (1989), P g 4 by ROSENBERG et al. (1991), pq5prp7and pp9by D. KIM and D. FRENDEWEY (personal communication), and pp8 and prplO-prpl4 by URUSHIYAMA et al. (1996). Of these, three p.p' genes have been characterized. The pp2' gene encodes an S. pornbe counterpart of mammalian U2AF65, which is an essential large subunit of a splicing factor U2AF ( POTASHKIN et al. 1993). The pq4' gene encodes a predicted serine/threonine kinase (ALAHARI et al. 1993), and the prpa' gene is identical to the cdc2a' gene, whose ts mutation causes cell cycle arrest in G2/M at the restrictive temperature (LUNDGREN et al. 1996). pp$/ cdc2a' encodes a member of the D M - b o x family of RNA dependent ATPases (LUNDGREN et al. 1996).

Recently, several observations suggesting a connection between pre-mRNA splicing and cell cycle progression have been reported, in addition to the case of prp8/cdc28of S. pombe. In S. cerevisiae, two dbf (dumbbell former) mutants, dbf3 and dbj3, showed a relationship between pre-mRNA splicing and cell cycle progression. dbfmutants were initially isolated as cell-cycle mutants

"

102 S. Urushiyama, T. Tani and Y. Ohshima

on the basis of the terminal cellular morphology or a dumbbell form UOHNSTON and THOMAS 1982a). The dbf3 and dbfs mutations, which caused defects in DNA synthesis and arrest in the S phase, were found to be allelic with p q 8 and pp3, respectively, whose gene products are involved in pre-mRNA splicing UOHNSTON and THOMAS 1982a,b; SHEA et al. 1994). In addition, the S. pombe misl 1 mutant, which displays high rates of minichromosome loss, is arrested at the G1 and G2 phases in the cell cycle. The misll’ gene was found to be identical with the p q z ‘ gene described above (TAKA- HASHI et al. 1994). Moreover, some newly isolated prp mutants in S. pombe also displayed defects in the cell cycle at a restrictive temperature, as did cdc mutants (URUSHIYAMA et al. 1996; D. KIM and D. FRENDEWEY, personal communication). In mammals, SRPKl kinase, which specifically phosphorylates the RS domain in members of the SR protein family of splicing factors, may play a role in the cell cycle-dependent intranuclear distribution of splicing factors (GUI et al. 1994). SRPKl is a human homologue of the S. pombe kinase Dsklp, the product of a multicopy suppressor of the disl mutation that affects sister chromatid separation during mitosis (OHKURA et d . 1988; TAKEUCHI and YANAGIDA 1993). Although the mechanism connecting pre-mRNA splicing and cell cycle progression remains to be elucidated, these findings imply the existence of factors or regulators that may play a role in both pre-mRNA splicing and cell cycle progression.

pvpl of S. pombe has a defect in both pre-mRNA splicing and pre-U6 snRNA splicing at a restrictive temperature, resulting in accumulation of mRNA precursors and the U6 snRNA precursor (POTASHKIN et al. 1989; POTASHKIN and FRENDEWEX 1989). pql -4 , a new allele of prpl, also has a defect in poly(A)+ RNA transport from the nucleus to the cytoplasm and exhibits chromosome condensation in some cells at a restrictive temperature (URUSHIYAMA et al. 1996). Here we report the characterization of the prpl’ gene and genetic interaction of Prplp with other splicing factors. The predicted Prplp contains 19 repeated TPR motifs and has high similarity with Prp6p of S. cermisiae, although their proteins cannot functionally substitute for one another. Synthetic lethal analyses suggest that Prplp interacts with Prp4p, Prp6p and Prpl3p of S. pombe. Unexpect- edly, the pql’ gene was found to be identical with the zerl+ gene involved in the regulation of the GO-Gl/GZ transition in the cell cycle (K OKAZAKI, H. MURAKAMI and H. OKAYAMA, personal communication). These result,~ suggest that Prplp/Zerlp may play a role, either directly or indirectly, in pre-mRNA splicing, cell cycle progression and poly(A)+ RNA transport.

MATERIALS AND METHODS

Yeast strains, media and genetic methods: The yeast strains used in this study are listed in Table 1. The complete media YPD or YE (GUTZ et al. 1974, SHERMAN et al. 1986) and mini- mum medium PM (MORENO et al. 1991) were used for stan-

dard cultures of S. pombe strains. Appropriate growth supple- ments (adenine, leucine, histidine or uracil, 75 mg/liter each) were added to PM, while 250 mg/liter of leucine was added to the plates for transformation of buI-32 strain (ALFA et al. 1993). SPA medium (GUTZ et al. 1974) was used for induction of mating and sporulation of S. pombe. The media used for S. cermisiae strains are W D as the complete medium, SD as the synthetic minimal medium, and a sporulation medium for the induction of mating and sporulation ( SHERMAN 1991). General genetic methods were previously described in GUTZ et al. (1974) for S. pombe and in GUTHRIE and FINK (1991) for S. cermisiae.

Molecular biological methods: The transformation of S. pombe was performed by the method of OKAZAKI et al. (1990). The recovery of plasmids, the preparation of chromosomal DNA from S. pombe and the genomic integration of a plasmid were done according to the methods of MORENO et al. (1991). The lithium acetate method was used for S. cereuisiae transformation as described in BECKER and GUARENTE (1991).

DNA staining and in situ hybridization: prpl and wild-type strains were grown at 26” to mid log phase. Half of each culture was then used as a sample at the permissive temperature, while the other half was shifted to 36” for 4 hr as a sample at the restrictive temperature. This incubation time (4 hr) after temperature shift was chosen as more suitable than 2 hr to observe hybridization signals, based on the results of other poly(A)+ RNA transport mutants (AZAD et al. 1997). Nuclear staining with DAPI (4’,6”diamidino-2-phenylindole dihydrochloride) and in situ hybridization using a biotin-labeled oligo (dT),, probe were performed as described by TANI et al. (1995). Signals of the hybridized poly(A)+ RNA were detected with FITC (fluorescein isothiocyanate)-avidin and observed by a fluorescence microscope.

Oligonucleotides: The following oligonucleotides were used in this study as probes or primers. UG-IN1, 5”TCGAAC CTTGGTAAATATTGS’, is complementary to the intron of S. pombe pre-U6 snRNA (TANI and OHSHIMA 1989). UG-EXS, 5’-CAGTGTCATCCTTGTGCAGG3’, is complementary to the second exon of S. pombe pre-U6 snRNA. TFII-IN1, 5’-GAA ATCTCGTGACATGGTAGS‘, is complementary to the first intron of S. pombe TFIID pre-mRNA (HOFFMANN et al. 1990). TFII-EX3, 5’-GAGCTTGGAGTCATCCTCGG3’, is complementary to the third exon of S. pombe TFIID pre-mRNA. P1-UO, 5’-CGAGCCATATATATTGCCGA-3’, and PI-U1, 5’- TGTAGTAAAGCTGCTAGGAG3’, have sequences in the 5’- flanking region of the S. pombe f l l gene. P1-Rl, 5”TAAGTG TTCAATCGAACTG3‘, and P1-R11, 5’-CTTGGGATGGCA ATTGTGA-3’, are complementary to a 3“flanking region of the S. pombe prpl gene. Pl-R7, 5’-CAAGTCGAGCAAGAGC TAAC-3’, is Complementary to S. pombe p q l mRNA. PRP6R1, 5‘-AACCATGCTGACAAGGTCGC-3’, is Complementary to S. cereuisiae PRP6 mRNA. The oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer (model 380A) or by Grainer Japan Co.

Preparation of RNA and Northern blot analysis: A yeast strain was grown in 10 ml of an appropriate medium to mid log phase at 26” to prepare a sample at the permissive temperature and then shifted to 36” for 2 hr to prepare a sample at the restrictive temperature. The cells were pelleted by centrif- ugation at O”, washed twice with ice-cold sterile water, and frozen at -20”. Total RNA was prepared by a previously described procedure (NISCHT et al. 1986).

For Northern blot analysis of U6 snRNA, 5 pg of total RNA from each mutant or wild-type strain was mixed with a dye solution (80% formamide, l x TBE, 0.1% bromophenol blue and 0.1% xylene cyanol) and electrophoresed for 1 hr at 300 V on an 8% polyacrylamide (acry1amide:bis-acrylamide = 19:1)/8.3 M urea gel. The RNAs were blotted onto nylon membranes (Biodyne A, PALL) in 2OX SSC by overnight c a p

-1' Gene in Fission Yeast

TABLE 1

Yeast strains used in this study

103

Strain Genotype Source

Schizosaccharomyces pombe 972 h- (wild type) U. LEUPOLD

#1686 h- cdcl0-129 NURSE et al. (1976) 975 h+ (wild type) U. LEUPOLD

SU60-3B h+ his2 This study SU59-1D h- h l - 3 2 This study UR250 h+ h l - 3 2 This study CH992 h- leul-32 his2 c. SHIMODA

SG18 h+ leu3 C. SHIMODA UR441 h- ura4-Dl8 This study UR443 h+ ura4Dl8 This study OR1 h- cut9 l ~ e l - 3 2 M. YANAGIDA NC102 h- nuc2-663 leul-32 HIRANO et al. (1988) KZ599-101 h- ~ m l - C 5 K. OKAZAKI et al. unpublished

results A60 h- ptrl-I h l - 3 2 AZAD et al. (1997) A60-2 h+ ptrl-1 ade6M216 AZAD et al. (1997) UR461 h- ptr2-I This study S13 h- ptr3-1 leul-32 AZAD et al. (1997)

SU345D h+ pPl-1 This study UR104 h+ prpl-4 This study UR107 h- Pql -4 This study UR100 h- pPl -4 leul-32 This study UR422 h- prpl-4 ura4D18 This study UR423 h+ prpl-4 ura4-Dl8 This study

s13-2 h+ ptr3-1 ade6-M216 &AD et al. (1997)

SU35-3A h+ pP2-1 This study SU50-5B h- pP2-2 This study SU13-1lB h- @3-3 This study

DK124 h+ pP4-2 h l - 3 2 ade6"210 D. FRENDE-

SU31-8B h- prp6 l This studyb

DK221 h- Pq7-1 D. F R E N D E W E ~

SU26-9B h- pP8-1 This studyb TS377 h- mPl D. FRENDEWE~ UR230 h- prpl0-1 This study

SU42-6C h- pP4-2 This study

SU38-12C h- Pq5-1 This studyb

SU31-11B h+ Pq6-1 This studyb

SU 100-2A h- pP12-1 This study su101-9c h- w l 3 - 1 This study UR550 h- Pql3-1 h I - 3 2 This study UDP6 h-/h+ ade6M21 O/ade6"216 h l - 3 2 / h 1 - 3 2 This study

UDP9 h-/h+ pPl::ura4+/+ ade6-M21O/ade6"216 This study ura4D18/ura4-D18

h l -32/h l -32 ura4-D18/ura4D18 Saccharomyces cereuisiae

X2180-1B MATO mal gal2 CUP1 Yeast Genetic Stock Center SpJ6.66 MATa pP6-1 adel lys2 ura3-52 LEGRAIN and ROSBASH (1989) Se 1 MATa pP6-1 adel leu2-3 ura3-52 This study Se3 MATa pp6-1 adel ura3-52 This study

a Unpublished mutant. Derived from an unpublished mutant isolated by D. KIM and D. FRENDEWEY.

illary blotting. For mRNA analysis, 20 pg of total RNA was fractionated for 5 hr at 100 V on 1% agarose/6% formaldehyde gels and transferred onto nylon membranes (Gene Screen, NEN) by the capillary blotting (SAMBROOK et al. 1989). The blots were cross-linked with a low dose of ultraviolet irra- diation (SAMBROOK et al. 1989). Hybridization with more than 5 X lo5 cpm/ml of an oligonucleotide probe end-labeled with 32P was performed at 42" overnight using standard methods

(SAMBROOK et al. 1989). The hybridized filters were exposed to Kodak X-ray film with an intensifylng screen at -70" for 2 or 3 days.

Cloning and sequencing: An S. pombe cosmid genomic library, which was constructed in pSSl0 with LEU2 marker (NA- KASEKO et al. 1986), was introduced into URlOO ( m l - 4 leul), and cosmids were recovered from transformants that grew at 36" on PM plates. Isolated cosmids were reintroduced into


A

lo6 Y 0 2 4 6 8 1 0 1 2

Time (hr)

B

'o20 L 2 4 6 8 10 12 l ime (hr)

C

-

0 2 4 6 8 1 0 Time (hr)

FIGURE 1.-Growth and morphological characteristics of the prpl-4 mutant at the restrictive temperature. prpl-4 (UR107) and the wild-type strain (972) were cultured in YPD medium at the permissive temperature (26") to mid log phase, and then were either maintained at 26" or transferred to the restrictive temperature (36"). (A) Growth curves. The total cell number was measured with a hemacytometer at the indicated times. (B) Relative viabilities. The results were obtained from the number of colonies grown at 26" after culturing at 26" or shifting to 36" for the indicated times. The symbols in A and B indicate the strains as follows: M, wild type maintained at 26"; 0, wild type shifted to 36"; A, prpl-4 maintained at 26"; A, prpl-4 shifted at 36". (C) Fraction of dumpy form cells in p p l - 4 at 36". The results were obtained as the ratio of the number of dumpy form cells to the total cell number as measured with a hemacytometer.

URlOO to confirm their ability to complement the growth defects of W l - 4 at the restrictive temperature. The gene in the -30-kb insert of the cosmid was subcloned into pSP1, which is an S. pombe arsl multicopy vector derived from pRS305 of S. cereuisiae (COTTAAREL et al. 1993), and a 4.2-kb Pstl-Sua fragment was also found to have the complementing activity.

To determine whether the complementing fragment contained the wild-type allele of the ppl gene, chromosomal integration mapping was performed. The 4.2-kb PstI-SUE fragment was recloned into multicloning sites of pSKur4, which was made by inserting an ura4' marker into Bluescript SK' vector (Stratagene). The resulting plasmid (pSKur4-4.2) was linearized with BamHI within the cloned gene and introduced into UR422 (prpl-4 uru4-DI8). Stable Ura+ transformants were isolated, and then precise integration of pSKur44.2 to the prpl locus was confirmed by PCR amplification of the prpl gene using P1-U1 and P1-R11 as primers. Ura' transformants were crossed with a wild-type strain (975), and then the diploids were dissected. All spore progenies from ten tetrads were Ts+.

For the sequencing of the 4.2-kb fragment, a series of over- lapping deletions of pSKpl-4.2, which was made by inserting the 4.2-kb PstI-SalI fragment into Bluescript SK' vector, was produced by unidirectional exonuclease I11 digestion (HENI- KOFF 1984). Nucleotide sequencing was performed for both strands using the AB1 PRISM Dye Terminator Cycle Sequenc- ing Ready Reaction Kit according to the attached protocol (Perkin Elmer) with an AB1 373A DNA sequencer.

To identify the mutation sites in two prpl alleles ( m l - 1 and pp1-4 ) , genomic DNA was prepared from the cells with each allele, and then the 3-kb fragment containing the entire region of the prpl open reading frame (OW) was amplified by PCR. Three independent amplification reactions for each allele were performed to exclude any possible nucleotide change generated during PCR. These fragments were inde- pendently cloned into pGEM'-T vector (Promega), and clones were sequenced with oligonucleotide primers, which

are complementary to the coding sequences of prpl, or with the M13 universal and reverse primers.

Disruption of the Irrpl' gene: A 2469-bp EcoRV fragment of the prpl' gene was removed from pSKpl-4.2 and replaced by the urd' gene. The EcoRV fragment contains most of the ppl' OW. The resulting plasmid (pSKpl::uru4+) was linearized by PstI-SulI digestion and introduced into the diploid strain, UDP6, in a one-step gene replacement method. Stable Ura+ diploid transformants were isolated. The disruption of one ppl' gene in the transformants was verified by agarose gel electrophoresis of the PCR fragment that was amplified from the genomic prpl gene. The resulting diploid strain (UDP9) was heterozygous for the disrupted gene.

Isolation of the double mutant haploids: The diploids obtained from genetic crosses were sporulated and tetrads were dissected. The dissected spores were allowed to germinate for a week at 22", which is lower than the usual permissive temperature of 26". The viable spore progeny were replica- plated and incubated at 36" to determine the temperature sensitivities. The double mutant strains for ppl and the other mutation were identified and isolated from tetrads that gave nonparental ditype or tetratype, based on the growth at 36". Identification of the double mutant in tetratype tetrads was confirmed by segregation of two mutations after crossing to the wild-type strain. We could obtain the double mutant haploids at 22" in all of the crosses, except for a Pql3 @lo double mutant. which was isolated at 26".

RESULTS

Time-course of growth and cell viability of Hl-4 after a temperature shift: The growth rate of the prpl- 4 was almost the same as that of a wild-type strain at the permissive temperature (26"), whereas the cell division cycle of prpl-4 was immediately arrested after shifting

prpl+ Gene in Fission Yeast 105

FIGURE 2.-Defects in poly(A)+ RNA transport at the restrictive temperature in prpl mutants. Wild-type strain (972), prp1-4 (URlOl) and @l-1 (SU345D) were grown to mid-log phase at the permissive temperature (26") and shifted to the restrictive temperature (36") for 4 hr. Fixed cells were subjected to in situ hybridization with a biotin labeled oligo (dT)50 probe to detect poly(A)+ RNA. The hybridized signals were detected by FITGavidin. The cells were treated with DAF'I to stain DNA and observed with a fluorescence microscope. Left two columns show the cells grown at 26" and right two columns show cells shifted to 36" for 4 hr. FITC and DAF'I columns at each temperature show the cells in the same fields. Size bar, 10 pm.

to the restrictive temperature (36"), and most of the cells (>94%) became inviable within 12 hr from shifting (Figure 1, A and B). We previously reported that some cells of the fn$ mutants, including W l , became remarkably shorter and rounder (see the cells indicated by arrowheads in Figure 2). We termed this phenotype as a dumpy form URUS^^ et al. 1996). About 50% of cells changed into this form within 2 hr after shifting to 36", and more than 80% of the cells became dumpy after 10 hr (Figure IC). The morphology of the dumpy cells is very similar to that of cells in the stationary phase.

p.Pl mutants have a defect in poly(A)+ RNA nuclear export: We previously described that only prPl-4, among the nine p p mutants isolated by us, also has a defect in poly(A)+ RNA nuclear export (URUSHIYAMA et al. 1996). To confirm whether this phenotype was allele-specific or not, we performed in situ hybridization with a biotin-labeled oligo (dT)BO probe on the ppl-1 mutant, which had previously been isolated by Po- TASHKIN et al. (1989). Strong hybridizing signals were detected in the nuclei of the p-pl-1 cells at 36" as well as for prPl4 cells, although the fractions of the cells that have strong signals in the nucleus were observed to differ between them (Figure 2). On the other hand, in wild-type cells, weak hybridizing signals were observed throughout the cells at both temperatures (Fig- ure 2). Therefore, we concluded that the defect of the

poly(A)+ RNA nuclear export in prP1-4 is not allele specific.

Time-courses of pre-mRNA accumulation and poly(A)+ RNA accumulation in -14 after a temperature shift: We performed a timecourse experiment to examine the order of pre-mRNA accumulation, nuclear poly(A)+ RNA accumulation and cell death in prPl-4. The accumulation of pre-U6 snRNA and TFIID pre- mRNA were detected by Northern blot analysis 30 min after shifting to the restrictive temperature and reached a peak at 1 or 2 hr, whereas nuclear hybridization signals of poly(A)+ RNA were detected only in less than €096 of the cells at 30 min or 1 hr ( d a t a not shown). Hybridization signals of poly(A)+ RNA were observed in -10-20% of the cells at 2 hr (data not shown) and in 50"70% of the cells at 4 hr (Figure 2). Furthermore, most of the cells (96%) were viable at 4 hr (Figure 1B). Taken together, in the p p l - 4 cells, the pre-mRNA splicing defect appears first, the blockage of poly(A)+ RNA transport occurs next, and most cell death take place later. The morphological change of ppi-4 cells shown in Figure 1C took place before nuclear poly(A) +

RNA accumulation and in a time course similar to that of pre-mRNA accumulation.

Genetic mapping of Ml: In the course of con- structing the ppl-4 leul-32 strain, we found that ppl is linked to leu1 on chromosome II. Furthermore, we found by tetrad analysis that Pgl is located distal to his2,

106 S. Urushiyama, T.

TABLE 2

Linkage analysis of prp1 and genetic map distances

No. of tetrads" Map distance

Gene pairs PD NPD T (cM)"

/rrj,l X le111 42 2 63 35.0 /@l X his2 2 5 0 32 28.1 pql X ptr2//1iml 49 1 14 16.1 p/ll X leu3 60 0 3 2.4

hi52 X ptr2/pirnI 42 1 18 19.7 h j l X ptr2/piml 47 0 12 10.2 h 1 3 X c d d o 49 1 8 12.1

p q 1 X crll-lo 35 0 9 10.2

" PD, parental ditype; NPD, nonparental ditype; T, tetratype.

"To determine the genetic map distances in centimorgans based on the tetrad data, we used the following formula: Map distance (in cM) = 50(T + 6NPD)/(PD + T + NPD) (PERI(ISS 1949).

Tani and Y. Ohshima

and not between I d and his2. Therefore, we crossed the w11-4 strain (UR104 or UR107) to each of three mutant strains (UR461; ptr2, SG18; h 3 , no. 1686; cdclo) , the mutations of which were located distal to his2, to determine the chromosome map position of # ] I . ptr2 was isolated as a L$ mutation, which has a defect in poly(A)' RNA transport, and is allelic with piml (Azm ~t nl. 1997). The linkage relationship among these genes obtained from tetrad analvsis are shown in Table 2. The j1tr2/PimI-his2 genetic distance (19.7 cM) obtained here is similar to the previously described piml-his2 distance (21 cM) (MATSUMOTO and BEACH 1991). From these results, we concluded that ppl is located between h 3 and cdc10 on the right arm of chromosome 11, and is 2.4 cM from h 3 and 10.1 cM from cdcl0.

Cloning and sequence analysis of the P g Z ' gene: To isolate the w)l' gene, we transformed the pqf11-4 strain (UR100) with an S. p o m h genomic libray in a cosmid vector. Cosmid clones that complemented the growth

A X P X S X P P P Sm

1 kb - P1 -Tr2 +

pSPl-18 - pSPl-11.7 pSP1-13 +

1 kb

B YPD

pSP14.2

I I ura4 +

PM+L PM+LU

+

FIGURE 3.-(A) A restriction map of the genomic DNA fragment containing the jnpl- gene. PI-Tr2 is the 29-kb genomic fragment in the cosmid clone, pP1-Tr2, that rescued the growth of /@I4 at 36". The number following pSPl of each suhcloned fragment indicates the length of the fragment in kilobascs. Complete rescue of the growth of M l - 4 at 36" is shown by +, and non-rescue by - for each fragment. The ORF for the wl+ gene is indicated by the broad arrow and the open box. The direction of OW is leftward. Restriction sites are abbreviated as follows: A, AntI; B, BnmHI; C , ClnI; E, EcoRV, P, PstI; S, Sun; Sm, SmnI; X, XIzoI. The EmRV fragment of the /@l+ gene was replaced by the urn4' gene for gene disruption. (R) Gene disruption of PPI+. In UDP6 diploid strain (trm4, h d , MI'), one of the hvo endogenous copies of the MI* gene was disrupted with the tm4' gene as shown in A (see MATTERIALS AND METHODS). The tetrads from the heterozygous diploid strain (UDP9) were dissected onto a YPD plate and incubated at 26". Grown colonies were subsequently replicated to a PM plate with adenine and leucine (PM+L) and a PM plate with adenine, leucine and uracil (PM+LU) at 26".

pr/11+ Gene in Fission Yeast 1 0 i

26^ 36" 26' 36" 26" 36" 26" 36" 26" 36"

C 26" 36"

FIGURE 4.-Complementation of the defects i n pre-mRNA splicing and pol!(X) * R S A transport in jnf~l-4 with pP1-TR2. (A) Northern blot analysis of U6 snRNA. Total RNA ( 5 pg) WAS prepared from the wild-type strain (972), pV11-4 strain (URIOI) and jnpl-4 strain transformed with pP1-Tr2 that have been incubated at the permissive temperature (26") or shifted to the restrictive temperature (36') for 2 hr. The fractionated RNAs on a denatured polyacrylamide gel were blotted to a membrane and hybridized with a mixture of ""P-labeled oligonucleotides UMN1 and UGEX2 (top). The arrowheads on the left indicate the unspliced precursor (P) and the mature U6 snRNA (M) . The lower panel shows 5s rRNA stained with ethidium bromide to indicate the quantity of total RNA in each lane. (R) Northern blot analysis of TFlID mRNA. Total RNAs (20 pg) prepared as in A were analyzed on a formaldehyde-agarose gel and then transferred to a nylon membrane. The blot was hybridized with :'YP-labeled oligonucleotides TFII-IN1 and TFII-EX3 (top). The arrowheads indicate unspliced precursor (P) and spliced mature TFlID mRNA ( M ) , respectively. 18s rRNAs were shown as a measure of the quantity of total RNA in each lane (bottom). (C) In sil7r hybridization of poly(A)+ RNA. MI-4 strain (URIOI) transformed with pP1-Tr2 was grown to the mid-log phase at 26" and shifted to 36" for 4 hr. Hybridization with oligo (dT),,, probe and DNA staining with DAPI were performed under the same conditions as for Figure 2. Size bar, 10 pm.

defect on PM plates at 36" were recovered from three transformants using Edzpn'chin coliJA226 strain. Restric- tion mapping revealed that the three clones contained the same DNA insert. After subcloning of this insert, the complementing activity was found to localize in a 4.2-kb Sn/"~t1 fragment (Fi<pre SA). We confirmed that the gene in this 4.2-kh suhclone was not an extragenic sup pressor of the fvpl mutation hv chromosomal integration mapping (see MATERIAIS AND METI-IODS).

To examine whether this cosmid clone (PPI-Tr2) could also complement the splicing defects, we prepared total RNA from the p ~ p I - 4 mutant and its transformant carrying PI-Tr2 and performed a Northern blot analysis. Pre-UG snRNA splicing and TFIID pre- mRNA splicing at the restrictive temperature were normal in the pP1-Tr2 transformants (Figure 4, A and R). ,&tubulin pre-mRNA splicing was also rescued (data not shown). The defect in poly(A)' RNA transport at the


CTATAAAGOCTGCAATTGAACA~ATCA~TAGAr~AAGARGAAGATATCGATCCTCGTTATCAGGATCCTGACAATGAAGTAGCGCT~TGCTACn;CCTATCCATATGA~ATG I K A A I E Q R K S E I E E E E D I D P R Y Q D P D N E V A L F A T A P Y D H E

AGGACGRADAAGCTGATAAAPITATATCAATCTGTAGAGGAACATTTAAGT~GRAG~TCCCAACGTGRAAAACAA~CAGCTACA~GRAAAATA~RAAAAG~CCCCA D E E A D K I Y Q S V E E H L S K R R K S Q R E K Q E Q L Q K E K Y E K E N P K

AGGTTTCTrrACRATTTGCGGACTT~CGT~TTATCCACTTTGACAGA~AA~CT~AATAATATCCCAGAACCC~GATCTAACTCGP~GGACTACAGCC~GAC V S S Q F A D L K R G L S T L T D E D W N N I P E P G D L T R K K R T K Q P R R

G P G A G A m T A T G C A A C R A G T G A m ~ T C T T G C A A G n ; C C T A T C G C A A T G ~ T C A A G C T A T C T C T A A m T G C A G T G G A T A C T C A A G C A G G A A C A G A C ~ C A ~ T A ~ R A ~ E R F Y A T S D F V L A S A R N E N Q A I S N F A V D T Q A G T E T P D M N G T

CRAAAACATTTTGTTGIUATTGGAGCmfiTCGTGACRAAGTTTTAGGTATCAAGTTAGCACAAGCTTCGTCGAATTTGACTTCACCATC~CTATAGATCCGAR~TAmRACTA K T N F V E I G A A R D K V L G I K L A Q A S S N L T S P S T I D P K G Y L T S

G T C T G A R T A G T A T G G T T C C A G C C A A T ~ C C n ; G G T G L N S M V P K N A N D L G D I R K A R K L L Q S V I E T N P K H A S G W V A A A

C G C G G C T T G A R G A G G T C G C G R A T A T T A T C T C A A G C A C A A T C T T T A A T T T T ~ ~ T T G C G ~ T T G T T C T C G C T C ~ A ~ T G T T T ~ T A ~ G C A A T A C G A C T A C A C C C ~ T G R L E E V A N K L S Q A Q S L I L K G C E N C S R S E D V W L E A I R L H P A A

C A G A A G C ~ G T T A T T A T T G C A A A T G C C G T T ~ T T A C C R A A G T C A G T ~ C G T T A T ~ T T ~ G C C G R A A A A C ~ ~ T C A A G C T C A G C A C ~ G G A T T A T ~ G E A K V I I A N A V K K L P K S V T L W L E A E K L E N Q A Q H K K R X I K K A

CTTTAGAAmAACCCGACTTCTGTAAGCTTA~AGRA~TGTATTTAG~GRAGRAGTGGATAATGCAAGGATACTATTAGCTCGn;CCTAC~GAATTAATACCCATGTCCATTG L E F N P T S V S L W K E A V N L E E E V D N A R I L L A R A V E L I P M S I D

A T T T A T G G T T A G C T C T T G C T C G A C T T G A A R C T T A T G A G A A L W L A L A R L E T Y E N A K K V L N K A R Q T I R T S H E V W I A A A R L E E

- 7 0 8

- 4 6 8 - 5 8 8

- 3 4 8 -228 - 1 0 8

13 5

1 3 3 4 5

2 5 3 85

373 1 2 5

4 9 3 1 6 5

613 205

7 3 3 2 4 5

2 8 5 853

973 325

1093 3 6 5

1213 4 0 5

1333 445

A A C A G C A A G G T A A T G T A T C A C G G G T G G ~ T R A ~ T C G T ~ T G T R A G ~ A G T T A C A ~ T A C ~ T G G T A T G T T A C R A C G C G A T C A G T ~ T T T C C G A ~ T ~ T G C G A 1453 Q Q G N V S R V E K I M A R G V S E L Q A T G G M L Q R D Q W L S E A E K C E T 4 8 5

CTGAAGGGGCAGTGATTACTGCACAAGCTATAATTAATACGTGCCTC~TGTT~ATTGGATGARGAAGATCAATTCGACACCT~TTAGATGATGCTCRATCTTTTATAGCTCGTRAAT 1 5 7 3 E G A V I T A Q A I I N T C L G V G L D E E D Q F D T W L D D A Q S F I A R K C 5 2 5

GTATAGATTGTGCGCGTGCTGTT~GCTTTTTCTTTRAGAGTCTATCC~GTGAGAC~~TGA~GCTGTTGAACTTG~GCTGTATGGTACAACAGAGPGmGTT 1 6 9 3 I D C A R A V F A F S L R V Y P K S E K L W L R A V E L E K L Y G T T E S V C S 5 6 5

CTATmAGRAAAAGCTGTAGAATCATGrrCTARRGCCGATmATGGTTGCTTTATGCTAGAGCG~CGTTAATGATATTGCTGGTGCTCGGAATATTCTT~AGAGCTT 1 8 1 3 I L E K A V E S C P K A E I L W L L Y A K E R K N V N D I A G A R N I L G R A F 6 0 5

A TTGAATATAATTCTAATAGCGAAGAGATAT~TGCTGC~TCAGRATTGAATTTOPGAACAATGARAl\TGAACGTGCCAGAGCTACTTGCCCGTGCTCGTATC~TCGGGAACTG 1 9 3 3

E Y N S N S E E I W L A A V R I E F V N N E N E R A R K L L A R A R I E S G T E 6 4 5 I l p r P l - 4 1

AACGAATATGGACTRAATCCATTTCGCTTGAACGAATTTT~ACGRAAAAGATC~GTTACAACTTTT~~TGCACTG~TTTATCCCCATTATGATAAGTTGTACAT~TGA 2053 R I W T K S I S L E R I L D E K D R A L Q L L E N A L K I Y P H Y D K L Y M M K 6 8 5

A A G G G T C A T T T T T G A A G A T A G A G C A ~ T T G 2 1 7 3

G Q I F E D K E Q I E L A R D A Y L A G T K V C P Y S I P L W L L L A K L E E K 7 2 5 D I p r p l - 1 )

AGCAGTCCGTTATACGCGCGAGAGTTGTGTGTTTGATA~T~~TTRAAAATCCTAAGAATGAATmTATGGCTTGAACTTATRAAAATGGAACTCCGGGC~RATAmCACAAG 2293 Q S V I R A R V V F D R A K V K N P K N E F L W L E L I K M E L R A G N I S Q V 7 6 5

T TTCGTGCAGCCCTTGCTAAGGCCTTGCAGGRATGCCATCTTCGGGTTTGCTGTGGACGGAGGCTATATGGCTTGAACCTCGCGCACAGCGT~CTCGTGCTACAGATGCTTTAAGGA 2413

R A A L A K A L Q E C P S S G L L W T E A I W L E P R A Q R K T R A T D A L R K 8 0 5 L ( p r p l - 4 1

AATGTGAAGGCAATGCACATTTACTTTGCACCATCGCCAGGATGCTGTGGCTGG~GCAGATAA~GCGTAGTTGGTTTTT~GCTGTTAAGGCTGACCAAGATAACGGAG 2 5 3 3 C E G N A H L L C T I A R M L W L E K K A D K A R S W F L K A V K A D Q D N G D 8 4 5

A T G T G T G G T G T T G G T ~ R T A A G T A T A G C T T A ~ G G C T G G G A T A 2653 V W C W F Y K Y S L E A G N E D Q Q K E V L T S F E T A D P H H G Y F W P S I T 8 8 5

CTAGATATCRAAAATTCTCGGAAAACTCCGCA~AACTATTACATCTTGCCATRAATGTGTTATGATTACGAC~TGATGACTGTAACATATTGGATCTATTATTTATCTT~ 2773 K D I K N S R K T P Q E L L H L A I N V L . 906

TTAATGTTTAAATTGGTATCGAA~ACTTTAGATATTTGTATTTAC~ACTTTTTAAT~GAACGAT~TACAGGTTCGATTGAACACTTAC~RAATCAACAAGART 2 8 9 3 A C C A G T T A A G A R A G C A G T A G T R A A C C R R R A C ? T T T 3013 GAG~WAAGTRACCCCTITTRRGTATATGTTGCCTTTACTCAGCAAC~ATTCCATTGTGAATATATATCCCAGCGACTAGATTC~TTGACGCGACACATATTTGACAA 3133 ATTGAAGGACACCTTCATCTTATRAACCAAAG~CATTCCAAGCGGC~GCAGAAAGC~TACTGTCATCTTGCTGATCACCCCTATACACAGCAGCATCTACATCAGGAT 3253 CTTCATCGCCAATCATATATATCATATGCACCAT~TCAGTATTCGCAAT~CT~CCCCGGAGATAAGCAAAGAGTAGCTTCTAGTRAAGCTGTGTCATTCGTAGTAGCAAGCGTAA 3373 AATCGGAACCAGGGCGGAATCGTCGAC 3400

FIGURE 5.-Nucleotide sequence of the @pl+ gene and flanking DNA. The predicted amino acid sequence is shown in the single letter code. The bases were numbered from the adenine residue of the initiation codon. An asterisk indicates the

ppl' Gene in Fission Yeast 109

restrictive temperature was also complemented by PPI- Tr2 (Figure 4C).

Sequence analysis of this 4.2-kb fragment revealed the prpl' gene to encode a protein of 906 amino acids with a calculated molecular mass of 103 kD (Figure 5). Prplp is similar to Prp6p of S. cerevzsiae (LEGRAIN and CHOULIKA 1990), which is a component of the U4/U6 snRNP required for spliceosome assembly (ABOWCH et al. 1990). The overall amino acid identity and similarity between Prplp and Prp6p are 29.6% and 50.6%, respectively (Figure 6). Prplp has many charged residues, 30.6% throughout and especially near the N-terminal region, as is the case for Prp6p.

The predicted Prplp contains 19 TPR motifs: S. cere- uzsiae Prp6p has nine PW motifs, a leucine repeat motif and cysteine/histidine motifs (LEGRAIN and CHOULIKA 1990). Among these motifs found in PrpGp, Prplp contains only the PW-like motifs. The PW motif consists of -30 residues including proline-X5-tryptophan in the middle of a unit. This motif is closely related to the TPR motif, which is a 34-amino acid motif found in tandemly repeated copies in a variety of proteins. The TPR motif contains eight loosely conserved consensus residues (Figure 6C), whose hydrophobicity and spacing are conserved (HIRANO et al. 1990; SIKORSKI et al. 1990; GOEBL and YANAGIDA 1991). Prplp contains 19 consecutively repeated TPR units (Figure 6B). In comparison with other TPR proteins, which have up to 16 repeats, Prplp contains more TPRs clustered toward the Gterminus. Moreover, some of highly conserved residues of the TPRs in Prplp do not match the eight consensus residues in the canonical TPR, even though the size, hydrophobicity and spacing in the TPRs of Prplp are consistent with the canonical TPR. The TPRs in Prplp contain two domains, A and B, that were proposed to form amphipathic a-helices (HIRANO et al. 1990; SIKORSKI et al. 1990) like other TPRs (Figure 6C). However, the TPRs in Prplp start from domain B whereas TPRs usually start with domain A. This reverse order of the domains is also observed in the PW motifs (TPR motifs) in Prp6p of S. cmevisiae (LEGRAIN and CHOULIKA 1990). Sequence comparison between S. pombe Prplp and S. cerevisiae Prp6p revealed that the number and the distribution of TPRs are conserved in the two proteins (Figure 6A), although proline and tryptophan residues are not present in some TPRs of Prp6p. The similarity of the amino acid sequence and arrangement of the TPR motifs between Prplp and Prp6p suggest that Prplp may be an S. pombe homologue of Prp6p of S. cerevisiae.

Identification of the mutation sites in the prpl gene and the expression of the ml' gene: We determined the mutation sites for two ppl alleles, prpl-I and prpl- 4, by sequencing PCR products from each mutant as

described in MATERIALS AND METHODS. pqibl-1 has a single nucleotide change: G at position 2114 was changed to A, resulting in replacement of glycine at 705 by aspar- tic acid (G705D). prpl-4 has two nucleotide changes: one is the change from C at 1846 to A, resulting in L6161, and the other is the change from C at 2336 to T, resulting in S779L (Figure 5). All of these mutations are located in the highly conserved residues of the TPR in Prplp (Figure 6, B and C). These results suggest that the TPRs in Prplp play a functionally important role, presumably in mediating protein-protein interactions.

We next examined the expression of the p q l gene in wild-type and prpl-4 strains. Total RNAs were prepared from each strain cultured at 26" or 36", and then North- ern blot analysis was done. In all cells, the prpl gene was expressed (data not shown) and there were no sig- nificant differences in the mRNA level between the two strains at either temperatures.

prpl' is an essential gene: We constructed a null mutant of p q l to determine whether the pq l ' gene is required for cell viability. The prpl::ura4+ diploid strain, UDP9, was sporulated and 40 azygotic asci were dissected. In all tetrads, only one or two viable spores were obtained, and all surviving progeny were ura- (Figure 3B). Therefore, the haploid spores of the prpl null allele were inviable, indicating that the prpl' gene is an essential gene for cell viability.

Functional complementation test between the ml' gene and the S. cerevisiae PW6 gene: Since Prplp is homologous to the Prp6p of S. cerevisiae, we tested whether or not the prpl' and PRP6genes could complement the prp6 and prpl mutations, respectively. pSPrpl carrying the prpl' gene (see MATERIALS AND METHODS) was introduced into an S. cerevisiaeprp6-1 strain (SEI). In addition, the 5.4kb ClaI fragment from PL1 (ho- VICH et al. 1990; LEGRAIN and CHOULIKA 1990), which contains the PRP6gene, was cloned in the pSPl vector. The resulting plasmid, pSPRP6, was then introduced into the S. pombeprpl-4 strain (UR100). Both leu+ transformants grown at 26" were streaked onto plates and incubated at 26", 30" and 36" for prpl transformants, and at 26", 33" and 36" for prp6 transformants. Semi- restrictive temperatures of prpl and prp6 were 30" and 33", respectively. In each mutant, transformants with a plasmid carrying the heterologous gene could not grow even at the semi-restrictive temperature (Figure 7 , A and B). Expression of the exogeneous gene in each transformant was confirmed by a Northern blot analysis (Figure 7, C and D). From these results, we concluded that the S. pornbe prpl' gene and the S. cermisiae PRP6 gene in the multicopy plasmid could not functionally complement prp6 and prpl, respectively.

Genetic interactions between @l and other mutations: To identify the proteins genetically interacting

termination codon. Mutations in the prpl-l and p p I - 4 are shown above the nucleotides and below the amino acids. The nucleotide sequence data of the ppl' gene reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with the accession number D83743.

110

A SP PrplP

Sc Prp6p

SP PrplP

Sc Prp6p

SP PrplP

Sc Prp6p

SP PrplP

Sc Prp6p

SP PrplP

Sc Prp6p

SF PrplP

Sc Prp6p

SP PrplP

Sc Prp6p

SP PrplP

Sc Prp6p

SP PrplP

Sc Prp6p

SP PrplP

Sc Prp6p

SP PrplP

Sc Prp6p

SP PrplP

Sc Prp6p

SP PrplP

Sc Prp6p

SP prpm

Sc Prp6p

SP prpw

Sc Prp6p

SP PrplP

Sc Prp6p

S. Urushivama, T. Tani and Y. Ohshima

MANFYPDFLNMQPPPNWAGLGRGATGF?TRSDLGPAQELPSQESIKAAIEQRKSEIEEE

MERPSFLWEPPAGWPGIGRGAn;FSTK------------------EKQWSNDDKG I I I : : I I : I I : I : I I I I I I I : I : I I : :

EDIDPRYQDPDNINALFATAPYDHEDEEADKIYQSVEEHLSKRRKSQREKQELQKEKYE 1 1 1 : : : I : I I ( I : J I :: : : I : I : : : I I : I : : :

RRIPKRYRE-NLNNHL-QSQPKDDEDDEKTLELKLAQKKK-KRA-NEK-DDDNS-

KENPKVSSQFADLKRGLSTLTD~IPEPGDLTRK-KRTK-QPRRERF-YATSDNLA : : I I I I I I I : ] : : : I : : I : I I : : : I 1 1 : 1 1 : I : I I I : : I :::

VDSSNVKRQFADLKEStESEWMDIPDATDFTRRNKRNRIQEQLNRKTYAAPDSLIP

SARN-ENQAISNFAVDTQAGTETPDMNCTKTN€VEIGAARDKVLGIKLAQASSNLTSPST . . . . . : I : : ::: : :: I I : . . . . . . : : I l l G--NVDLN?CLTE-EREKLLQSQ-ID~QLTK-NASNPIQVNKF'NAATDALSYLKDL~

60

40

120

94

177

154

236

209

IDPKGYLTSLNWPILGDIR

-------DRVNSL-S-DAT-LEDLQ : I I : : : I I I :

TPRtA TPRttE; . . . . . . . ...... 414

379

TPR#6 47 1

L L ~ . : 4 3 9

TPR# TPR#10 : !I:

_""""""""_ VCSILXKA- 570

LAFFQELLFQTKNSDDI-P 558

m--EP-RAQ-RIITRA 799 I"1P 794 I I : :: :I [

KA--PSLEAG 858

!ZRYGDlWWLFR"YA-WX; 853 I l l 1 1 I : I

E-LLHLAINVL 906

899

FIGURE 6.-(A) Comparison of the amino acid sequences between S. pornbe Prplp and S. cmmisiw Prp6p. The alignment was done with GENElYX-Mac Ver. 8.0 (Software Development Co.) and refined bv inspection. Identical amino acids are marked by vertical lines while amino acids that have similar physiochemical properties are marked by colons. The TPR motifs are numbered. (B) Alignment of the TPR motifs of the Prplp. The 19 TPRs were aligned with the Prplp consensus sequence, which identified the most frequently occurring amino acid at each position in the 19 repeats. Individual TPR units are numbered at left.

prpl' Gene in Fission Yeast 111

with Prplp, we produced haploid double mutants by crosses of prpl with various other mutants, and examined whether or not they exhibited synthetic lethality. The following mutant strains that share common phenotypes or protein motifs with Prplp were selected as the strains to be crossed with prpl:j@6 mutants, which have defects in pre-mRNA splicing; ptrl-1, ptr2-I and ptr3-I, which have defects in poly(A)+ RNA transport (ptr2-l is allelic with piml and results in chromosome condensation and splicing defect as does ml) (AZAD et al. 1997); and cut9 and nuc2, whose wild-type gene products have repeated TPR motifs (HIRANO et aZ. 1990; GOEBL and YANACIDA 1991). Most of the double mutants could grow at 22" (see MATERIALS AND METHODS). However, in the double mutants prpl-4 w 4 - 2 , prpl-4 p 9 6 - I and prpl-4 ml3-1, spore progeny showed extremely slow germination and growth even at 22". All the double mutants were streaked onto YE plates together with the wild-type haploid strain and each single mutant haploid strain as control. They were then incubated at 26", a permissive growth temperature for all of the single mutants (Table 3). Most of the double mu-

B

PrPl P

TPR #1 TPR #2 TPR #3 TPR #4 TPR #5 TPR #6 TPR #7 TPR #8 TPR #9 TPR #10 TPR #11 TPR #12 TPR #13 TPR #14 TPR #15 TPR #16 TPR #17 TPR #18 TPR #19

C

tants could grow at 26", but H I - 4 prp6-1 and prpl-4 @l3-I double mutants were inviable and m I - 4 p q 4 - 2 was lethal after a few cell-divisions at 26" (Table 4). These results suggest that Prpl has functional relationships with Prp4p, Prp6p and Prpl3p.

We also examined whether the genetical interactions of P g I - 4 with those splicing mutations were allele-specific or not by using another allele, p q l - I , as well (Table 5). Subtle differences in growth characteristics were observed between the two alleles. prpl-4 prpl3-1 double mutant was inviable at 26", whereas the prpl-1 prPl3-1 double mutant formed extremely small microcolonies after 5 days at 26". The PPI-4 prp6-1 double mutant was inviable at 26", whereas prpl-1 p~p6-I was viable at 26". Notably, prpl-I grew at 30" whereas prpl-4 did not (Ta- ble 3). The effects of PgI-4 were more severe than those of p q l - I .

Furthermore, to test for possible interactions among the pq4-2, prpdl and m13-l mutations, the double mutant haploids were produced at 22" from crosses of all possible combinations and their growth properties were analyzed. All double mutants grew well at 26" (Ta-

'EK

.TD

IM

A

(Positions)

(263-296) (297-326) (327-357) (358-388) (389-418) (419-457) (458-491) (492-528) (529-562) (563-596) (597-630) (631-663) (664-697) (698-730) (731-'764) (765-802) (803-828) (829-862) (863-896)

* * *

* *

*

*

*

AR-L--A----P-s--LWL-A--LE-------- Prplp consensus

FIGURE 6.- Continued Residues identical with the Prplp consensus are shown in reverse print, and similar residues were shown in bold as classified into groups of L, I and V D and E; K, R and H; W and Y. The positions are numbered from the initial methionine. The TPRs with a single asterisk require insertions indicated by dashes, while that with two asterisks requires removal of three contiguous residues, FDTat 510-513, to optimize the alignment. (C) Comparison among the TPRconsensus sequences. Consensus residues of TPR motifs in S. pornbe Prplp and S. cereuisiae PrpGp (LEGRAIN and CHOULICA 1990) are those found in at least 40% of the repeats. The canonical TPR consensus was derived from the TPRs in five fungal proteins (SIKORSKI et al. 1990). Identical residues at each position are indicated with vertical lines. The highly conselved structural domains A and B are underlined.

112 S . Urushiyama, T. Tani and Y. Ohshima

II

I - lESrRNA

FK;I.W 7.-Complemcntation analysis hctuxwl thr S. p~dw p r p l ' gcnc and the S. wrnJi.yiur PRP6 gene. S. pornbr prj,l-4 strain (CRIOO) antl S. cnwkiccr prj16-1 strain (Sel) ~vere transformccl with either a vector plasmid (pSPI) o r plasmid with the wild-type pr/11+ gcnr (pSPrpl) or IW'6 gcnc (pSPRP6). An approximately equal number of cells of each transformant and an untrans- formed strain (ClilOi o r ScJ) were streaked i n each sectorial area of the selective agar plates as shown on the left, and were incuhatetl for 2 days at each temperature (A and R). For both prl, mutants, 26" and 86" arc the permissive and restrictive temperatures, respectively. 30" antl 33" are the semi-restrictive temperature for prpl-4 antl p r p 6 - I , respectively. A Northern hlot analysis was clone to confirm expression of the prpl+ ( C ) or PRP6 (D) genes in each cell. The cells were cultured i n the selective medium at 26" up to ;I mid-log phasc. Total RNAs were prepared from them, fractionated on a formalclehyde-agarose gel and hlotted to a membranes. The hlots were subjected to hyhridization with '"P-labeled oligonucleotides, PRPCRI or PI-Ri (C and D, top panels). PRP(iRI, which specifically detects PRP6 mRNA, was used for hvhridization of RNA from prj,l-4, and PI-Ri, which specifically detects prpl mRNA, was used for hyhridization of RNA from p p 6 - I . The hottom panels in C and D show 18s rRNA stained w i t h ethidium hromicle to show the amount of total RSA in each lane.

ble 4). These viabilities did not change even at 30" (data not shown).

Because Prpl3p was found to interact genetically with Prplp, we examined possible genetic interactions of prpI?-I with the other pqi' mutations. All double mutant spore progeny could germinate and grow at 22", except for j ) q ) I ? - l p q ) I O - l (see MATERIALS ASD METI-I- ODS). The viahilitics of these double mutants at 26" are also shown in Table 4. j n p I 3 - 1 p7p2-2 and pq)l?-l m7- 1 were.just as inviable at 26" as jq+)1-4 /npI?-1. p r p I 3 - I pq15-1 was dead after a few cell-divisions at 26", and j)q)13-1 p 7 ) I O - I showed extremely slow growth and formed microcolonies after incubation at 26" for a week. The jn7)I?-l pp2-I double mutant was also constructed and analyzed. It formed microcolonies at 26", whereas it was lethal at SO" (Table 5). These results suggest that the j)q)l?-l mutation physically and/or SF-

ergistically interacts with the jwpl-4, jnp2-2, p j 5 - I , pq)7- I and pq)lO-l mutations. zerl is allelic with prlbl and causes defects in both

cell cycle control and pre-mRNA splicing: Recently, K. OLAZAKI r / NI. isolated an 5'. pornbr tcmperature sensitive mutant, wrl-C5, which was unable to start either meiosis or a mitotic cycle and exhibited tvpical GO phenotypes at the restrictive temperature. They cloned and characterized the wrl* gene (K. OK.VL,\KI, H. MCRAKAMI and

H. OKAYAMA, personal communication). Unexpectedly, the wrl' gene was found to be identical with the pq))I+ gene since genetic analysis showed that zoI-C5 was allelic to iqi)l. In addition, w I - C 5 accumulated unspliced U6 snRNA precursor and mRNA precursors at the restrictive temperature (data not shown). Also pp1-4 en- ters the GO phase at the restrictive temperature (K. OKAZAKI, H. MURAWMI and H. OKAYAMA, personal communication). A putative function of Zerlp in cell cycle control seems to he the regulation of GO-Gl/GZ transi- tions (K. OKAZAKI, H. MURAKAMI and H. OLIYAMA, personal communication). Prplp/Zerlp may play a role directly or indirectly in both pre-mRNA splicing and cell cycle control.

DISCUSSION

Phenotypes of prlbl mutants at a restrictive temperature: The lq+l mutant exhibit3 pleiotropic phenotvpes at a restrictive temperature. The splicing defects of pre- mRNAs and pre-U6 snRNA in jnpl result in the accumulation of their precursors at 36". This result suggests that the j)q)l+ gene product is required for an early stage in the pre-mRNA splicing as previously described (POTASHIKIN and FRENDEWEY 1989; POTMHKIS r/ 01. 1989; URUSI-IIYAMA P/ 01. 1996). The rapid morphologi-

ml' Gene in Fission Yeast 113

TABLE 3 ate protein-protein interaction based on the predicted

Growth of single mutants used for the construction secondary structure (HIRANO et al. 1990; SIKORSKI et al. of double mutants 1990; GOEBL ~ ~ ~ Y A N A G I D A 1991). There is no evidence

for any direct interaction between proteins via TPRs. Growth at

Mutant 26" 30" 36"

Prpl-1 Prpl-4 PP2-1 Prp2-2 Prp3-3 Prp4-2 Prp5-1 m6-1 Prp 7-1 m 8 - l m 9 - 1 PrpIo-1 prp12-1 prpl3-I ptrl-I ptr2-1 ptr3-1 cut9 nuc2-663

~

+ + + + + + + + + + + + + + + + + + +

+ - + -

+/- + +

+/- + + +

+/- + + +

tal change in pibl cells at 36" may imply that Prplp is also involved in cell cycle progression. Indeed, the Ppl' gene is identical to the zerl' gene, whose product seems to be involved in the control of GO-transition in the cell cycle control (K. OKAZM, H. MURAKAMI and H. OKAY-, personal communication). Furthermore, both prpl alleles caused defects in poly(A)+ RNA transport at 36", thus suggesting that Prplp is also involved in poly(A)+ RNA transport, either directly or indirectly.

Role of TPRs in Prplp: Prplp contains 19 tandemly repeated TPR motifs. The TPR is found in both prokary- otic and eukaryotic proteins involved in very diverse processes, including cell cycle progression, transcrip tion repression, stress response, protein import into mi- tochondria and peroxisomes, neurogenesis and protein kinase inhibition (see reviews, GOEBL and YANAGIDA 1991; LAMB et al. 1995). Though the biochemical function for TPR has yet to be defined, the TPRs have been suggested to form two amphipathic a-helices and medi-

However, CdclGp, Cdc23p and Cdc27p, which are TPR- containing proteins of S. cereuisiae, do form a complex (LAMB et al. 1994). In addition, evidence indicating that the TPRs mediate interactions with non-TPR proteins have also been reported: for example, the TPRs of the transcriptional repression protein Ssn6p (Cyc8p) interact with Tuplp (TZAMARWS and STRUHL 1995) and/or a 2 (SMITH et al. 1995), and the TPRs of Cdc23p interact with the chromatin protein Sinlp (SHPUNGIN et al. 1996).

The three mutations in the two p . p l alleles changed conserved residues in TPRs in Prplp. In addition, prpl- 4, which has a mutation in each of two TPRs, shows more severe temperature sensitivity than that of Wl-1, which has a mutation in one TPR. These results strongly suggest that TPRs play an important role in the function(s) of Prplp, though the precise role of TPRs in Prplp function has yet to be elucidated.

Prplp is similar to PrpSp of S. cerarisiae: The predicted amino acid sequence and the arrangement of TPR motifs of Prplp are similar to those of Prp6p in S. cereuisiae. We could not find any similar proteins other than Prp6p in SGD (Saccharomyces Genome Data- base), and therefore Prplp seems to be an S. pombe homologue of Prp6p in S. cereuisiae, although these two proteins could not functionally substitute for each another. Minor differences in the amino acid sequences of the TPRs between the two proteins might reduce their ability to mediate interactions with the target proteins in the heterologous organism.

Genetic interactions of Prplp with other factors: In this study, we performed synthetic lethal analyses to define functional relationships of Prplp with other proteins. Based on these results, we found that Prplp functionally interacts with Prp4p, Prp6p and Prpl3p. The Prp4p of S. pombe is a putative serine/threonine kinase (ALAHARI et al. 1993), although neither the function in pre-mRNA splicing nor the substrate protein(s) of this kinase are known. Prplp might be one of the substrates of Prp4p. Unfortunately, the pib6' and pqbl? genes

TABLE 4

Growth of haploid double mutants at 26"

prp2-2 prp3-3 m4-2 m5-1 pq6-1 pq7-1 pq8-1 pq9-1 prpl0-I m 1 2 - 1 H l 3 - 1

Prpl-4 + + -/+ + + + + + + -

m13-I - + + -/+ + + + +/- + / prP4-2 + + / nt + nt nt nt n t nt /

- -

~

ptrl-I ptr2-1 ptr3-l cut9 nuc2-663

prpl-4 + + + + + PrpI-I + nt + + nt

+, normal growth; +/-, extremely slow growth and microcolonies after incubation for a week; -/+, lethal after a few cell divisions; -, no growth; nt, not tested.


TABLE 5

Growth of double mutants at three temperatures showing allelic differences in prp1 and prp2

prP4-2 pp6-1 ppl3-1

22" 26" 30" 22" 26" 30" 22" 26" 30"

ppl -1 + -/+ - + + - + +/- f l l - 4 +/- -/+

-

- +/- - - - - +/- p p 2 1 Prp2-2

22" 26" 30" 22" 26" 30"

prpl3-1 + +/- - +/- - -

+, normal growth; +/-, extremely slow growth and microcolonies after a week; -/+, lethal after a few - cell divisions; -, no growth.

have not been characterized yet. Since pfp6 and prpl3 mutants accumulate unspliced mRNA precursors (pfp6, D. KIM and D. FRENDEWEY, personal communication; m13, URUSHIYAMA et al. 1996), Prplp, Prp6p and Prpl3p may interact with one another at an early stage of spliceosome assembly. Furthermore, we found that some of the haploid double mutants with pfpl or prpl3 are inviable or show an extremely slow growth even at permissive temperature.

What is the function of Prplp/Zerlp? The prpl mutation affects a variety of intracellular processes including splicing of pre-mRNh, poly(A)+ RNA transport and cell cycle control. Time-course experiments showed that pre-mRNA splicing defect and morphological change occurred before the blockage of poly(A)+ RNA transport. It is possible that the defect in pre-mRNA splicing in prpl gives rise to defective poly(A)+ RNA transport. Judging from the sequence analysis, Prplp/ Zerlp may be a homologue of S. cerevzszae PrpGp, which is a component of the U4/U6 snRNP (probably U4 snRNP) in the spliceosome (ABOVICH et al. 1990). How- ever, we cannot conclude that S. pombe Prplp/Zerlp is a mere spliceosomal protein nor that the blockage of poly(A)+ RNA transport is a secondary effect of the pre-mRNA splicing defect, because the blockage of the poly(A)+ RNA transport could not be detected in any other fwp mutants of S. pombe (URUSHIYAMA et al. 1996). The possibility that the splicing defect causes the defect in cell cycle progression may be less likely based on the time-course experiments and the fact that zerl /prpl and cdc2 alleles interact (K OW, H. MURAKAMI and H. OKAYAMA, personal communication). A more fascinat- ing hypothesis for the function of Prplp/Zerlp is that this protein is a mediator connecting pre-mRNA splicing and cell cycle control: for example, individual TPRs of Prplp/Zerlp may play important roles in the interactions with different target proteins, either simultane- ously or individually. Further studies will be necessary to define the function of Prplp/Zerlp and the role of the TPRs in this protein. This may lead us to a better understanding of the relationship between pre-mRNA processing and cell cycle progression.

We thank K. O m , H. MURAKAMI and H. OKAYM (The OKAY- AMACell Switching Project, ERATO, JRDC.) and D. FKENDEWEY (New York University School of Medicine) for providing strains and sharing their results with us before publication as well as for valuable discussions. We also thank T. MATSUMOTO for the S. pombegenomic library, M. ROSBASH for the p p 6 strain of S. cerevisiae and PRP6 plasmid, M. YANAGIDA and K. KUMADA for the nuc2 and cut9 mutants, C. SHIMODA for the marker strains and A. K. AZAO for the ptr mutants, T. O m and members of our laboratory for valuable discussions. This research was supported by grants from the Ministry of Education, Science and Culture of Japan.

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Communicating editor: P. G. YOUNG

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