Nucleic Acids Research

This Article Abstract Print PDF (320K) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (15) Request Permissions Commercial Re-use Guidelines for Open Access NAR Content Google Scholar Articles by Dahan, O. Articles by Kupiec, M. Search for Related Content PubMed PubMed Citation Articles by Dahan, O. Articles by Kupiec, M. Social Bookmarking          What's this?

Nucleic Acids Research, 2002, Vol. 30, No. 20 4361-4370
© 2002 Oxford University Press

Mutations in genes of Saccharomyces cerevisiae encoding pre-mRNA splicing factors cause cell cycle arrest through activation of the spindle checkpoint

Orna Dahan and Martin Kupiec^*

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel

*To whom correspondence should be addressed. Tel: +972 3 640 9031; Fax: +972 3 640 9407; Email: martin{at}post.tau.ac.il

Received July 14, 2002; Revised and Accepted August 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Previous work has identified a group of genes whose products play important roles in two seemingly unrelated processes: cell cycle progression and splicing. The products of these genes show a network of physical and genetic interactions suggestive of the existence of a protein complex, the cell cycle and splicing complex (CSC). Here we analyze the genetic interactions between ISY1, SYF2 and NTC20, three non-essential components of the CSC. We show that mutations in ISY1 cause lethality in the absence of Ntc20p, and that the double mutant isy1 syf2 shows a temperature-dependent cell cycle arrest. This arrest is due to lower levels of -tubulin, a protein encoded by TUB1 and TUB3, two intron-containing genes. We show that the low levels of -tubulin in isy1 syf2 trigger activation of the spindle checkpoint, causing cell cycle arrest. Thus, our results have uncovered an unexpected role for pre-mRNA splicing in the maintenance of the fidelity of chromosome transmission during cell division.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The eukaryotic cell cycle is a collection of ordered events in which the initiation of late processes depends upon the completion of early ones. Coordination of the timing and order of these events is critical for the high-fidelity transmission of genetic information. Work in several laboratories has demonstrated that this coordination is genetically controlled by surveillance mechanisms, termed checkpoints. Checkpoints are pathways that affect cell cycle progression in response to genotoxic agents or alteration of particular structures such as the spindle (reviewed in 1,2).

Removal of introns from pre-mRNA is an important component of the regulation of gene expression in eukaryotes. Pre-mRNA splicing requires a large number of trans-acting factors, which together constitute the spliceosome (3,4). Biochemical studies have demonstrated that the spliceosome consists of a large number of specific proteins associated with functional small nuclear ribonucleoprotein particles (snRNPs) composed of small nuclear RNA molecules (U1, U2, U4, U5 and U6 snRNA). In addition, a large number of non-snRNP proteins are either transiently associated with, or constitute an integral part of, the spliceosome (3). Genetic screens have identified many proteins involved in splicing in the yeast Saccharomyces cerevisiae (5).

The CDC40/PRP17 gene encodes a protein that plays a role during the second step of the splicing reaction. Mutations in this gene result in accumulation of splicing intermediates (6). This gene was also independently found to function in the mitotic and meiotic cell cycles. At the restrictive temperature, cdc40/prp17 strains arrest as large-budded cells with one undivided nucleus containing 2N content of DNA (7,8). Thus, the CDC40/PRP17 gene is involved in two seemingly unrelated processes: pre-mRNA splicing and progression through the G₂/M phases of the cell cycle. For simplicity, we will refer to the gene as CDC40.

Previously, we have implemented a screen to identify mutants that are synthetically lethal in the absence of Cdc40p (9,10). This screen identified several genes (SYF, synthetic lethal with forty). When some of the SYF genes were used as baits in two-hybrid screens, we uncovered a complex network of interactions between a group of splicing factors (including Prp8, Slu7, Prp22, Slt11, Syf1, Syf2, Syf3/Clf1, Cef1, Isy1 and Ntc20). Most of these splicing factors are involved in cell cycle progression and, when mutated, cells arrest at the G₂/M phase of the cell cycle (9,10). Based on these results, we have suggested the existence of a protein complex [cell cycle and splicing complex (CSC)] that plays a role in both splicing andS.cerevisiae and Schizosaccharomyces pombe has confirmed the existence of protein complexes containing several of the CSC proteins (11,12).

In this paper, we investigate the genetic interactions between three non-essential components of the CSC, and we report the characterization of the double mutant isy1 syf2. Our results demonstrate that the double mutant is temperature sensitive and arrests in the cell cycle. Moreover, we show that the G₂/M arrest phenotype observed in this mutant is due to the low levels of -tubulin protein encoded by TUB1 and TUB3, two intron-containing genes. The low levels of -tubulin activate the spindle checkpoint, leading to cell cycle arrest. Our results point at an unexpected role played by pre-mRNA splicing in guarding the stability of the genome during cell division.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Strains, media, growth conditions and general procedures
The S.cerevisiae strains used in the present work are as follows. YR4, MATa ade2-1 his3-RS21 leu21 lys2-801 trp11 ura3-52 can1-100. YOD84, MATa/MAT CDC40/cdc40::URA3 SYF2/syf2::TRP1 ade2-1/ade2-101 his3-RS21/his3-RS21 leu21/leu21 lys2-801/lys2-801 trp11/trp11 ura3-52/ura3-52. YOD19, MATa/MAT CDC40/cdc40::LEU2 ISY1/isy1::HIS3 ade2-1/ade2-1 his3-RS21/his3200 leu21/leu2-3,112 LYS2/lys2-801 trp11/trp11 ura3-52/ura3-1 CAN1/can1-100. YOD7, MATa/MAT ISY1/isy1::HIS3 SYF2/syf2::TRP1 ADE2/ade2-1 his3200/his3-RS21 leu2-3,112/leu21 LYS2/lys2-801 trp11/trp11 ura3-1/ura3-52 CAN1/can1-100. YOD15, MAT isy1::HIS3 ade2-1 his3 leu2 lys2-801 trp11 ura3. YOD18, MATa syf2::TRP1 ade2-1 his3 leu2 lys2-801 trp11 ura3. YOD13, MATa isy1::HIS3 syf2::TRP1 ADE2 his3 leu2 lys2-801 trp11 ura3. YOD43, cdc40::URA3 ntc20::KanMX6 ade2-101 his3-RS21 leu21 lys2-801 trp11 ura3-52 can1-100. YOD44, MATa syf2::TRP1 ntc20::KanMX6 ade2-1 his3 leu2 lys2-801 trp11 ura3. YOD78, MATa/MAT SYF2/syf2::TRP1 ISY1/isy1::HIS3 NTC20/ntc20::kanMX6 ade2-1/ ade2-1 his3/his3 leu2/leu2 lys2-801/lys2-801 trp11/trp11 ura3/ura3. YOD96, MATa isy1::HIS3 syf2::TRP1 mad1:: KanMX6 bub2::KanMX6 his3 leu2 ura3. YOD97, MATa mad1::KanMX6 bub2::KanMX6 his3 leu2 ura3. The disruption of the coding sequence of ISY1 with HIS3, of CDC40 with either URA3 or LEU2 and the replacement of SYF2 with TRP1 have been described (9,10,13). Disruption of the entire coding sequence of NTC20 with KanMX6 was carried out using NTC20-specific primers according to the method described (14).

Yeast cells were grown in YEPD (1% yeast extract, 2% Bacto peptone, 2% dextrose) or SD media (0.67% yeast nitrogen base, 2% dextrose and the appropriate nutrients added). In order to induce expression from the GAL promoter, cells were grown on YEPGal plates (1% yeast extract, 2% Bacto peptone, 2% galactose), or SGal media (0.67% yeast nitrogen base, 2% galactose and the appropriate nutrients added). Bacto Agar (1.8%) was added for solid media. Selective media lacking one nutrient are designated SD-nutrient (e.g. SD-Ura). Ura^– colonies were selected on SD complete medium with uracil (50 mg/l) and 5-fluoroorotic acid (5-FOA, 0.8 g/l). Sporulation was carried out in SPO medium (1% potassium acetate, 0.1% yeast extract, 0.05% dextrose).

Nocodazole (Sigma, St Louis, MO) was diluted in YEPD or YEPGal agar at the concentrations indicated from a stock solution of 5 mg/ml in dimethyl sulfoxide. Standard molecular biology procedures such as restriction enzyme and Southern blot analysis were carried out as described (15). Yeast media and molecular biology procedures (transformations, DNA preparations, etc.) were done as described (16).

Cell synchronization
Exponentially growing cells were arrested in G₁ by incubation in the presence of 4 µM -factor (Sigma, St Louis, MO) for 3 h at 25°C. In order to release cells from G₁ arrest, cells were washed three times and resuspended in fresh YEPD.

Microscopic and flow cytometry analysis
To visualize nuclei, cells were fixed with 70% methanol overnight at 4°C, washed twice with phosphate-buffered saline (PBS), and resuspended in 1 µg/ml 4',6'-diamidino-2-phenylindole (DAPI) in PBS. To determine nuclear position, the distance from the edge of the nucleus to the bud neck was measured for each budded cell, as well as the diameter of the mother cell. The ratio between these two distances was calculated. A ratio of 0.4 or larger was considered to be indicative of nuclear localization failure. Most wild-type large-budded cells exhibited a ratio of 0.25 or lower. At least 200 cells were analyzed for each strain.

Anti-tubulin antibody staining was performed as described (16). For -tubulin staining we used YOL1/34 antibodies (Harlan Sera-lab Ltd). The secondary antibody was FITC-conjugated anti-rat IgG (Jackson Immunoresearch Laboratories, Inc.). Flow cytometry analysis was carried out as described (10). Cells were classified as having long spindles if the stained spindle encompassed more than two-thirds of the combined length of mother and daughter cells.

Screening for temperature sensitivity suppressors
YOD13 cells (isy1 syf2) were transformed with a cDNA expression library, regulated by the GAL1 promoter on the URA3-CEN vector pRS316 (17). The transformation mixture was plated on SGal-Ura plates at 25°C. After 12 h, cells were shifted to 35°C for 3–4 days until the appearance of colonies. Colonies were then streaked on YEPGal plates and replicated on YEPGal and 5-FOA plates at 25 and 35°C. Colonies that were able to grow on YEPGal plates at 25 and 35°C and on 5-FOA plates at 25°C but not at 35°C were further analyzed. Plasmids were extracted, re-checked for their ability to support growth at 35°C and then their cDNA insert was sequenced.

Microcolony formation assays
Exponentially growing cultures were grown at 25°C, transferred to 37°C for 2.5 h, briefly sonicated and streaked on a YEPD plate. Cells were picked using a dissection needle and placed at marked spots on the plates, which were incubated at 37°C. The number of cells in each microcolony was counted at different time points. For assessing growth in the presence of nocodazole, cells were picked and placed on YEPD plates containing 7.5 µg/ml nocodazole.

Western blot analysis
For immunoblot analysis, protein extracts were made as follows: 5 x 10⁸ cells were harvested and the pellets were washed and resuspended in lysis buffer [20% glycerol, 20 mM HEPES (pH 7.9), 20 mM NaCl, 5 mM EDTA (pH 8), 1 mM DTT]. After the addition of protease inhibitors (Complete^TM; Boehringer Mannheim, Mannheim, Germany) and glass beads, cells were disrupted by vortexing. Lysates were clarified by microcentrifugation. Proteins were fractionated by SDS–PAGE and detected using anti--tubulin antibody (YOL1/34) or anti-Vma5p, followed by peroxidase- conjugated anti-rat IgG antibody in the case of -tubulin and peroxidase-conjugated anti-guinea pig IgG antibody in the case of Vma5p. Signals were detected using ECL (NEN Life Science Products, Inc.) according to the manufacturer’s instructions, and visualized by film exposure.

RT–PCR analysis
RNA was extracted from 5 x 10⁷ cells using the RNeasy kit (Qiagen, Inc.) according to the manufacturer’s instructions. Prior to the RT–PCR step, genomic DNA was degraded by RQ1 RNase-free DNase (Promega, Inc.). Complete removal of contaminating DNA was verified by negative control PCRs with a specific set of primers. One microgram of total RNA was used as a template for cDNA synthesized using the Expand^TM Reverse Transcriptase Kit (Boehringer Mannheim) and 500 ng of oligo (dT)₁₅ as a primer. One quarter of each cDNA preparation was used as a template in a PCR using specific primers spanning the intron of either TUB1, TUB3 or RPL25.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Genetic interactions between ISY1, SYF2, NTC20 and CDC40
The SYF2 gene was previously identified in our screen for mutants unable to grow in the absence of Cdc40p. It encodes a small protein with no obvious structural motifs (9,10). ISY1 and NTC20 also encode two small proteins that lack any clear structural features. Neither gene was identified in our screen for mutations that were synthetically lethal with cdc40, but both were found as two-hybrid interactors with Syf1p and Syf3/Clf1p (9,10,13). Syf2p, Isy1p and Ntc20p form part of a large protein complex, the CSC (9,10). Although mutations in most of the genes encoding the CSC proteins cause cell cycle arrest at the G₂/M transition, single mutations in SYF2, ISY1 and NTC20 have no apparent viability problems or cell cycle phenotype. In order to study the relationship between these three genes and CDC40, we sought to create double mutant strains.

Null alleles of ISY1, SYF2 and NTC20, in which the ORFs were replaced with DNA fragments containing HIS3, TRP1 or KanMX, respectively, were created. These strains were individually crossed to cdc40::LEU2 or cdc40::URA3 strains. The double heterozygous strains were subjected to meiosis and tetrad analysis. As expected from our previous results (9,10), we were unable to obtain any syf2 cdc40 double mutant spores among 40 tetrads dissected, indicating a synthetic lethality between mutations in these genes. Similarly, no isy1 cdc40 spores were obtained in 38 tetrads analyzed. In contrast, double mutants ntc20 cdc40 were obtained in the numbers expected and grew well at 25°C, showing no synthetic phenotypes. Thus, mutations in SYF2 and ISY1, but not NTC20, are lethal in the absence of Cdc40p (Fig. 1).

View larger version (13K): [in this window] [in a new window]   Figure 1. Genetic interactions between CDC40, ISY1, SYF2 and NTC20. The following interactions were observed between full deletion mutants: s.l., synthetic lethality; t.s., temperature sensitivity; n.g., normal growth.

 
To investigate the genetic interactions between the three non-essential genes of the CSC, we carried out crosses in all possible combinations between the genes ISY1, SYF2 and NTC20. Tetrad analysis showed that isy1 is synthetically lethal in combination with ntc20: no viable double mutant spores were observed among 25 tetrads dissected. In contrast, isy1 syf2 and syf2 ntc20 double mutants were obtained at the expected numbers. Whereas the latter strain is viable at all temperatures and shows no obvious synthetic phenotypes, isy1 syf2 is temperature sensitive, being unable to grow at 37°C (Figs 1 and 2A). Below we describe a detailed analysis of the phenotypes of the isy1 syf2 strain.

View larger version (58K): [in this window] [in a new window]   Figure 2. Temperature sensitivity of the isy1 syf2 mutant and accumulation of large-budded cells at the restrictive temperature. (A) Deletion of both ISY1 and SYF2 confers a temperature-sensitive phenotype. Growth of isogenic wild-type, isy1, syf2 and isy1 syf2 strains at 25 and 37°C is shown. (B) Altered cell cycle distribution in the isy1 syf2 strain. Double mutant or wild-type cells were grown at 25°C, synchronized at the G1 stage with -factor and then released at either 25 or 37°C. After the release, samples were analyzed by flow cytometry. (C) The isy1 syf2 strain accumulates as large-budded cells at the restrictive temperature. (D) The percentages of large-budded cells in wild-type and isy1 syf2 strains incubated in either 25 or 37°C are shown. Large-budded cells were scored if the bud was greater than two-thirds of the size of the mother cell.

 
The isy1 syf2 double mutant exhibits cell cycle arrest at the non-permissive temperature
To study if the temperature sensitivity of the isy1 syf2 strain is related to defects in the cell cycle, we examined whether the cells exhibit the typical phenotype of cdc mutants at restrictive temperatures. To this end, isy1 syf2 cells were synchronized at the G₁ phase of the cell cycle with -factor and then released at either permissive (25°C) or restrictive (37°C) temperatures. At different time points, samples were analyzed by flow cytometry. Figure 2B shows that isy1 syf2 cells released from G₁ at the restrictive temperature replicated their DNA and subsequently accumulated with a 2N DNA content. Light microscopy of samples taken 3 h after release to 37°C showed that almost all the population was composed of large-budded cells (Fig. 2C and D). Staining the cells with DAPI and -tubulin antibodies demonstrated that the arrested cells had undivided nuclei and short spindles (see below). We thus conclude that isy1 syf2 cells are unable to proceed through the G₂/M stage at the restrictive temperature.

The cell cycle arrest phenotype of isy1 syf2 cells can be suppressed by TUB1 or TUB3 cDNA
Since splicing constitutes a key cellular mechanism, genes encoding splicing factors are often essential. Many temperature-sensitive splicing mutants have been isolated; most of them arrest at the restrictive temperature without any defined cell cycle morphology (10). We reasoned that the cell cycle-specific arrest of the isy1 syf2 double mutant strain might be caused by inefficient processing of a small number of intron-containing genes particularly dependent on Isy1p and Syf2p for their pre-mRNA splicing. If this were the case, we expected that introduction of the relevant target gene(s) in the form of cDNA should be sufficient to bypass the need for Isy1p and Syf2p and therefore should allow growth at the restrictive temperature. To test this hypothesis, we conducted a screen to isolate cDNAs able to suppress the temperature sensitivity of isy1 syf2 cells. Strain YOD13 (isy1 syf2) was transformed with a cDNA library and screened for cell growth at 35°C. Table 1 shows that in addition to the ISY1 and SYF2 genes, two intron-containing genes, TUB1 and TUB3, were isolated. TUB1 is the major gene encoding the -tubulin subunit in S.cerevisiae. TUB3 is a second gene encoding the -tubulin subunit. It is expressed at lower levels than TUB1. The protein products of TUB1 and TUB3 are very similar, showing 90% sequence identity (18,19). TUB1 and TUB3 cDNA showed better suppression than three other cDNAs isolated, supporting growth of isy1 syf2 cells at temperatures up to 35°C (although not at 37°C). The three additional cDNA clones (encoding MOT2, RPN5 and YOL162w) were isolated only once each and allowed only weak growth at 35°C. They encode, respectively, a general transcription factor, a proteasome subunit and an unknown ORF. Since their effect seemed relatively weak, these cDNAs were not further analyzed.

View this table: [in this window] [in a new window]   Table 1. cDNAs able to suppress the cell cycle arrest of an isy1 syf2 strain at 35°C

 
The double mutant isy1 syf2 exhibits sensitivity to the microtubule-disrupting agent nocodazole
Increased sensitivity of cells to anti-tubulin drugs is a common phenotype of tubulin mutants (18,19). To establish whether the phenotypes of the isy1 syf2 mutant were related to tubulin, we checked the ability of the double mutant cells to grow in the presence of increasing nocodazole concentrations. As shown in Figure 3, at a semi-restrictive temperature (30°C), the isy1 syf2 double mutant displays increased sensitivity to nocodazole relative to single mutants or to the wild-type control. Such sensitivity could not be seen at the permissive temperature. Figure 3B also shows that nocodazole sensitivity was completely suppressed by TUB1 or TUB3 cDNA expression. The fact that both the temperature sensitivity and nocodazole sensitivity of isy1 syf2 cells can be suppressed by TUB1 or TUB3 cDNA suggests a common cause for these phenotypes.

View larger version (32K): [in this window] [in a new window]   Figure 3. The isy1 syf2 strain is sensitive to nocodazole. (A) Nocodazole sensitivity of wild-type, isy1, syf2 and isy1 syf2 strains. Serial dilutions of cells were spotted onto YEPD plates containing 0 or 7.5 µg/ml nocodazole and incubated at the permissive (25°C) and semi-permissive temperature (30°C). (B) Suppression of the nocodazole sensitivity by expression of TUB1 and TUB3 cDNA. isy1 syf2 cells carrying ISY1, TUB1 or TUB3 cDNA under the inducible GAL1 promoter, or the vector alone (pRS316) were spotted onto YEPGal plates containing 0 or 7.5 µg/ml nocodazole at the semi-permissive temperature (30°C).

 
Isy1p and Syf2p are required for the maintenance of the mitotic spindle
In S.cerevisiae, spindle pole body duplication and spindle assembly take place concomitantly with initiation of DNA replication (20). Thus, during the S, G₂ and early M phases, a short spindle is observed, whereas spindle elongation occurs during anaphase. Since TUB1 and TUB3 encode the -tubulin subunit, which is a major component of the mitotic spindle, we considered the possibility that lower expression of TUB1 and TUB3 in the isy1 syf2 double mutant strain incubated at the restrictive temperature may disrupt the mitotic spindle and therefore cause cell cycle arrest. To test this hypothesis, we carried out a careful analysis of the spindle length of isy1 syf2 cells at the permissive and non-permissive temperatures. The cells were grown at 25°C and then shifted to the restrictive temperature (37°C). At different time points samples were taken for tubulin staining by indirect immunofluorescence with anti--tubulin antibodies. At the permissive temperature, the isy1 syf2 cells contained spindles of all sizes, as did the wild-type control. However, after 2.5 h of incubation at the non-permissive temperature, mutant cells exhibited either a short spindle or a dot of -tubulin material. Following a longer incubation at 37°C, the mitotic spindle eventually disappeared, and after 5 h incubation at the non-permissive temperature almost no staining was detected (Fig. 4). These results indicate that Isy1p and Syf2p are required for maintenance of the mitotic spindle.

View larger version (39K): [in this window] [in a new window]   Figure 4. The isy1 syf2 mutant cells arrest at G2/M with unstable microtubules. (A) Asynchronous cultures of wild-type or isy1 syf2 cells were shifted to the restrictive temperature and stained for microtubules using an anti--tubulin antibody followed by a FITC-conjugated secondary antibody. The same samples were also examined by Nomarski microscopy. (B) Graphic presentation of spindle morphology in isy1 syf2 cells incubated at the restrictive temperature. Percent of cells with long or short spindle, or without anti-tubulin signal, are shown.

 
Nuclear migration phenotypes of isy1 syf2 mutant cells
In wild-type cells, nuclear migration takes place early in the cell cycle. Therefore, in large-budded cells the nucleus is almost always seen near, or extended through, the bud neck. This process of nuclear migration to the bud neck requires extranuclear microtubules (21). In tubulin mutants, the nucleus does not divide; moreover, some tubulin mutants also exhibit defects in nuclear migration (22). To test the effect of Isy1 and Syf2 on these tubulin-dependent events, we followed nuclear migration and nuclear division in isy1 syf2 mutant and in wild-type cells. As we have shown (Fig. 2C and D), the isy1 syf2 strain accumulated large-budded cells when incubated at the non-permissive temperature. DAPI staining showed that more than 95% of these cells contained one undivided nucleus. Under these conditions, a significant nuclear migration defect is observed: in 50% of the arrested cells the nucleus is located away from the bud neck (Fig. 5). This number may be an underestimate, as a very conservative classification limit was used: only cells in which the distance between the edge of the nucleus to the neck was at least 40% of the diameter of the mother cells were classified as having a nucleus away from the neck. Similar defects, although less profound, can be seen in isy1 syf2 cells incubated at the permissive temperature. This is in stark contrast to the wild-type control cells, in which more than 95% of the large-budded cells had the nucleus localized to the bud neck (Fig. 5B). We therefore conclude that isy1 syf2 cells are defective in nuclear migration.

View larger version (31K): [in this window] [in a new window]   Figure 5. isy1 syf2 cells are defective in both nuclear division and nuclear migration. (A) The isy1 syf2 cells were grown at 37°C, DAPI stained and examined under the microscope by UV light. As a comparison, ISY1 SYF2 cells grown at 37°C are shown. (B) The percentage of large- budded cells with one nucleus away from its position near the bud neck was determined in both isy1 syf2 and wild-type cells after 3 h of incubation at 25 or 37°C.

 
Efficient splicing of TUB1 and TUB3 requires ISY1 and SYF2
To test the possibility that the efficient splicing of TUB1 and TUB3 pre-mRNA is dependent on Isy1p and Syf2p, we examined the splicing efficiency of the TUB1 and TUB3 pre-mRNA by RT–PCR analysis. Total RNA from isy1 syf2 and wild-type strains was isolated before and after the cells were shifted to the non-permissive temperature. RNA was subjected to RT–PCR analysis using primers specific to either TUB1, TUB3 or, as a control, to the intron-containing ribosomal gene, RPL25. As shown in Figure 6, when cells deleted for ISY1 and SYF2 were incubated at the non-permissive temperature, an accumulation of unspliced TUB1 and TUB3 was observed. Such an accumulation was not seen in cells incubated at the permissive temperature, or in wild-type cells at either temperature. No pre-mRNA accumulation was seen using primers specific to the ribosomal gene RPL25 (data not shown). These results indicate that the splicing of TUB1 and TUB3 pre-mRNA is highly dependent on Isy1 and Syf2 proteins.

View larger version (30K): [in this window] [in a new window]   Figure 6. Isy1p and Syf2p are required for efficient splicing of the TUB1 and TUB3 introns. RT–PCR analysis of isy1 syf2 and wild-type cells using TUB1- or TUB3-specific oligonucleotides spanning the intron. The level of products for spliced and unspliced transcripts of TUB1 and TUB3 was quantified, after gel electrophoresis, using the program NIHimage (v. 1.62). For each strain/temperature, the ratio unspliced/spliced is shown.

 
isy1 syf2 cells have less -tubulin protein at the restrictive temperature
The disappearance of the mitotic spindle observed in double mutant isy1 syf2 cells at elevated temperatures might be due to a reduction in -tubulin protein levels caused by defects in TUB1 and TUB3 pre-mRNA splicing. We therefore measured directly the levels of -tubulin by immunoblotting. As a control we measured the amount of Vma5p, the c subunit of the vacuolar H (+) ATPase, which is also encoded by an intron-containing gene. As shown in Figure 7, -tubulin protein levels decreased rapidly when isy1 syf2 cells were incubated at the non-permissive temperature. In contrast, constant amounts of -tubulin were detected in isy1 syf2 cells incubated at the permissive temperature or in wild-type cells. The reduction in -tubulin levels is not due to a general decrease in protein stability in isy1 syf2 cells at the non-permissive temperature, since the level of other proteins (such as Vma5) remained constant under all conditions (Fig. 7).

View larger version (42K): [in this window] [in a new window]   Figure 7. The level of -tubulin is reduced in isy1 syf2 mutant cells incubated at 37°C. (A) Asynchronous cultures of wild-type or isy1 syf2 strains were incubated at either 25 or 37°C. At different time points total protein was extracted and subjected to western blot analysis using anti- -tubulin antibody. As a control, anti-Vma5p antibody was used. (B) Quantitative analysis of -tubulin in isy1 syf2 cells. The western blots shown in (A) were quantified using the program NIHimage (v. 1.62) and the calculated ratio -tubulin/Vma5 is shown.

 
The cell cycle arrest of isy1 syf2 strains is mediated by the spindle checkpoint
When the mitotic spindles are defective (due to the action of drugs or to mutated spindle components) cells delay the onset of chromosome segregation by activating the spindle checkpoint (reviewed in 2). This mechanism is dependent on the activity of two groups of genes, the mad genes (23) and the bub genes (24). Cell cycle arrest is achieved by inhibiting the anaphase-promoting complex-dependent degradation of specific proteins. Sister chromatid separation requires proteolysis of Pds1p (25), whereas assembly of replication-competent complexes on origins (to ensure DNA replication in the next S-phase) requires degradation of B-type cyclins, such as Clb2 (26). Recent work has shown that prevention of Pds1 degradation depends on the Mad1, Mad2, Mad3 and Bub1 checkpoint proteins, whereas the prevention of Clb2 proteolysis is mediated by Bub2 (27). Since mutations in genes encoding tubulin activate the spindle checkpoint (28), we tested whether the inefficient splicing of TUB1 and TUB3 transcripts causes cell cycle arrest by activating this control mechanism. We reasoned that if that were the case, mutations that inactivate the spindle checkpoint would prevent cell cycle arrest and would lead to catastrophic cell divisions and to cell death. We therefore created isy1 syf2 strains in which the MAD1 and BUB2 genes were deleted.

The isy1 syf2 double mutant strain and the quadruple isy1 syf2 mad1 bub2 mutant strain were grown to mid-logarithmic phase, and were then transferred to the non-permissive temperature. Individual cells were monitored (under the microscope) for their ability to undergo cell cycle arrest. Figure 8 shows that whereas the double mutant arrested as large-budded cells in response to the restrictive temperature, the quadruple mutant continued to divide and created microcolonies that did not continue to develop (Fig. 8). Although the quadruple isy1 syf2 mad1 bub2 mutant strain was able to undergo several divisions in non-permissive conditions, the cells rapidly lost viability, as can be seen by their reduced ability to recover when returned to permissive conditions (Fig. 8C). Similar results were observed when tubulin was depolarized by nocodazole: whereas the wild-type strain arrested for several hours as large-budded cells, the mad1 bub2 mutant strain failed to arrest and produced inviable microcolonies (Fig. 8B).

View larger version (31K): [in this window] [in a new window]   Figure 8. Quantification of cell cycle arrest and survival of mad1 bub2 deleted strains. (A) Double mutant isy1 syf2 and quadruple mutant isy1 syf2 mad1 bub2 cells were grown at 37°C for 2.5 h, spotted on YEPD plates and incubated at 37°C. At different time points the cells were examined by light microscopy and the number of cell divisions in each microcolony was determined. (B) Wild-type MAD1 BUB2 and double mutant mad1 bub2 cells were placed on YEPD plates containing 7.5 µg/ml nocodazole, and incubated at 30°C. At different time points the cells were examined by light microscopy and the number of cell divisions in each microcolony was determined. (C) Double mutant isy1 syf2 and quadruple mutant isy1 syf2 mad1 bub2 cells were grown at 37°C, at timely intervals samples were taken, briefly sonicated, diluted and spotted onto YEPD plates. Colonies were counted after 3 days of incubation at 25°C.

 
These results indicate that the absence of the Isy1 and Syf2 proteins leads to reduced tubulin levels, which trigger cell cycle arrest by activating the spindle checkpoint.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell cycle progression and pre-mRNA splicing are two apparently independent processes in eukaryotic cells. However, a growing body of evidence has recently accumulated indicating that these processes may be connected. The CDC40/PRP17 gene, for example, encodes a protein essential for G₂/M transition (8), which also plays a role in splicing (29–31). Using synthetic lethal and two-hybrid screens we have previously identified a putative protein complex that genetically interacts with Cdc40p (i.e. CSC). The CSC contains proteins involved in both cell cycle progression and in splicing (9,10,13). Most of the genes encoding proteins of this complex are essential; however, the CSC also contains (at least) three proteins encoded by non-essential genes: ISY1, SYF2 and NTC20. In this paper we have dissected the genetic relationships between these genes, which exhibit a complex set of interactions. While deletion of ISY1 is lethal in the absence of NTC20 and is temperature sensitive in combination with a deletion of SYF2, the syf2 ntc20 double mutant shows no viability or growth defect (Fig. 1).

The nature of the interactions observed between the CSC genes is still under investigation; currently the results could be interpreted in two alternative ways. (i) The three proteins could be part of one large complex with a role in both cell cycle progression and pre-mRNA splicing. The different gene interactions observed among the non-essential members of this complex could be due to different effects on the stability or function of the complex. For example, it is possible that for the protein complex to be fully stable/active, only the Isy1 protein is needed. In its absence, however, Ntc20p can provide some degree of stability, such that an isy1 syf2 strain is only temperature sensitive, and not completely lethal. In contrast, Syf2p alone is insufficient for CSC stability and thus the isy1 ntc20 double mutant is inviable. (ii) An alternative view posits that the elaborate interactions observed are due to the existence of several alternative protein complexes with a similar core, each containing different non-essential proteins. These complexes may act redundantly or together as mediators that link splicing to cell cycle progression. For example, functional complexes may exist in the cell, some carrying Isy1p, some carrying Ntc20p. In the absence of both proteins, no active complex can be formed, and the cells die.

The nature of the connection between CDC40 and the group of interacting proteins is still unclear. We were unable to detect physical or two-hybrid interactions between Cdc40p and any of the CSC proteins. Furthermore, protein complexes similar to the CSC have been recently purified biochemically from S.cerevisiae (11,32) and S.pombe (12). In neither case was Cdc40p found among the proteins isolated. On the other hand, in recent studies of a large-scale analysis of protein complexes of yeast, Cdc40p was identified in association with some (but not all) of the complexes precipitated with antibodies directed against members of the CSC (33,34). This may indicate that Cdc40p is loosely associated with some of the CSC components. Thus, Cdc40p may act alone, or in conjunction with other proteins, to affect the same cellular activity as the CSC. Alternatively, Cdc40 may associate transiently with the CSC, to facilitate the access or activity of other proteins. This process could also take place in the absence of Cdc40, provided Syf2 or Isy1 are functional.

When shifted to the restrictive temperature, the isy1 syf2 double mutant cells arrest as large-budded cells with a short spindle. We isolated TUB1 and TUB3 cDNAs as suppressors of the cell cycle arrest of isy1 syf2. RT–PCR analysis showed that isy1 syf2 cells incubated at the restrictive temperature accumulate TUB1 and TUB3 pre-mRNA at a higher level than other pre-mRNAs, indicating that the dependence on Isy1p and Syf2p for the removal of the TUB1 and TUB3 introns is specific. The TUB1 and TUB3 introns may contain unique features that make their pre-mRNA splicing highly dependent on the CSC. Dix et al. (13) have previously shown that Isy1p is required in vivo for the splicing of introns carrying suboptimal consensus splicing sequences. The need for specific splicing factors for the efficient splicing of a subset of genes is a well known phenomenon. For example, the splicing factor Slu7 is required for the efficient splicing of introns in which the distance between the 3'-splice site and the branch point is larger than 7 bp (35). The fact that TUB1 and TUB3 cDNA support growth of isy1 syf2 cells up to 35°C, but not at 37°C, indicates that under these conditions another transcript(s) becomes rate limiting. This putative transcript may contain introns that share some characteristics with those of TUB1 and TUB3.

Microtubules are ubiquitous cytoskeletal structures, consisting of - and ß-tubulin hetrodimers (36). In S.cerevisiae, as in all eukaryotic cells, microtubules are necessary for chromosome movement. Unlike most other eukaryotes, however, in yeast the only other functions known to depend on microtubules are the movement of nuclei to the bud neck before mitosis and the fusion of nuclei after mating (21,37). Most tub1 and tub3 mutants show several common phenotypes such as supersensitivity to spindle disrupting agents, accumulation of large-budded cells, and defects in nuclear division, nuclear migration or both (18,19,22,38). We have shown that the double mutant isy1 syf2 cells exhibit similar phenotypes when incubated at non-permissive or semi-permissive conditions. The sensitivity to nocodazole, as well as the cell cycle arrest, could be fully suppressed by the presence of intronless -tubulin cDNA, indicating that the phenotypes observed are truly due to lack of -tubulin protein. Furthermore, the syf2 ntc20 strain, which grows well at all temperatures, was not sensitive to nocodazole (data not shown). These results strongly imply that the nocodazole sensitivity phenotype of isy1 syf2 cells is connected to the cell cycle arrest phenotype.

The mechanism that links cell cycle progression and splicing exhibits an exquisite sensitivity to the level of pre-mRNA splicing. Our RT–PCR results show a 3-fold increase in accumulation of TUB1 and TUB3 pre-mRNAs in isy1 syf2 cells at the restrictive temperature (Fig. 6). Although splicing of the TUB1 and TUB3 transcripts is only reduced and not eliminated, a strong decrease in the levels of -tubulin is seen. It is not yet clear why accumulation of unspliced transcripts has such a strong effect on the level of expression. Unspliced intermediates may exert their negative effect by affecting RNA stability or the efficiency of translation. Likewise, -tubulin levels, as detected by immunoblot experiments, are strongly reduced; the protein, however, is not totally eliminated. It has been well established that the ratio between - and ß-tubulin is of crucial importance for microtubule stability in yeast. Microtubule function is sensitive to very small changes in tubulin stoichiometry. Several reports have demonstrated that gene dosages of ß-tubulin in excess of -tubulin, or overexpression of ß-tubulin, are not tolerated and result in rapid depolymerization of normal microtubules and loss of viability (39,40). Thus, the small effect in splicing of the -tubulin-encoding genes is amplified, resulting in a strong destabilization of the microtubules.

The spindle checkpoint of S.cerevisiae is able to sense a multitude of spindle defects and halt cell cycle progression before the exit from mitosis. The genes in this pathway are necessary for the cell cycle arrest phenotype observed in the presence of spindle-depolarizing drugs: spindle checkpoint mutants fail to arrest in the presence of benomyl or nocodazole (Fig. 8B) (23,24,41). We have shown that the cell cycle arrest of isy1 syf2 cells as a consequence of inefficient TUB1 and TUB3 splicing is not direct (i.e. the cells do not arrest with a short spindle simply because they are unable to elongate it in order to proceed in the cell cycle). Instead, the defect in microtubule structure is detected by the spindle checkpoint, which initiates a cascade of events leading to cell cycle arrest. In contrast to the isy1 syf2 strain, the quadruple isy1 syf2 mad1 bub2 mutant (defective in the two branches of the spindle checkpoint) continues to divide at the non-permissive conditions in the presence of damaged spindles resulting in loss of viability (Fig. 8). Our results thus show that pre-mRNA splicing plays an important role in promoting the fidelity of chromosome segregation and cell cycle progression. While this work was in progress, Burns et al. (42) reported that TUB1 pre-mRNA splicing is also defective in temperature-sensitive cef1 mutants. Similar to results presented here, TUB1 cDNA could also suppress the cell cycle arrest of these mutants. These results further support our findings, since Cef1 is one of the components of the CSC. It is very likely that defects in CEF1 lead to cell cycle arrest by activating the spindle checkpoint, as we have shown here for Isy1 and Syf2.

Maintaining genome stability is essential for all eukaryotes. The survival of cells and organisms requires accurate chromosomal segregation. The cell has developed several regulatory networks to maintain genome stability. Recent studies have identified some CSC components as important for sister chromatid separation and segregation (41,43). Thus, one of the possible roles of the CSC could be to ensure proper chromosome segregation by linking pre-mRNA splicing to spindle integrity.

The mechanism unveiled here is probably evolutionarily conserved. Orthologs for most of the CSC proteins exist in other eukaryotes, and mutations in many splicing mutants in S.pombe cause cell cycle arrest (44,45). Interestingly, a recent report (45) has shown that mutations in some genes encoding splicing factors in S.pombe can lead to a reduction in -tubulin levels and to defects in microtubule integrity. The splicing defects result in unequal chromosome separation and in polyploidy. These findings suggest that the link between pre-mRNA splicing and spindle integrity in both S.cerevisiae and S.pombe may represent a common mechanism in eukaryotes.

Are all splicing factors required for maintaining the stability of the genome? As mentioned above, mutants defective in several components of the CSC cause cell cycle arrest by a similar mechanism. Cells lacking the Cdc40 protein also arrest at the G₂/M transition upon the shift to the restrictive temperature. As in the case of isy1 syf2, prolonged incubation at the restrictive conditions induces spindle depolymerization (8). However, unlike the isy1 syf2 strain, introduction of TUB1 or TUB3 cDNA does not suppress the temperature-sensitive phenotype of cdc40 cells (data not shown). Furthermore, TUB1 and TUB3 pre-mRNA do not accumulate in the cdc40 strain (data not shown) (46). These results suggest that the CSC and CDC40 may be differentially required for the efficient splicing of two subsets of transcripts involved in cell cycle progression. Control of cell cycle progression via pre-mRNA splicing appears to be a complex process involving several regulatory pathways.

	ACKNOWLEDGEMENTS

 
We thank Anat Krauskopf and all the members of the Kupiec Laboratory for encouragement and useful discussions. This work was supported by grants to M.K. by the AICR, the ICRF and the Israeli Cancer Association. O.D. was a recipient of a travel scholarship provided by the Constantiner Institute for Molecular Genetics.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Elledge,S.L. (1996) Cell cycle checkpoints: preventing an identity crisis. Science, 274, 1664–1672.[Abstract/Free Full Text]

2. Rudner,A.D. and Murray,A.W. (1996) The spindle assembly checkpoint. Curr. Opin. Cell Biol., 8, 773–780.[Web of Science][Medline]

3. Madhani,H.D. and Guthrie,C. (1994) Dynamic RNA–RNA interactions in the spliceosome. Annu. Rev. Genet., 28, 1–26.[Medline]

4. Will,C.L. and Luhrmann,R. (1997) Protein functions in pre-mRNA splicing. Curr. Opin. Cell Biol., 9, 320–328.[Web of Science][Medline]

5. Beggs,J.D. (1995) Yeast splicing factors and genetic strategies for their analysis. In Lamond,R.G. (ed.), Pre-mRNA Processing. Landes Company, Austin, TX, pp. 79–95.

6. Vijayraghavan,U., Company,M. and Abelson,J. (1989) Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev., 3, 1206–1216.[Abstract/Free Full Text]

7. Kassir,Y. and Simchen,G. (1978) Meiotic recombination and DNA synthesis in a new cell cycle mutant of Saccharomyces cerevisiae. Genetics, 90, 49–58.[Abstract/Free Full Text]

8. Vaisman,N., Tzoulade,A., Robzyk,K., Ben-Yehuda,S., Kupiec,M. and Kassir,Y. (1995) The role of Saccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance. Mol. Gen. Genet., 277, 123–136.

9. Ben-Yehuda,S., Dix,I., Russell,C.S., McGarvey,M., Beggs,J.D. and Kupiec,M. (2000) Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae. Genetics, 156, 1503–1517.[Abstract/Free Full Text]

10. Russell,C.S., Ben-Yehuda,S., Dix,I., Kupiec,M. and Beggs,J.D. (2000) Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae. RNA, 6, 1565–1572.[Abstract]

11. Chen,C.H., Tsai,W.Y., Chen,H.R., Wang,C.H. and Cheng,S.C. (2001) Identification and characterization of two novel components of the Prp19p-associated complex, Ntc30p and Ntc20p. J. Biol. Chem., 276, 488–494.[Abstract/Free Full Text]

12. McDonald,W.H., Ohi,R., Smelkova,N., Frendewey,D. and Gould,K.L. (1999) Myb-related fission yeast cdc5p is a component of a 40S snRNP containing complex and is essential for pre-mRNA splicing. Mol. Cell. Biol., 19, 5352–5362.[Abstract/Free Full Text]

13. Dix,I., Russell,C.S., Ben-Yehuda,S., Kupiec,M. and Beggs,J.D. (1999) The identification and characterisation of a noval splicing protein, Isy1p, of Saccharomyces cerevisiae. RNA, 5, 360–368.[Abstract]

14. Longtine,M.S., McKenzie,A.,III, Demarini,D.J., Shah,N.G., Wach,A., Brachat,A., Philippsen,P. and Pringle,J.R. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast, 14, 953–961.[Web of Science][Medline]

15. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory, Plainview, NY.

16. Rose,M., Winston,F. and Hieter,P. (1990) Methods in Yeast Genetics. A Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

17. Liu,H., Krizek,J. and Bretscher,A. (1992) Construction of GAL1-regulated yeast cDNA expression library and its application to the identification of genes whose overexpression causes lethality in yeast. Genetics, 132, 665–673.[Abstract]

18. Schatz,J.P., Pillus,L., Grisafi,P., Solomon,F. and Botstein,D. (1986) Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins. Mol. Cell. Biol., 6, 3711–4059.[Abstract/Free Full Text]

19. Schatz,J.P., Solomon,F. and Botstein,D. (1986) Genetically essential and nonessential -tubulin genes specify functionally interchangeable proteins. Mol. Cell Biol., 6, 3722–3733.[Abstract/Free Full Text]

20. Pringle,J. and Hartwell,L.H. (1981) The Saccharomyces cerevisiae cell cycle. In Strathern,J.N., Jones,E.W. and Broach,J.R. (eds), The Molecular Biology of the Yeast Saccharomyces cerevisiae: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 97–142.

21. Huffaker,T.C., Thomas,J.H. and Botstein,D. (1988) Diverse effect of beta-tubulin mutations on microtubule formation and function. J. Cell Biol., 106, 1997–2010.[Abstract/Free Full Text]

22. Richards,K.L., Anders,K.R., Nogales,E., Schwartz,K., Downing,K.H. and Botstein,D. (2000) Structure–function relationships in yeast tubulins. Mol. Biol. Cell, 11, 1887–1903.[Abstract/Free Full Text]

23. Li,R. and Murray,A.W. (1991) Feedback control of mitosis in budding yeast. Cell, 66, 519–531.[Web of Science][Medline]

24. Hoyt,M.A., Totis,L. and Roberts,B.T. (1991) S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell, 66, 507–517.[Web of Science][Medline]

25. Cohen-Fix,O., Peters,J.M., Kirschner,M.W. and Koshland,D. (1996) Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev., 15, 3081–3093.

26. Nasmyth,K. (1996) At the heart of the budding yeast cell cycle. Trends Genet., 12, 405–412.[Web of Science][Medline]

27. Alexandru,G., Zachariae,W., Schleiffer,A. and Nasmyth,K. (1999) Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage. EMBO J., 18, 2707–2721.[Web of Science][Medline]

28. Hardwick,K.G., Li,R., Mistrot,C., Chen,R., Dann,P., Rudner,A. and Murray,A.W. (1999) Lesions in many different spindle components activate the spindle checkpoint in the budding yeast Saccharomyces cerevisiae. Genetics, 152, 509–518.[Abstract/Free Full Text]

29. Ben-Yehuda,S., Dix,I., Russell,C.S., Levy,S., Beggs,J.D. and Kupiec,M. (1998) Identification and functional analysis of hPRP17, the human homologue of the PRP17/CDC40 yeast gene involved in splicing and cell cycle control. RNA, 4, 1304–1312.[Abstract]

30. Ben-Yehuda,S., Russell,C.S., Dix,I., Beggs,J.D. and Kupiec,M. (2000) Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression. Genetics, 154, 61–71.[Abstract/Free Full Text]

31. Boger-Nadjar,E., Vaisman,N., Ben-Yehuda,S., Kassir,Y. and Kupiec,M. (1998) Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing. Mol. Gen. Genet., 260, 232–241.[Web of Science][Medline]

32. Stevens,S.W., Ryan,D.E., Ge,H.Y., Moore,R.E., Young,M.K., Lee,T.D. and Abelson,J. (2002) Composition and functional characterization of the yeast spliceosomal penta-snRNP. Mol. Cell, 9, 31–44.[Web of Science][Medline]

33. Gavin,A.C., Bosche,M., Krause,R., Grandi,P., Marzioch,M., Bauer,A., Schultz,J., Rick,J.M., Michon,A.M., Cruciat,C.M., Remor,M., Hofert,C., Schelder,M., Brajenovic,M., Ruffner,H., Merino,A., Klein,M., Hudak,M., Dickson,D., Rudi,T., Gnau,V., Bauch,A., Bastuck,S., Huhse,B., Leutwein,C., Heurtier,M.A., Copley,R.R., Edelmann,A., Querfurth,E., Rybin,V., Drewes,G., Raida,M., Bouwmeester,T., Bork,P., Seraphin,B., Kuster,B., Neubauer,G. and Superti-Furga,G. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature, 415, 141–147.[Medline]

34. Ho,Y., Gruhler,A., Heilbut,A., Bader,G.D., Moore,L., Adams,S.L., Millar,A., Taylor,P., Bennett,K., Boutilier,K., Yang,L., Wolting,C., Donaldson,I., Schandorff,S., Shewnarane,J., Vo,M., Taggart,J., Goudreault,M., Muskat,B., Alfarano,C., Dewar,D., Lin,Z., Michalickova,K., Willems,A.R., Sassi,H., Nielsen,P.A., Rasmussen,K.J., Andersen,J.R., Johansen,L.E., Hansen,L.H., Jespersen,H., Podtelejnikov,A., Nielsen,E., Crawford,J., Poulsen,V., Sorensen,B.D., Matthiesen,J., Hendrickson,R.C., Gleeson,F., Pawson,T., Moran,M.F., Durocher,D., Mann,M., Hogue,C.W., Figeys,D. and Tyers,M. (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature, 415, 180–183.[Medline]

35. Brys,A. and Schwer,B. (1996) Requirements for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3' splice site. RNA, 2, 707–717.[Abstract]

36. Hyams,J.S. and Lloyd,C.W. (1994) Microtubules. In Harford,J.B. (ed.), Modern Cell Biology. Wiley-Liss, New York, Vol. 13, pp. 1–439.

37. Jacobs,C.W., Adams,A.E., Szaniszlo,P.J. and Pringle,I.R. (1988) Functions of microtubules in the Saccharomyces cerevisiae cell cycle. J. Cell Biol., 107, 1409–1426.[Abstract/Free Full Text]

38. Neff,N.F., Thomas,I.H., Grisafi,P. and Botstein,D. (1983) Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo. Cell, 33, 211–219.[Web of Science][Medline]

39. Burke,D., Gasdaska,P. and Hartwell,L. (1989) Dominant effects of tubulin overexpression in Saccharomyces cerevisiae. Mol. Cell. Biol., 9, 1049–1059.[Abstract/Free Full Text]

40. Weinstein,B. and Solomon,F. (1990) Phenotypic consequence of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin. Mol. Cell. Biol., 10, 5295–5304.[Abstract/Free Full Text]

41. Hwang,L.H. and Murray,A.W. (1997) A novel yeast screen for mitotic arrest mutants identifies DOC1, a new gene involved in cyclin proteolysis. Mol. Biol. Cell., 8, 1877–1887.[Abstract/Free Full Text]

42. Burns,C.G., Ohi,R., Mehta,S., O’Toole,E.T., Winey,M., Clark,T.A., Sugnet,C.W., Ares,M.,Jr and Gould,K.L. (2002) Removal of a single alpha-tubulin gene intron suppresses cell cycle arrest phenotypes of splicing factor mutations in Saccharomyces cerevisiae. Mol. Cell. Biol., 22, 801–815.[Abstract/Free Full Text]

43. Biggins,S., Bhalla,N., Chang,A., Smith,D.L. and Murray,A.W. (2001) Genes involved in sister chromatid separation and segregation in the budding yeast Saccharomyces cerevisiae. Genetics, 159, 453–470.[Abstract/Free Full Text]

44. Habara,Y., Urushiyama,S., Shibuya,T., Ohshima,Y. and Tani,T. (2001) Mutation in the prp12+ gene encoding a homolog of SAP130/SF3b130 causes differential inhibition of pre-mRNA splicing and arrest of cell-cycle progression in Schizosaccharomyces pombe. RNA, 7, 671–681.[Abstract]

45. Ochotorena,I.L., Hirata,D., Kominami,K., Potashkin,J., Sahin,F., Wentz-Hunter,K., Gould,K.L., Sato,K., Yoshida,Y., Vardy,L. and Toda,T. (2001) Conserved Wat1/Pop3 WD-repeat protein of fission yeast secures genome stability through microtubule integrity and may be involved in mRNA maturation. J. Cell Sci., 114, 2911–2920.[Medline]

46. Clark,T.A., Sugnet,C.W. and Ares,M.,Jr (2002) Genomewide analysis of mRNA processing in yeast using splicing-specific microarrays. Science, 296, 907–910.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				P.-C. Chu, T.-Y. Wang, Y.-T. Lu, C.-K. Chou, Y.-C. Yang, and M.-S. Chang Involvement of p29 in DNA damage responses and Fanconi anemia pathway Carcinogenesis, October 1, 2009; 30(10): 1710 - 1716. [Abstract] [Full Text] [PDF]

				K.-J. Chang, H.-C. Chen, and S.-C. Cheng Ntc90 is required for recruiting first step factor Yju2 but not for spliceosome activation RNA, September 1, 2009; 15(9): 1729 - 1739. [Abstract] [Full Text] [PDF]

				M. Hebeisen, J. Drysdale, and R. Roy Suppressors of the cdc-25.1(gf)-associated intestinal hyperplasia reveal important maternal roles for prp-8 and a subset of splicing factors in C. elegans RNA, December 1, 2008; 14(12): 2618 - 2633. [Abstract] [Full Text] [PDF]

				I. Kuraoka, S. Ito, T. Wada, M. Hayashida, L. Lee, M. Saijo, Y. Nakatsu, M. Matsumoto, T. Matsunaga, H. Handa, et al. Isolation of XAB2 Complex Involved in Pre-mRNA Splicing, Transcription, and Transcription-coupled Repair J. Biol. Chem., January 11, 2008; 283(2): 940 - 950. [Abstract] [Full Text] [PDF]

				M. Shimada, C. Namikawa-Yamada, M. Nakanishi, and H. Murakami Regulation of Cdc2p and Cdc13p Is Required for Cell Cycle Arrest Induced by Defective RNA Splicing in Fission Yeast J. Biol. Chem., September 23, 2005; 280(38): 32640 - 32648. [Abstract] [Full Text] [PDF]

				R. J. GRAINGER and J. D. BEGGS Prp8 protein: At the heart of the spliceosome RNA, May 1, 2005; 11(5): 533 - 557. [Abstract] [Full Text] [PDF]

				S.-W. Wang, K. Asakawa, T. Z. Win, T. Toda, and C. J. Norbury Inactivation of the Pre-mRNA Cleavage and Polyadenylation Factor Pfs2 in Fission Yeast Causes Lethal Cell Cycle Defects Mol. Cell. Biol., March 15, 2005; 25(6): 2288 - 2296. [Abstract] [Full Text] [PDF]

				O. Dahan and M. Kupiec The Saccharomyces cerevisiae gene CDC40/PRP17 controls cell cycle progression through splicing of the ANC1 gene Nucleic Acids Res., May 7, 2004; 32(8): 2529 - 2540. [Abstract] [Full Text] [PDF]

				G. Chawla, A. K. Sapra, U. Surana, and U. Vijayraghavan Dependence of pre-mRNA introns on PRP17, a non-essential splicing factor: implications for efficient progression through cell cycle transitions Nucleic Acids Res., May 1, 2003; 31(9): 2333 - 2343. [Abstract] [Full Text] [PDF]

This Article Abstract Print PDF (320K) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (15) Request Permissions Commercial Re-use Guidelines for Open Access NAR Content Google Scholar Articles by Dahan, O. Articles by Kupiec, M. Search for Related Content PubMed PubMed Citation Articles by Dahan, O. Articles by Kupiec, M. Social Bookmarking          What's this?

Online ISSN 1362-4962 - Print ISSN 0305-1048

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics