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Vol. 15, Issue 11, 4960-4970, November 2004

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Schizosaccharomyces pombe RanGAP Homolog, SpRna1, Is Required for Centromeric Silencing and Chromosome Segregation

Ayumi Kusano ^*, Tomoko Yoshioka, Hitoshi Nishijima, Hideo Nishitani, and Takeharu Nishimoto 

Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Submitted January 28, 2004; Revised July 22, 2004; Accepted August 3, 2004
Monitoring Editor: Tony Hunter

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We isolated 11 independent temperature-sensitive (ts) mutants of Schizosaccharomyces pombe RanGAP, SpRna1 that have several amino acid changes in the conserved domains of RanGAP. Resulting Sprna1^ts showed a strong defect in mitotic chromosome segregation, but did not in nucleocytoplasmic transport and microtubule formation. In addition to Sprna1⁺ and Spksp1⁺, the clr4⁺ (histone H3-K9 methyltransferase), the S. pombe gene, SPAC25A8.01c, designated snf2SR⁺ (a member of the chromatin remodeling factors, Snf2 family with DNA-dependent ATPase activity), but not the spi1⁺ (S. pombe Ran homolog), rescued a lethality of Sprna1^ts. Both Clr4 and Snf2 were reported to be involved in heterochromatin formation essential for building the centromeres. Consistently, Sprna1^ts was defective in gene-silencing at the centromeres. But a silencing at the telomere, another heterochromatic region, was normal in all of Sprna1^ts strains, indicating SpRna1 in general did not function for a heterochromatin formation. snf2SR⁺ rescued a centromeric silencing defect and clr4⁺ was synthetic lethal with Sprna1^ts. Taken together, SpRna1 was suggested to function for constructing the centromeres, by cooperating with Clr4 and Snf2SR. Loss of SpRna1 activity, therefore, caused chromosome missegregation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Ran, a small GTPase, plays a key role in the nucleocytoplasmic transport of macromolecules, the mitotic spindle formation and the postmitotic nuclear envelope assembly (Moore, 2001; Dasso, 2002; Hetzer et al., 2002; Weis, 2003). RanGAP, RanGTPase activating protein, is primarily cytosolic, whereas RCC1 (Kai et al., 1986), mammalian RanGEF (Ran-GDP/GTP exchange factor; Bischoff and Ponstingl, 1991), is a chromosomal protein (Ohtsubo et al., 1989). Ran-GTP concentration, thus, is high in the nucleus and low in the cytoplasm (Kalab et al., 2002). The Ran-GTP/Ran-GDP gradient is currently thought to be critical for Ran-mediated cellular processes. Proteins carrying a nuclear export signal (NES), for instance, make a complex with the nuclear export receptors by the aid of Ran-GTP in the nucleus, and they enter the cytoplasm where Ran-GTP is hydrolyzed to Ran-GDP by the aid of RanGAP, releasing NES-cargo proteins into the cytoplasm (Weis, 2003). The high local concentration of Ran-GTP in the vicinity of mitotic chromosomes promotes an assembly of mitotic spindle and a fusion of the nuclear membrane vesicles, by the aid of RanGAP (Hetzer et al., 2001, 2002).

Although RanGAP is exclusively localized to the cytoplasm, Saccharomyces cerevisiae RanGAP, Rna1, possesses a novel type of nuclear localization signal (NLS) and two of the classical NES, and the endogenous Rna1 location is dependent on the nuclear export receptor, Xpo1/Crm1 (Feng et al., 1999). In Drosophila melanogaster, a naturally occurring meiotic drive system of the Segregation Distorter (SD; Lyttle, 1991), which shows preferential transmission of the SD chromosome from SD/SD⁺ heterozygous males, is caused by the mutated RanGAP, referred to as Sd-RanGAP that is an enzymatically active protein with a truncated NES (Kusano et al., 2001, 2002). Sd-RanGAP, therefore, accumulates in the nucleus. The SD phenotype was thus suggested to be induced by abolishing the Ran-GTP/Ran-GDP gradient (Kusano et al., 2001). Indeed, overexpression of Ran rescues the SD phenotype (Kusano et al., 2001). Taken together, RanGAP obviously enters the nucleus. However, it is unknown whether nuclear RanGAP participates in a complete RanGTPase nuclear cycle or serves a novel nuclear function (Feng et al., 1999). To address this issue, we chose RanGAP.

A series of temperature-sensitive (ts) mutants of Schizosaccharomyces pombe RanGAP homolog, Sprna1⁺, were isolated using the error-prone polymerase chain reaction (PCR) as described previously (Oki et al., 1998). Resulting Sprna1^ts showed a defect in the chromosome segregation, rather than the mitotic spindle formation and the nucleocytoplasmic transport. Subsequently, we isolated the multicopy suppressors of Sprna1^ts that encoded Clr4, a methyltransferase specific for histone H3-K9 (Rea et al., 2000; Bannister et al., 2001; Nakayama et al., 2001), and Snf2SR, a member of chromatin remodeling factors, Snf2 family that have DNA-dependent ATPase activity (Havas et al., 2001; Becker and Horz, 2002, Lusser and Kadonaga, 2003), in addition to SpRna1 and SpKsp1, S. pombe homolog of S. cerevisiae Ksp1 (Fleischmann et al., 1996). Consistent with the previous reports that both Clr4 and Snf2-homolog function for heterochromatin formation in Arabidopsis thaliana (Jeddeloh et al., 1999; Gendrel et al., 2002; Johnson et al., 2002), most of Sprna1^ts strains showed a silencing defect at the innermost repeat domain and one mutant showed a clear silencing defect at the outer repeat domain of the centromeres where Swi6, S. pombe homolog of mammalian heterochromatin protein 1 (HP1)-dependent heterochromatin is formed (Bannister et al., 2001). A silencing at the telomeres, another heterochromatic chromosomal region, was normal in all of Sprna1^ts. SpRna1, therefore, in general did not function for heterochromatin formation. A half of Sprna1^ts also showed a significant defect at the central core of the centromeres. Especially one mutant showed a slight, but significant, silencing defect only at the central core. Taken together with the report that mammalian RanGAP1 is localized on kinetochores in mitotic cells (Joseph et al., 2004), SpRna1 was suggested to have a novel function that is required for constructing the centromeres.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Yeast Media and Strains
The S. pombe strain was grown in rich medium (YE5S) or Edinburgh minimal medium (EMM) with appropriate supplements. Sprna1-A1 strain: 478 (Table 1), was provided by S. Sazer (Baylor College of Medicine; Matynia et al., 1996). To isolate a haploid derivative that carries the Sprna1::ura4⁺ allele, Sprna1-A1 was transformed first with pREP81X-Sprna1⁺ which carries the open reading frame (ORF) of wild-type Sprna1⁺ and LEU⁺ marker (Matynia et al., 1996). Sprna1-A1 [pREP81X-Sprna1⁺] sporulated on ME plates, and spores were treated with -glucuronidase (Sigma, St. Louis, MO) and replated for germination. The strains possessing Ura⁺ and Leu⁺ markers were identified as Sprna1::ura4⁺ cells carrying pREP81X-Sprna1⁺. The strains used in this experiment are listed in Table 1.

Isolation of Sprna1^ts
Sprna1^ts were generated through the error-prone PCR method as described previously (Oki et al., 1998). Briefly, the Sprna1⁺ genomic DNA fragment, which covers an entire ORF of Sprna1⁺ along with about 1 kilobase pair (kbp) of both flanking regions derived from the upstream and the downstream of Sprna1⁺ ORF, was amplified by PCR using the following primers: the 5' primer, 5'-CAC AGT AAT GAA TAA GAA ATG, and the 3' primer, 5'-AGC ACT TTC ATC CAA ACA CTC, in the presence of 0.2 mM Mn²⁺. Amplified DNA fragments were introduced into the haploid Sprna1::ura4⁺ cells carrying pREP81X-Sprna1⁺ through electroporation. Transfected cells were grown for four generations in EMM medium supplemented with leucine, uracil, and adenine and then incubated in the presence of 1.5 mg/ml 5FOA (5-fluoroorotic acid) at 26°C. Resulting cells were replica-plated at 26 and 35°C. Cell lines that grew at 26°C (permissive temperature), but not at 35°C (restrictive temperature), were identified as ts mutants.

Each Sprna1^ts strain was crossed to clr4 strain, gifted from R. Allshire (University of Edinburgh; Bannister et al., 2001), to generate the double mutants of Sprna1^ts clr4.

Isolation of Multicopy Suppressors for Sprna1^ts
S. pombe genomic library provided from M. Yanagida (Kyoto University), was introduced into Sprna1^ts with electroporation. Transformed cells were plated and incubated at either 34 or 35°C for 5 d. Cells that grew on plates at 35°C were selected for further analysis. Plasmids retained in ts⁺ transformants of Sprna1^ts were recovered in Escherichia coli, and their nucleotide sequences of S. pombe genomic DNA carried on plasmids were determined using the ABI Genetic Analyzer 3100 (Applied Biosystems, Tokyo, Japan).

Construction of NLS-NES-GFP and NLS-NES^P12-GFP
pREP3X-NLS-NES-GFP and pREP3X-NLS-NES^p12-GFP constructs were created by introducing a DNA fragment that encodes NLS-NES-GFP or NLS-NES^p12-GFP, derived from pKW430 or pKW431 (Stade et al., 1997), into the pREP3X vector.

Immunofluoresence Microscopy
DNA. Cells were spotted onto a glass slide, fixed on 70°C heat block, and then mounted in VECTASHIELD with DAPI (Vector Laboratories, Burlingame, CA) to visualize chromosomes.

Microtubules. Cells were fixed with 3.3% formaldehyde in phosphate-buffered saline, stained first with the mouse anti-Tat1 antibody, gifted from K. Gull (University of Manchester; Sherwin and Gull, 1989), followed by the fluorescein isothiocyanate–conjugated goat anti-mouse immunoglobulin G (IgG, Jackson ImmunoResearch Laboratories, West Grove, PA).

Spindle Pole Body (SPB). Cells fixed as described above, were first stained with the rabbit anti-Sad1 antibody gifted from I. Hagan (University of Manchester; Hagan and Yanagida, 1995), followed by the Alexa Fluor 568–conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR).

Images were visualized with the Zeiss Axioskop Fluorescence Microscope (Carl Zeiss, Thornwood, NY), and processed with Adobe Photoshop (Adobe Systems, San Jose, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Isolation of Sprna1^ts
The Sprna1⁺ genomic DNA fragment that contains the entire ORF of Sprna1⁺ and 1 kb of flanking regions was amplified by PCR in the presence of 0.2 mM of Mn²⁺ as described previously (Oki et al., 1998). Amplified DNA fragments were introduced into the haploid strain; Sprna1::ura4⁺ carrying pREP81X-Sprna1⁺ and then temperature-sensitive (ts) cells were isolated as described in Materials and Methods.

Initially, 57 of ts cells were independently isolated. The Sprna1⁺-ORF of the resulting ts cells was sequenced to confirm that the temperature-sensitive lethality of cells was caused by the mutation of SpRna1. In parallel, pREP81X-Sprna1⁺ was introduced into resulting ts cells to examine whether a temperature-sensitive lethality of isolated strains could be rescued by this plasmid. According to the sequencing data, 11 lines of Sprna1^ts strains were finally chosen for further analyses (Table 2). Most of them had a single point mutation, which was mainly localized either in the conserved leucine-rich repeats (LRRs) domain (Hillig et al., 1999) or in the Ran-interacting domain of SpRna1 (Seewald et al., 2002; Figure 1).

View larger version (42K): [in this window] [in a new window]   Figure 1. Mutation sites in Sprna1ts mutants. Mutation sites of Sprna1ts were localized on a ribbon presentation of the structure of SpRna1 (modified from Hillig et al., 1999). Red ribbons and green arrows represent -helixes and -sheet structures, respectively. Yellow lines show hinge region between -helix and -sheet. Mutations at the Ran interaction site are indicated in green, and the mutations at the conserved leucine residues of the LRRs domain are indicated in red. Mutations at other amino acid residues are indicated in blue. The number in parentheses indicates the number of times that the same mutation was independently isolated. As for Sprna1-15ts, Sprna1-47ts, and Sprna1-82ts, which have two to three amino acid changes (see Table 2), the mutation site in the Ran-interaction or the LRRs domains was shown.

The cellular amount of mutated SpRna1 was not reduced in most of Sprna1^ts, except for Sprna1-47^ts and Sprna1-82^ts, after incubation for 5 h at 35°C, the restrictive temperature (unpublished data). Both Sprna1-1^ts and Sprna1-86^ts have a mutation at the amino acid in the NES region estimated from S. cerevisiae Rna1, but a corresponding S. cerevisiae Rna1 mutant has no effect on the nuclear export (Feng et al., 1999). The other Sprna1^ts mutants have no mutation in the putative SpRna1 NES. Expectedly, all of mutated SpRna1 proteins were mainly localized in the cytoplasm, even after incubation at the restrictive temperature (unpublished data).

Mitotic Chromosome Segregation Was Defective in Sprna1^ts
RanGTPase is involved in several cellular processes (Moore, 2001; Dasso, 2002; Hetzer et al., 2002; Weis, 2003), which were then examined in Sprna1^ts isolated.

First, the nucleocytoplasmic transport of proteins was examined. To address this issue, GFP-fused reporter proteins appended with NLS and NES (NLS-NES) were expressed in Sprna1^ts. The representative results are shown in Figure 2. Because the activity of the used NES is stronger than that of NLS, the expressed GFP was localized in both the cytoplasm and the nuclear rim of wild-type cells, both at 26 and at 37°C, as reported (Stade et al., 1997; Figure 2, A and C). On the other hand, GFP-fused reporter proteins appended with NLS and an inactive NES (NLS-NES^p12), which are mainly localized in the nucleus (Figure 2, B and D), were used to estimate the nuclear-protein import activity of Sprna1^ts. Most of Sprna1^ts strains did not show obvious defects in the nucleocytoplasmic transport (Figure 2, E and G, F and H). Only both Sprna1-82^ts and Sprna1-87^ts showed a defect in the nuclear protein import at 37°C. In these mutants, there was no obvious nuclear accumulation of GFP-fused NLS-NES^p12 proteins, compared with Sprna1-86^ts and wild-type cells (Figure 2, J and L, N and P).

View larger version (54K): [in this window] [in a new window]   Figure 2. Nucleocytoplasmic transport in Sprna1ts mutants. Cultures of indicated cells were incubated at 26°C, and then half of cultures were incubated at 37°C for 4 h. Images of wild-type, Sprna1-86ts, Sprna1-82ts, and Sprna1-87ts cells expressing the indicated GFP-fused NLS-NES or NLS-NESp12 proteins were shown as representatives. Left columns of the indicated NLS-NES and NLS-NESp12 show GFP localization, and right columns show merged images with DAPI-staining. Scale bar, 5 µm.

Second, the mitotic spindle formation and the localization of SPB stained with the anti-Tat1 and the anti-Sad1 antibodies, respectively, were examined in all of Sprna1^ts. The representative results are shown in Figure 3. Both the mitotic spindle formation and the localization of SPB were indistinguishable from those of wild-type cells, even after incubation at 37°C, in all of Sprna1^ts. When DNA of the same samples was stained with DAPI, however, chromosomal DNA was dislocated from SPB on elongated mitotic spindles in most of Sprna1^ts, and the images were similar to the sim mutants (Pidoux et al., 2003), indicating the uneven segregation of mitotic chromosomes occurred in Sprna1^ts at 37°C. To further confirm this issue, Sprna1^ts was grown at 35°C for 4 h in liquid medium and then fixed to stain with DAPI. Apparent uneven chromosome segregation was detected in all of Sprna1^ts. The representative results are shown in Figure 4. Cells marked with arrows and arrowheads showed an aberrant chromosome segregation as indicated in Figure 4D. Compared with wild-type cells, uneven chromosome segregation was apparent in Sprna1^ts at 35°C.

View larger version (32K): [in this window] [in a new window]   Figure 3. Mitotic spindle formation in Sprna1ts mutants. Cultures of indicated cells were incubated at 26°C, and then half of cultures were incubated at 37°C for 4 h. Images of wild-type, Sprna1-86ts, and Sprna1-87ts are shown as representatives. Mitotic spindle and SPB were detected with anti-Tat1 (green) and anti-Sad1 (red) antibodies, respectively. DNA was stained with DAPI (blue). Left columns show an anti-Tat1 staining pattern and right columns show an anti-Tat1 staining image merged with anti-Sad1 for SPB (red) and DAPI (blue), respectively, at the indicated temperature. Scale bar, 5 µm.

View larger version (52K): [in this window] [in a new window]   Figure 4. Sprna1ts mutants showing aberrant mitotic chromosome segregation. Cultures of indicated cells were grown in liquid medium at 26°C, and then half of cultures were incubated at 35°C for 4 h. Images of wild-type (A), Sprna1-86ts (B), and Sprna1-87ts (C) cells are shown as representatives. (D) The frequency of the indicated patterns of aberrant chromosome segregation that are shown by black and white arrowheads and an arrow. Chromosomal DNA was stained with DAPI. Scale bar, 5 µm.

Clr4 and Snf2SR Rescued a Temperature-sensitive Lethality of Sprna1^ts
To elucidate the molecular mechanism of uneven chromosome segregation, S. pombe genomic library was screened for the multicopy suppressor genes of Sprna1^ts that rescue a temperature-sensitive lethality of Sprna1^ts. In addition to Sprna1⁺ and Spksp1⁺, both clr4⁺ and the S. pombe gene, SPAC25A8.01c, encoding a member of Snf2 family proteins (Lusser and Kadonaga, 2003), designated as snf2SR⁺-like Suppressor of Sprna1^ts), were frequently obtained from ts⁺ transformants of several Sprna1^ts that grew on plates at 35°C (Table 3).

The ability of clr4⁺ and snf2SR⁺ to rescue a temperature-sensitive lethality of Sprna1^ts was well correlated (Figure 5). On overexpression of clr4⁺ or snf2SR⁺ genes, Sprna1^ts such as Sprna1-1^ts, Sprna1-8^ts, Sprna1-15^ts, Sprna1-48^ts, and Sprna1-86^ts, grew at the restrictive temperature (34 and 35°C), but not the other Sprna1^ts such as Sprna1-11^ts, Sprna1-47^ts, Sprna1-50^ts, Sprna1-55^ts, Sprna1-82^ts, and Sprna1-87^ts. The frequency of uneven chromosome segregation was also significantly reduced in Sprna1-1^ts, Sprna1-8^ts, Sprna1-15^ts, Sprna1-48^ts, and Sprna1-86^ts, but not in Sprna1-11^ts, Sprna1-47^ts, Sprna1-50^ts, Sprna1-55^ts, Sprna1-82^ts, and Sprna1-87^ts by overexpression of clr4⁺ or snf2SR⁺ (representative results are shown in Figure 6). The ability to rescue a temperature-sensitive lethality of Sprna1^ts by clr4⁺ or snf2SR⁺ was well correlated with the ability to achieve proper chromosome segregation, suggesting that Sprna1^ts became lethal by uneven chromosome segregation at the restrictive temperature.

View larger version (39K): [in this window] [in a new window]   Figure 5. Expression of clr4+or snf2SR+ rescued the temperature-sensitive lethality of Sprna1ts mutants. (A) Multicopy suppression of Sprna1-8ts, Sprna1-15ts, and Sprna1-87ts by clr4+ or snf2SR+ are shown as representatives. Left and right columns show cells grown at 26 and 34°C, respectively. Clones are shown in the following clockwise order: wild-type, Sprna1ts cells possessing a vector alone, clr4+ or snf2SR+ as indicated. (B) Allele specificity of multicopy suppression by clr4+ or snf2SR+. (+), suppressed; (-), not suppressed. The number of (+) indicated the efficiency of suppression.

View larger version (72K): [in this window] [in a new window]   Figure 6. Aberrant mitotic chromosome segregation of Sprna1ts was rescued by clr4+ or snf2SR+. Cultures of Sprna1ts mutants expressing no suppressors (left), clr4+ (middle), or snf2SR+ (right) grown in liquid medium at 26°C, were incubated at 35°C for 4 h and then stained with DAPI. Images of Sprna1-86ts (A) and Sprna1-87ts (B) are shown as representatives. Scale bar, 5 µm. Black and white arrowheads indicate aberrant chromosome segregation as described in Figure 4. (C) Summary of A and B. The vertical line indicates a frequency of cells showing an aberrant mitosis (%). At least 300 cells were counted for each strain.

Sprna1^ts Was Defective in Centromeric, but not in Telomeric, Silencing
Proper chromosome segregation is carried out by interaction between mitotic spindle and kinetochores. Because mitotic spindle formation and SPB distribution seemed to be normal (Figure 3), the uneven chromosome segregation suggested that the kinetochore-centromere function that ensures an interaction between mitotic spindle and kinetochores became defective at the restrictive temperature. Clr4, which methylates specifically lysine 9 of histone H3 (Rea et al., 2000; Bannister et al., 2001; Nakayama et al., 2001), is essential for constructing heterochromatin. Snf2SR is a member of the Snf2 family (Havas et al., 2001; Becker and Horz, 2002; Lusser and Kadonaga, 2003). The Snf2-like protein is required for heterochromatin formation and maintenance in A. thaliana (Gendrel et al., 2002; Johnson et al., 2002). The fact that both suppressors of Sprna1^ts are involved in constructing heterochromatin, which is important for the centromeric function (Jeddeloh et al., 1999; Pidoux et al., 2003; Appelgren et al., 2003), indicated that the centromeres might become defective in Sprna1^ts at the restrictive temperature. To address this issue, Sprna1^ts was examined for a gene-silencing activity at the centromere, based on previous reports that the expression of the euchromatic gene inserted into the centromere is repressed (Nakagawa et al., 2002; Pidoux et al., 2003).

We assayed the silencing of the ura4⁺ gene inserted at three different domains of Chromosome I centromere: the outer repeat (otr1R::ura4⁺), the innermost repeat (imr1R::ura4⁺), and the central core (cnt1::ura4⁺), as reported previously (Nakagawa et al., 2002). Expression of the inserted ura4⁺ gene was monitored by plating serially diluted cells of indicated each strain on the nonselective (N/S), the selective (Ura^-), or the counterselective (5FOA) plates (5FOA is a toxigenic substrate for the Ura4 protein). As expected, ura4⁺ cells grew on Ura^- plates, but not on 5FOA plates. In contrast, ura4^- cells grew on 5FOA plates, but did not on Ura^- plates (Figure 7, D and E, h⁺ 975 and h⁺ ura4-D18). In Sprna1⁺ (wt) cells, the ura4⁺ gene expression was repressed at the outer repeat (otr1R::ura4⁺) and the innermost repeat (imr1R::ura4⁺), but it was partially repressed at the central core (cnt1::ura4⁺; Figure 7, A–C, wt, compare Ura^- and 5FOA plates), as previously reported (Pidoux et al., 2003). After incubation at 30°C for 5 d, a silencing defect at the centromeres was observed, as shown in Figure 7. Sprna1-1^ts, Sprna1-11^ts, Sprna1-47^ts, and Sprna1-86^ts showed a significant silencing defect at the central core (Figure 7A, 5FOA plate). Except Sprna1-86^ts that has a silencing defect only at the central core, all of the other Sprna1^ts strains also showed a silencing defect at the innermost repeat. In addition to the innermost repeat, a clear silencing defect at the outer repeat domain where Swi6-mediated heterochromatin is formed (Bannister et al., 2001) was observed only in Sprna1-87^ts. Consistently, the distribution of Swi6, which binds to the outer repeat domain (Bannister et al., 2001), was slightly changed in this mutant, whereas it seemed to be normal in other Sprna1^ts strains, after incubation at the restrictive temperature (Supplementary Figure 1).

View larger version (54K): [in this window] [in a new window]   Figure 7. Gene silencing activity of Sprna1ts mutants. Top panel, a schematic representation of the cen1 and the ura4+ insertion sites within the cen1. (A–D) Centromeric silencing. Sprna1ts strains possessing the ura4+ gene at the indicated domain are spotted (A–C). wt: AK906 (A); AK89 (B); AK90 (C). Sprna1ts [imr1R::ura4+] strains expressing snf2SR+ (D). (E) Telomeric silencing. Sprna1ts strains possessing the ura4+ gene at the telomere (Nimmo et al., 1998) are spotted as indicated. wt: tlm+. The h+975 and h+ura4-D18 were used as the control strains of either Ura4+ or Ura4- cells, respectively. The dotted marks of these strains in D indicate that they have no plasmids. Each strain was grown to 1.0 x 107 cells/ml in YE5S. Serial dilution (1:5) of the indicated cultures were spotted onto nonselective (N/S), selective (Ura-), or counterselective (5FOA) plates and incubated at 30°C for 5 d. The highest density spots contained 1 x 104 cells.

To address the question whether a defect of SpRna1 affects gene silencing at the heterochromatic domains other than the centromeres, the expression of the ura4⁺ gene inserted into the telomere was examined. It was clearly repressed in all of Sprna1^ts strains examined (Figure 7E), indicating that SpRna1 in general did not function for heterochromatin formation.

The snf2SR⁺ rescued a centromeric silencing defect of Sprna1^ts mutants. Representative results at the innermost repeat domain (imr1R::ura4⁺) were shown (Figure 7D). When Clr4 was overexpressed, the gene silencing ability at three domains of the centromeres became defective in Sprna1⁺ (wt) cells. So, we could not determine whether Clr4 rescues the centromeric silencing defect of Sprna1^ts or not. In the supplemental figure, we showed the representative results at the innermost repeat domain of the centromeres. Curiously, Clr4 rescued a silencing defect of some Sprna1^ts strains at this domain, although it abolished a silencing at the same domain of Sprna1⁺ (wt) cells (Supplementary Figure 2, 5FOA).

Sprna1^ts Was Synthetically Lethal with clr4
To confirm a functional relationship between SpRna1 and Clr4, the clr4⁺ gene of each Sprna1^ts was disrupted. The temperature-sensitive lethality was enhanced by a disruption of the clr4⁺ gene, in most of Sprna1^ts strains (Figure 8). Among them, Sprna1-11^ts, Sprna1-47^ts, and Sprna1-87^ts, which temperature-sensitive lethality could not be rescued by clr4⁺ or snf2SR⁺ (Figure 5), showed severe synthetic lethality with clr4. In contrast, Sprna1-1^ts, Sprna1-^8ts, Sprna1-15^ts, Sprna1-48^ts, and Sprna1-86^ts in which temperature-sensitive lethality was well rescued by overexpression of clr4⁺ or snf2SR⁺ (Figure 5), showed less severe synthetic lethality with clr4. Thus, SpRna1 was suggested to have some functional relationship with Clr4. Sprna1-50^ts, Sprna1-55^ts, and Sprna1-82^ts did not show the synthetically lethality at the temperature examined.

View larger version (58K): [in this window] [in a new window]   Figure 8. Synthetic lethality of Sprna1ts clr4 double mutants. (A) Cells with indicated genotypes were grown on YE5S plates at 26°C (left columns), 30°C (middle columns), or 33°C (right columns). Cell lines are shown in the following clockwise order: wild-type, clr4, Sprna1ts, and Sprna1tsclr4. Images of Sprna1-47ts and Sprna1-86ts are shown as representatives. (B) Lethality of each Sprna1tsclr4 double mutant. The temperature, at which each sprna1ts mutant showed synthetic lethality with clr4 is indicated as asterisks. Temperature-sensitive (ts) lethality is shown as ts lethal.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We have isolated 11 Sprna1^ts strains that have one to three amino acid alterations in SpRna1. Except for Sprna1-48^ts and Sprna1-50^ts, all of the other Sprna1^ts have a mutation in the amino acid residues that are conserved from yeast to mammalian RanGAP (Hillig et al., 1999). Specifically 5 of 11 mutants have a mutation in leucine residues of the RanGAP LRR domains, suggesting that these regions have a key role for the RanGAP activity.

Sprna1^ts showed a little defect in the nucleocytoplasmic transport, but not in the mitotic spindle formation and the SPB localization. They showed a strong defect in the chromosome segregation, after incubation at the restrictive temperature. For proper chromosome segregation, both the kinetochores and the centromeres that underlie kinetochores are important, in addition to mitotic spindle and SPB. Although both mitotic spindle and SPB localization were normal after incubation at the restrictive temperature, chromosomal DNA was dislocated from SPB on elongated mitotic spindle, similar to the sim4^-, which has a defect in the centromeres (Pidoux et al., 2003). Indeed, a gene-silencing defect was observed at the centromeres of Sprna1^ts. The uneven chromosome segregation of Sprna1^ts, therefore, could be caused by a defect of centromere-kinetochore function that disrupts an interaction between mitotic spindles and kinetochores. The fact that Clr4 and Snf2SR, a member of Snf2 family, were isolated as suppressors of Sprna1^ts suggested that SpRna1 is required for building the centromeres, by the unknown mechanism, in which Clr4, Snf2SR, and probably another suppressor, SpKsp1, might be involved.

Clr4 performs histone H3 methylation at the 9th lysine (Nakayama et al., 2001), which is essential for constructing heterochromatin. Snf2SR, another multicopy suppressor of Sprna1^ts, is a member of Snf2 family with DNA-dependent ATPase, comprising the chromatin-remodeling complex (Lusser and Kadonaga, 2003). SpKsp1 is an S. pombe homolog of S. cerevisiae Ksp1, which has been isolated as a multicopy suppressor of prp20 (S. cerevisiae ts mutant of RCC1 homolog; Fleischmann et al., 1996). It encodes Ser/Thr protein kinase. The A. thaliana Snf2-family protein, DDM1, has been reported to function for histone H3-K9 methylation and maintenance by cooperating with A. thaliana Clr4 homolog, KRYPTONITE (Gendrel et al., 2002; Johnson et al., 2002; Soppe et al., 2002). The fact that both Clr4 and Snf2SR were frequently obtained as the multicopy suppressor of Sprna1^ts mutants, therefore, indicated that SpRna1 might be required for heterochromatin formation. However, all of Sprna1^ts strains did not show a silencing defect at the telomere, which is another heterochromatic domain of the chromosomes (Nimmo et al., 1998). Thus, SpRna1 in general was not required for constructing heterochromatin.

SpRna1 seemed to have a tight functional relationship with Clr4, because Clr4 suppressed Sprna1^ts and clr4 is synthetic lethal with Sprna1^ts. Preliminarily, we found that recombinant SpRna1 binds histone H3 and activates the methylase activity of recombinant Clr4 without S. pombe Ran, Spi1, although SpRna1 was not coimmunoprecipitated with Clr4 (Nishijima et al., unpublished results), indicating that there is a functional interaction between SpRna1 and Clr4. It is currently being investigated how SpRna1 interacts with Snf2SR and SpKsp1.

The centromeres of S. pombe are composed of two domains: the central core and the outer repeat domains (Appelgren et al., 2003), both of which are required for a full function of the centromeres (Nakagawa et al., 2002; Pidoux et al., 2003). The central core, which has a chromatin structure distinct from the outer repeat, possesses the histone H3-variant Cnp1 (S. pombe CENP-A homolog). It actually underlies the kinetochores. The gene silencing activity at the central core is maintained by several centromere-associated proteins. For instance, there are two proteins: the centromere protein Sim4 (Pidoux et al., 2003) and the kinetochore protein Mis6 (Takahashi et al., 2000), both of which colocalize at the centromeres (Pidoux et al., 2003). The inactivation of either Sim4 or Mis6 causes a silencing defect at the central core but not at the outer repeat domains (Pidoux et al., 2003), in contrast to loss of Swi6, which causes a silencing defect at the outer repeat but not at the central core domains (Nakagawa et al., 2002). Swi6 is supposed to maintain heterochromatin structure by binding to histone H3 methylated at Lys 9 (Bannister et al., 2001). Six of eight Sprna1^ts strains showed a silencing defect at the innermost repeat and only one mutant, Sprna1-87^ts, showed a clear silencing defect at the outer repeat domains of the centromeres where Swi6-mediated heterochromatin is formed (Bannister et al., 2001). Consistently, the Swi6-staining pattern was slightly changed in Sprna1-87^ts compared with that of Sprna1-86^ts, which showed a silencing defect only at the central core. The level of methylated histone H3-K9 might not be reduced enough for Swi6 to maintain heterochromatin structure, resulting in a gene silencing defect. In contrast, 4 Sprna1^ts strains showed a silencing defect at the central core. Sprna1-86^ts especially showed a silencing defect only at the central core domain where the histone H3 variant Cnp1 is present. Whether SpRna1 interacts with Cnp1, Sim4, or Mis6 remains to be investigated. Preliminary, we found that SpRna1 binds histone H3 (Nishijima et al., unpublished results). It will be an intriguing question whether SpRna1 also binds to Cnp1 in order to construct the central core. Taken together, SpRna1 might act with Clr4, Snf2SR, histone H3, and perhaps other centromere-associated proteins throughout the centromere, not just at the Swi6-mediated heterochromatic region of the centromeres.

In mammalian cells, RanGAP is reported to be concentrated at the kinetochores and to function for microtubule-kinetochore interaction (Joseph et al., 2004). This report is consistent with our present observation of Sprna1^ts, although we do not know whether SpRna1 is localized at S. pombe kinetochore. A defect of centromere function observed in Sprna1^ts might impair the spindle checkpoint, similar to sim mutants (Pidoux et al., 2003), resulting in uneven chromosome segregation, with few lagging chromosomes (Figure 4D). Indeed, Sprna1^ts did not exhibit a strong arrest at metaphase.

There are two formal possibilities regarding how SpRna1 functions to form the centromeres. First, SpRna1 participates in nucleocytoplasmic transport, so that proteins required for building centromeres, such as Snf2SR and Clr4, could be assembled onto the centromeres, by the aid of SpRna1. The second possibility is that SpRna1 is required to construct the centromeres, as a novel function of SpRna1. In the first case, overexpression of Spi1 (S. pombe Ran), should suppress Sprna1^ts, as has been shown in the SD system (Kusano et al., 2001). Because Sprna1-87^ts has a defect in the nuclear protein import, a clear silencing defect of Sprna1-87^ts at the outer repeat domain of Chromosome 1 centromere could be caused by a defect in the nuclear protein import. However, the spi1⁺ gene was not isolated as the multicopy suppressor of Sprna1^ts strains including Sprna1-87^ts. We obtained a lot of the spi1⁺ gene as a multicopy suppressor of the pim1^ts mutants (Hirose et al., unpublished results), using the same S. pombe genomic library as the presently used one. Furthermore, the spi1⁺ gene carried on the same vector as the clr4⁺ gene could not rescue a temperature-sensitive lethality of Sprna1^ts (unpublished data). Taking together with the fact that the nucleocytoplasmic transport and the mitotic spindle formation seemed to be normal in most of Sprna1^ts strains, the second possibility is currently reasonable. SpRna1, therefore, may have a novel function in the nucleus in order to properly segregate chromosome. Currently, it is unknown how SpRna1 functions to construct the centromeres. Our present finding will pave the way to addressing this question.

Because independently isolated Sprna1^ts showed a similar silencing defect at the centromere, it could be concluded that our findings reflect a nuclear function of SpRna1. Because the concentration of Ran-GTP is high in the nucleus for nucleocytoplasmic transport, we reason that there must be some inhibitor(s) or regulator (s) of RanGAP activity for SpRna1 properly functioning in the nucleus, otherwise the nuclear SpRna1 may abolish the Ran-GTP concentration gradient from the nucleus to the cytoplasm. Furthermore, the mechanism of how SpRna1 enters the nucleus and is exported from the nucleus, is as yet unknown.

In conclusion, our present findings suggest that SpRna1 has a novel nuclear function that is required for constructing the centromeres.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank J. Fukumura and Dr. E. Hirose (Kyushu University) for S. pombe genomic library amplification and library transformation. Also, we thank Drs. S. Sazer (Baylor College of Medicine) for the rna1-A1 strain; K. Weis (University of California, Berkeley) for the NLS-NES-GFP constructs; K. Gull (University of Manchester) for the anti-Tat1 antibodies; I. Hagan (University of Manchester) for the anti-Sad1 antibodies; M. Yanagida (Kyoto University) for S. pombe genomic library; Y. Murakami (Kyoto University), F. Ishikawa (Kyoto University), and R. Allshire (University of Edinburgh) for the strains for silencing assay; and R. Allshire (University of Edinburgh) for clr4 and Y. Watanabe for the eswi6-GFP construct (University of Tokyo). We thank A. Wittingfoher (Max-Plank Institute) for letting us modify the schematic drawing of the SpRna1 crystal structure shown in Figure 1. This work is supported by Grants-in-Aid for Specially Promoted Research from the Ministry of Education, Science, Sports, and Culture of Japan. A.K. acknowledges the Hayashi Memorial Foundation for Female Natural Scientists. The English used in this manuscript was revised by Professor David Maruyama (Long Beach City College, California).

	Footnotes

 
Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E04–01–0067. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E04–01–0067.

The online version of this article contains supplementary material accessible at http://www.molbiolcell.org.

^* Present address: Research Organization of Information and Systems, National Institute of Genetics, Genetics Strains Research Center, Laboratory for Frontier Research, 1111 Yata Mishima, 411-8540, Japan.

Corresponding author. E-mail address: tnishi{at}mailserver.med.kyushu-u.ac.jp.

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