The Saccharomyces cerevisiae Small GTPase, Gsp1p/Ran, Is Involved in 3' Processing of 7S-to-5.8S rRNA and in Degradation of the Excised 5'-A0 Fragment of 35S Pre-rRNA, Both of Which Are Carried Out by the Exosome

Corresponding author: Takeharu Nishimoto, Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maedashi, Higashiku, Fukuoka 812-8582, Japan., tnishi{at}molbiol.med.kyushu-u.ac.jp (E-mail)

Communicating editor: A. G. HINNEBUSCH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Dis3p, a subunit of the exosome, interacts directly with Ran. To clarify the relationship between the exosome and the RanGTPase cycle, a series of temperature-sensitive Saccharomyces cerevisiae dis3 mutants were isolated and their 5.8S rRNA processing was compared with processing in strains with mutations in a S. cerevisiae Ran homologue, Gsp1p. In both dis3 and gsp1 mutants, 3' processing of 7S-to-5.8S rRNA was blocked at three identical sites in an allele-specific manner. In contrast, the 5' end of 5.8S rRNA was terminated normally in gsp1 and in dis3. Inhibition of 5.8S rRNA maturation in gsp1 was rescued by overexpression of nuclear exosome components Dis3p, Rrp4p, and Mtr4p, but not by a cytoplasmic exosome component, Ski2p. Furthermore, gsp1 and dis3 accumulated the 5'-A0 fragment of 35S pre-rRNA, which is also degraded by the exosome, and the level of 27S rRNA was reduced. Neither 5.8S rRNA intermediates nor 5'-A0 fragments were observed in mutants defective in the nucleocytoplasmic transport, indicating that Gsp1p regulates rRNA processing through Dis3p, independent of nucleocytoplasmic transport.

A Ras-like small GTPase, Ran, is regulated by the GTPase-activating protein RanGAP1 and the GDP/GTP exchange protein RCC1 (AZUMA and DASSO 2000 ; NISHIMOTO 2000 ). A set of Ran-binding proteins was found to be involved in nucleocytoplasmic transport of macromolecules. Those are proteins of the importin-ß family, RanBP1, RanBP2, p10/Ntf2p, and a novel protein, Mog1p (OKI and NISHIMOTO 1998 ; reviewed by MELCHIOR and GERACE 1998 ; WOZNIAK et al. 1998 ; GORLICH and KUTAY 1999 ; NAKIELNY and DREYFUSS 1999 ). Curiously, two Ran-binding proteins, human RanBPM (NAKAMURA et al. 1998 ) and Saccharomyces cerevisiae Dis3p (NOGUCHI et al. 1996 ), have no clear functional relationship to nucleocytoplasmic transport. Studies of RanBPM first showed that Ran is required for microtubule assembly in Xenopus mitotic extracts (reviewed by KAHANA and CLEVELAND 1999 ; NISHIMOTO 1999 ).

Recombinant S. cerevisiae Dis3p binds directly to Gsp1p, the S. cerevisiae Ran homologue, and Schizosaccharomyces pombe Dis3p is in a complex in vivo with S. pombe Ran and RCC1 homologues (NOGUCHI et al. 1996 ). Independently, S. cerevisiae Dis3p was identified as Rrp44p, a subunit of the exosome (MITCHELL et al. 1997 ), which is composed of 11 subunits (ALLMANG et al. 1999 ). The exosome carries out not only 3' processing of 7S-to-5.8S ribosomal RNA (rRNA), but also 3' 5' degradation of S. cerevisiae mRNA (MITCHELL et al. 1996 ; JACOBS ANDERSON and PARKER 1998 ). The exosome that is conserved from yeast to human (ALLMANG et al. 1999 ) is therefore an important RNA processing/degradation machine in eukaryotic cells (VAN HOOF and PARKER 1999 ). The exosome is localized in both nucleus and cytoplasm (ALLMANG et al. 1999 ).

Other proteins of the RanGTPase cycle were defined as temperature-sensitive (ts) mutants defective in RNA metabolism. Rna1p, the S. cerevisiae homologue of RanGAP1 (BISCHOFF et al. 1995 ), was identified as rna1-1, which is defective in RNA synthesis (HUTCHISON et al. 1969 ). rna1-1 also has defects in both tRNA splicing and rRNA processing (HOPPER et al. 1978 ). Prp20p, the S. cerevisiae RCC1 homologue, was identified as prp20-1, which is defective in mRNA splicing (AEBI et al. 1990 ). These phenotypes were thought to be indirect consequences of defects in nucleocytoplasmic transport (GORLICH and KUTAY 1999 ; NAKIELNY and DREYFUSS 1999 ), since both small nuclear and nucleolar RNPs are required for RNA metabolism, the maturation of which involves nucleocytoplasmic transport (VEGVAR and DAHLBERG 1990 ; CHENG et al. 1995 ; LAFONTAINE and TOLLERVEY 1995 ; MAXWELL and FOURNIR 1995 ; YU et al. 1999 ). For instance, Xenopus U8 snRNP is required for the endonucleolytic cleavage of 12S pre-rRNA at both 5' and 3' ends (PECULIS and STEITZ 1993 ). The resulting 12S pre-rRNA is processed to 5.8S rRNAs by exonucleolytic cleavages at both the 5' and 3' ends. In yeast, while 5' 3' trimming of pre-rRNA is carried out by Xrn1p and Rat1p (HENRY et al. 1994 ), 3' 5' trimming is performed by the exosome, a subunit of which is Dis3p/Rrp44p (MITCHELL et al. 1997 ).

To investigate the functional relationship between the exosome and the RanGTPase cycle, we isolated a series of ts mutants of S. cerevisiae DIS3/RRP44 (henceforth referred to as DIS3) by error-prone PCR and compared their rRNA processing with that of gsp1 alleles that had been isolated by OKI et al. 1998 . Both dis3 and gsp1 mutants showed defects in 3' processing, but not 5' processing, of the 5.8S rRNA precursor, as do other exosome mutants. 3'-end processing of 5.8S rRNA was blocked at three identical sites in both dis3 and gsp1, in an allele-specific manner. Furthermore, the 5'-A0 fragment, which is degraded by the exosome (DE LA CRUZ et al. 1998 ), accumulated in both gsp1 and dis3. Taken together with the fact that Dis3p binds directly to Gsp1p (NOGUCHI et al. 1996 ), it is therefore likely that Gsp1p regulates the exosome through Dis3p.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and plasmids:
S. cerevisiae strains and plasmids used in this study are described in Table 1 and Table 2. Transformation of S. cerevisiae was performed by a modified LiCl method using dimethyl sulfoxide (DMSO; HILL et al. 1991 ). Selection against Ura⁺ strains was carried out by culturing on solid synthetic media containing 1 mg/ml 5'-fluoroorotic acid (5'-FOA; BOEKE et al. 1984 ).

Construction of plasmids:
The EcoRI-ApaI fragment of YCp5072 (NOGUCHI et al. 1996 ) was introduced into the EcoRI/ApaI site of pBluescript IISK(+), resulting in pTKSDIS3P. The SacI-ApaI fragment of pTKSDIS3P was introduced into the SacI/ApaI site of pUC28, resulting in pUCDIS3P. The NspV-NotI fragment of pUCDIS3P was introduced into the NspV/NotI site of pRS314, resulting in p314DIS3P. The XhoI fragment of p314DIS3P was introduced into the XhoI site of pRS316, resulting in p316DIS3P. The EcoRI-BglII fragment of YCp5072 (NOGUCHI et al. 1996 ) was inserted into the EcoRI/BamHI site of pBluescript IISK(+), resulting in pSKEB2.0. The EcoRI-SpeI fragment of pSKEB2.0 was inserted into the EcoRI/SpeI site of pRS404, resulting in p404DIS3-5'. The PstI-NotI fragment of p314dis3ts was inserted into the PstI/NotI site of p404DIS3-5', resulting in p404dis3ts. The PstI-EcoRI fragment of pTKSDIS3P containing the N-terminal noncoding and the coding region of DIS3 was cut out and exchanged with the PstI-EcoRI fragment of pTKSURA3, resulting in pTKSURA3-3'. Subsequently, the BamHI-PstI fragment of pSKEB 2.0 was inserted into the BamHI/PstI site of pTKSURA3-3', resulting in pTKSdis3::URA3. Finally, the PstI-EcoRI fragment of pTKSdis3::URA3 was exchanged with the NsiI-EcoRI fragment of pSKHIS3, resulting in pNS3 (pTKSdis3::HIS3). The SacI-XhoI fragment of pUCDIS3P was inserted into the SacI/SalI site of YEplac195, resulting in p195DIS3P.

The DNA fragment containing the C-terminal open reading frame (ORF) of mtr4-1 and the 3' noncoding region of MTR4 was amplified from the DNA of pRMts (LIANG et al. 1996 ), using as the primers MTR4-6 (GAA ACC TGT CGA CCC TAC CTT AC) and MTR4-8 (AAG ATA CTA GTC TGG ATT CTG G), digested with the restriction enzymes SalI and SpeI, and then introduced into the SalI/SpeI site of pRS405, resulting in p405mtr4CT.

Disruption of DIS3 gene:
DNA of the plasmid pNS3 (dis3::HIS3) was digested with the restriction enzymes SacI and ApaI and introduced into the S. cerevisiae diploid strain N43, resulting in the strain YSN2, into which p316DIS3P was introduced. The resulting strain YSN2[p316DIS3P] was sporulated and a haploid segregant YSN2T-1 was isolated and mated with the YPH499. The resulting diploid was sporulated and a haploid strain YSN3T-1b was isolated.

Mutagenesis of DIS3 gene:
The N-terminal and C-terminal parts of the S. cerevisiae Dis3p ORF carried on p314DIS3P were separately amplified by the error-prone PCR (BECKMAN et al. 1985 ; LEUNG et al. 1989 ) as described by OKI et al. 1998 using two sets of primers: the N-terminal set, ATT CAG TAG CAC ATG GCG GAA AAG and AGA ACA CAG GTC GGT ACC TAG AAG [amino acids (aa) 1–614], and the C-terminal set, GAT CCA CAA AGC AGT AGT ACA CAG and TGA AAG CGC GCA AGT GGT TTA GTG (aa 413–1001). Site-directed mutagenesis of DIS3 was performed using p314DIS3P by the site-directed mutagenesis system Mutan-K (TaKaRa, Kyoto, Japan).

Extraction of total RNA:
Cultures of 10 ml sampled at the indicated time points were centrifuged at 10,000 rpm for 5 min. Cell pellets were washed with ice-cold ddH₂O containing diethyl pyrocarbonate (DEPC; 0.1%) and suspended in 0.2 ml of lysis buffer (0.5 M NaCl, 0.2 M Tris-HCl, pH 7.6, 0.01 M EDTA, 1% SDS). After addition of glass beads and 0.2 ml of chloroform-saturated phenol, cells were vortexed for 3 min and then received 0.3 ml of lysis buffer and 0.3 ml of chloroform-saturated phenol. After further vortexing for 3 min, the mixture was centrifuged at 10,000 rpm for 5 min, and the supernatant received 0.3 ml of chloroform-saturated phenol and was vortexed and then centrifuged at 10,000 rpm for 5 min. Sodium acetate was added to 0.3 M, followed by 1 ml of 100% ethanol, and the mixture was kept at -20° overnight and then centrifuged at 10,000 rpm for 15 min. The precipitate was washed twice with 100% ethanol and dried. Afterward, the precipitates were suspended in TE buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA) containing 0.1% DEPC at the final RNA concentration (2.0 mg/ml) and stored at -80°. All procedures were carried out at 4° except where otherwise indicated.

Northern hybridization:
In the 5.8S rRNA processing analysis, 4 µl of total RNA for each lane was electrophoresed at 250 V, 16 mA for 12 hr in TBE buffer containing 8.3 M urea, 5.0% Long Ranger (FMC, Rockland, ME), 0.05% ammonium persulfate (APS), and 0.07% TEMED (N,N,N',N'-tetramethylethylenediamine) and electrotransferred onto Hybond-N (Amersham Pharmacia Biotech) in 60 mM sodium acetate buffer at 120 mA for 15 hr as described (MITCHELL et al. 1996 ).

In the mRNA analysis, 2 µl of total RNA was electrophoresed at 50 V, 20 mA for 8 hr in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer containing 1% agarose, 1x MOPS (20 mM MOPS, 10 mM CH₃COONa · 3H₂O, and 0.5 M EDTA), and formamide (16%) and transferred onto Hybond (Amersham Pharmacia Biotech, Arlington Heights, IL) in 20x SSC.

The prepared RNA filters were prehybridized twice with 100 µg/ml of single-strand salmon sperm DNA at 50° for 2 hr in buffer containing 0.5% SDS, 50% formamide, 6x SSC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), and 5x Denhardt's solution and were then incubated with ³²P-labeled oligodeoxyribonucleotides for 8 hr. After hybridization, filters were washed in the following manner: once in 2x SSC plus 0.1% SDS for 10 min at room temperature, once in 1x SSC plus 0.1% SDS for 10 min at 42°, and twice in 1x SSC plus 0.1% SDS for 10 min at 42°. Finally, filters were dried and analyzed by Fuji Bioimage analyzer.

The oligodeoxyribonucleotide probes 1, 2, 3, 4, 5, 6, and 7 that were used for 5.8S rRNA analysis were as follows: 1, CGA ACG ACA AGC CTA CTCG; 2, CAT GGC TTA ATC TTT GAG AC; 3, CCA GTT ACG AAA ATT CTTG; 4, TTT CGC TGC GTT CTT CATC; 5, TGA GAA GGA AAT GAC GCT; 6, GGC CAG CAA TTT CAA GTTA; and 7, GAA CAT TGT TCG CCT AGA, as described by DE LA CRUZ et al. 1998 . The 1.2 kb of CRY1 genomic DNA (from 175,773 to 176,958 on S. cerevisiae chromosome III) containing an intron was amplified by PCR, using as the 5' primer GGA AAG CTT ACA AGT TCT GGT ATA TTCTAT and as the 3' primer AAC TCA TAA GCT TCT ACC TCT TCT ACC, to be used as a probe for mRNA analysis.

Pulse-chase labeling experiments:
dis3, gsp1, and, as a control, wild-type Dis3 strains were grown to OD₆₆₀ = 0.2 in 100 ml of YPD medium and harvested by centrifugation. Cells were resuspended in SD medium lacking uracil at a density of 20 OD₆₆₀/ml in a total volume of 1 ml. The culture was preincubated for 15 min at 37° with shaking, 0.8 mCi of prewarmed [5.6-³H]uracil (TRK408; Amersham Pharmacia Biotech) was added to the culture, and it was incubated for 20 min (SACHS and DAVIS 1990 ; TOLLERVEY et al. 1993 ). Following centrifugation, cells were suspended in 100 ml of prewarmed SD medium containing 0.24 mg/ml of uracil (final concentration) and incubated at 37°. Every 10 min, 20 ml of culture was sampled and total RNA was extracted as described above. Total RNAs of 20,000 cpm were resolved on 5.75% polyacrylamide gel containing 7.0 M urea (TaKaRa Long Ranger single pack 373; 34 cm), using 12-cm-long, 1-mm-thick glass, at 10 mA (constant current) at 4° for 400 min. After electrophoresis, gels were fixed with 10% methanol, 10% CH₃COOH, and then RNA was transferred to Hybond (Amersham Pharmacia Biotech). The membranes were sprayed with ENLIGHTNING (New England Nuclear Life Science Products, Boston) and were exposed to X-ray films for 96–120 hr at -80° (DE LA CRUZ et al. 1998 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Construction of temperature-sensitive S. cerevisiae dis3 strains:
To introduce mutations into the S. cerevisiae DIS3 gene, the ORF of Dis3p was divided into an N-terminal half (aa 1–614) and a C-terminal half (aa 413–1001). Subsequently, each region was separately amplified by error-prone PCR. The amplified DNA fragments were introduced into the strain 37C19 (dis3::HIS3 [YCp5072]; Table 1), along with the plasmid p314DIS3P (DIS3, CEN, TRP1), from which a part of the DIS3 gene had been removed using either PstI and ClaI (N-terminal half) or BamHI and EcoRI (C-terminal half), as shown in Fig 1A. Transfected cells were plated onto synthetic medium lacking tryptophan and uracil at 26°. Of 200,000 Trp⁺, Ura⁺ colonies, 2000 grew on synthetic medium containing 1 mg/ml of 5'-FOA. Colonies were replated onto YPD plates and incubated either at 26°, the permissive temperature, or at 37°, the nonpermissive temperature. A total of 12 ts dis3 alleles were obtained (Table 3). Some dis3 alleles possessed multiple amino acid changes. In these cases, the amino acid changes of conserved residues (underlined in Table 3) were introduced into the wild-type DIS3 gene by site-directed mutagenesis, to identify single amino acid changes responsible for the ts phenotype. Finally, 4 ts dis3 alleles had single amino acid changes (Table 3). To characterize in a uniform genetic background, all of the ts dis3 genes listed in Table 3 and, as a control, the wild-type DIS3 were inserted into the haploid strain YSN3T-1b (dis3::HIS3[p316DIS3P]) by homologous recombination, and colonies (Trp⁺, Ura⁺) were incubated in synthetic medium containing 1 mg/ml of 5'-FOA. All of the resulting dis3 strains ceased to grow after incubation at 37° for 12 hr in liquid medium (representative results are shown in Fig 1B). Southern hybridization analysis revealed that the dis3 alleles were correctly integrated into the chromosome (data not shown) and all of the dis3 mutant strains could be complemented by the wild-type DIS3 gene.

View larger version (50K): In this window In a new window Download PPT slide   Figure 1. Construction and growth of ts dis3 strains. (A) Localized mutagenesis of the DIS3 gene. The DIS3 gene was amplified by mutagenic PCR using two sets of primers, DIS3NTN/DIS3NTC and DIS3M/DIS3C. The resulting PCR products were cotransformed into the strain 37C19 with the plasmid p314DIS3P treated with restriction enzymes as shown. Transformants (Ura+, Trp+) were selected for ts growth as described in MATERIALS AND METHODS. *, mutagenized DIS3 gene. The number with an asterisk (*) is the nucleotide number on S. cerevisiae chromosome XV, and the number without an asterisk is the arbitrary number of the nucleotide on the cloned DIS3 DNA fragment. (B) Growth curve of DIS3 and dis3 strains. Overnight cultures of DIS3 and ts dis3 strains were diluted into YPD medium and grown to OD660 nm = 0.1. Cultures (5 ml) were centrifuged, suspended in the same volume of prewarmed (37°) YPD medium, and then incubated at 37° (solid line). Another 5-ml culture was incubated at 26° (dotted line). At the indicated times (hr), the OD660 nm was measured. For dis3 strains, only the growth curve at 37° is shown.

3' end processing of 5.8S rRNA is blocked at three distinct sites:
A series of the dis3 strains and, as a control, the wild-type DIS3 strain (dis3-WT) were cultured at 26° to OD₆₆₀ = 0.8 and then incubated at 37°. After incubation for 2, 4, and 8 hr, total RNA was extracted, resolved by polyacrylamide gel electrophoresis, and hybridized with oligonucleotide 4, which is complementary to the mature 5.8S rRNA (Fig 2A, bottom). In the DIS3 strain, two bands corresponding to 7S and 5.8S rRNAs were detected. Additionally, one or two fragments intermediate in length between 7S and 5.8S rRNA appeared in 12 out of 13 dis3 strains upon incubation at 37° (Table 3; representative results are shown in Fig 2A).

View larger version (82K): In this window In a new window Download PPT slide   Figure 2. dis3 strains have defects in 3' end processing of 5.8S rRNA, similar to other exosome mutants. (A) Ladders of intermediate 5.8S rRNA fragments observed in dis3. Cultures (50 ml) of dis3 strains and, as a control, the wild-type strain DIS3 were grown at 26° to OD660 = 0.8, centrifuged, and resuspended in the same volume of prewarmed (37°) YPD medium. After incubation at 37° for 0, 2, 4, and 8 hr as indicated, 10 ml was centrifuged and the extracted total RNA was subjected to Northern blot analysis with oligonucleotide 4 (bottom). Mutants showing a representative pattern are shown. Arrows indicate the position of 7S pre-RNA and 5.8S rRNA, and arrowheads indicate the long (L), middle (M), and short (S) intermediates. The bottom indicates the position of the nucleotide probes in the 35S rRNA. (B) 3' end processing was blocked in dis3 mutants. Total RNAs (10 µl) extracted from dis3-1, -4, and -9 strains were mixed, and then 5 µl of the mixture was electrophoresed, transferred to filters, and hybridized with oligonucleotide 4, 5, or 6 as indicated. Arrows indicate the position of 7S pre-RNA and 5.8S rRNA, and arrowheads indicate the long (L), middle (M), and short (S) intermediates. (C) Cultures (100 ml) of strains Gal10::RRP4 and GAL10::RRP44 were grown at 26° to OD660 = 0.8 in synthetic medium without uracil containing 2% galactose, centrifuged, and resuspended in the same volume of a synthetic medium containing raffinose. After incubation for 0, 2, 4, 8, 12, and 24 hr, 10 ml was centrifuged. For comparison, 50 ml of the strain rrp4-1, grown at 26° to OD660 = 0.8, was centrifuged and resuspended in the same volume of prewarmed (37°) YPD medium. After incubation at 37° for 0, 2, 4, and 8 hr, 10-ml samples were collected by centrifugation. Total RNA was analyzed by Northern hybridization with oligonucleotide 4 (A, bottom). Lane M contains the mixture of total RNA of dis3-1, -4, and -9 strains. Arrows indicate the position of 7S pre-RNA and 5.8S rRNA, and arrowheads indicate the long (L), middle (M), and short (S) intermediates.

To determine whether the observed intermediates are 3' end-extended forms of 5.8S rRNA, total RNAs of dis3-1, -4, and -9 strains, which contain the middle, long, and short intermediate fragments, respectively, were mixed and hybridized with the oligonucleotides 3, 4, 5, or 6. The positions of these oligonucleotides in pre-rRNA are shown in the bottom of Fig 2A. As expected, probe 4 hybridized with all three intermediate fragments, in addition to both 7S and 5.8S rRNA (Fig 2B, probe 4). Probe 5, which is partially complementary to both the 3' end of 5.8S rRNA and the 5' end of the ITS2 spacer, hybridized with all three intermediate fragments and with 7S rRNA, but not with the mature 5.8S rRNA, as previously reported (MITCHELL et al. 1996 ) (Fig 2B, probe 5). On the other hand, probe 6, which is complementary to a site in the ITS2 50 nucleotides farther 3' from the mature 5.8S rRNA, hybridized with 7S rRNA and the two long (L) and middle (M) intermediate fragments (Fig 2B), but not with the short (S) intermediate fragment (Fig 2B, probe 6). No 5.8S rRNA fragments hybridized with probe 3 (data not shown), which is complementary to a site in ITS1 proximal to the 5' end of 5.8S rRNA. These observations prove that the fragments of 5.8S rRNA intermediate in length between 7S and 5.8S rRNAs are 3' extended forms of 5.8S rRNA.

To learn whether loss of another exosomal component also inhibits 5.8S rRNA maturation in a similar pattern, the strains GAL10::rrp4, rrp4-1, and GAL10::rrp44 were precultured at 26° in synthetic medium containing 2% galactose or YPD medium and then incubated under nonpermissive conditions. At the indicated times, total RNAs were extracted, coelectrophoresed with the mixture of total dis3 RNAs, and hybridized with oligonucleotide 4. As reported (MITCHELL et al. 1996 , MITCHELL et al. 1997 ), ladders of 5.8S rRNA fragments appeared in GAL10::rrp4 and GAL10::rrp44, both after 8–24 hr of incubation in the absence of galactose, and also in rrp4-1 after 2 hr of incubation at 37° (Fig 2C). Strikingly, the lengths of 5.8S rRNA fragments observed were identical to those found in dis3 mutants (Fig 2C, compare with lane M), indicating that the 3' end processing of 5.8S rRNA from 7S rRNA was inhibited at the same three sites by loss of exosome function.

Northern analysis cannot show whether 5.8S rRNA maturation was terminated at these three distinct sites, as opposed to being paused for an extended period. In the latter case, intermediate fragments will ultimately be processed to mature 5.8S rRNA. To address this issue, several dis3 mutants and, as a control, wild-type DIS3 strains were labeled in vivo with [5.6-³H]uracil for 20 min at 37° and then chased with an excess of cold uracil for 60 min. Every 10 min, total RNA was extracted and low molecular weight RNA species were analyzed as described (DE LA CRUZ et al. 1998 ). We chose dis3 strains dis3-1, dis3-4, dis3-9, and dis3-8, which accumulate one or two intermediate fragments at 37° (Table 3). In DIS3 cells, mature 5.8S rRNA appreared immediately after 20-min pulse labeling. On the other hand, in the dis3 mutants, fragments intermediate in length between 7S and 5.8S rRNA appeared before labeling of 5.8S rRNA was seen. Representative results are shown in Fig 3A. Even after a 60-min chase, the amount of the intermediate species did not decrease (Fig 3B). To compare the number and length of labeled intermediates, total RNA extracted after a 60-min chase was analyzed (Fig 3B). Strikingly, labeled intermediates showed three distinct lengths. For instance, dis3-1 accumulated a single M-intermediate while dis3-8 accumulated two intermediates (L and S; Fig 3B, lanes 1 and 6), consistent with the Northern analysis (Fig 2A). These results indicate that 5.8S rRNA maturation is blocked at three distinct sites in an allele-specific manner.

View larger version (132K): In this window In a new window Download PPT slide   Figure 3. In both dis3 and gsp1 mutants, 3' end processing of 5.8S rRNA was blocked at three distinct sites. Cultures of dis3-WT(DIS3), dis3-1, dis3-4, dis3-8, dis3-9, and gsp1-1260 were grown in YPD medium, transferred to SD medium lacking uracil, labeled with [5.6-3H]uracil for 20 min, and then chased with an excess of cold uracil for 60 min. Immediately after labeling (0), and then after chasing for 10, 20, 30, and 60 min, total RNA was extracted, separated on 5.75% polyacrylamide-7.0 M urea gels, transferred to nylon membrane, and visualized by fluorography. Approximately 20,000 cpm was loaded in each lane. (A) Representative results of time-course experiments. (B) Total RNAs extracted from indicated strains after a 60-min chase were analyzed for comparison. The positions of the 7S, 5.8S, and 5S rRNA are indicated by arrows. Arrowheads indicate the long (L), middle (M), and short (S) intermediates.

gsp1 has a defect in 3' end but not 5' end processing of 5.8S rRNA, similar to dis3:
The maturation of 5.8S rRNA was examined in 25 gsp1 strains, to learn whether there is any functional relationship between the Ran-GTPase and the exosome. Total RNAs extracted from gsp1 strains after incubation for 2, 4, and 8 hr at 37° were analyzed by Northern hybridization with oligonucleotide 4. In 20 out of 25 gsp1 strains, intermediate 5.8S rRNA fragments appeared (Table 4). Representative results are shown in Fig 4A. Remarkably, three intermediates seen in gsp1 mutants have the same lengths as those of dis3 mutants (Fig 4A, compare with lane M), suggesting that 3' end processing of 5.8S rRNA is inhibited in a similar manner in both gsp1 and dis3 mutants. This is also the case after chasing of [5.6-³H]uracil-labeled RNA with an excess of cold uracil. gsp1-1260 accumulated both L- and M-length intermediates (Fig 3A and Fig B), as observed by Northern analysis (Table 4). Thus, 5.8S rRNA maturation is blocked at three distinct sites in gsp1 mutants at 37°, as in dis3 mutants.

View larger version (112K): In this window In a new window Download PPT slide   Figure 4. gsp1 strains have a defect in 3' end, but not 5' end, processing of 5.8S rRNA, similar to dis3. (A) gsp1 mutations blocked 3' processing of 5.8S rRNA at three sites. Cultures (50 ml) of gsp1 strains indicated (gsp1-1819, gsp1-1894, gsp1-1518, gsp1-1260, and gsp1-1582) were prepared as for dis3 and samples of total RNA extracted after incubation at 37° for 0, 2, 4, and 8 hr were analyzed by Northern hybridization with oligonucleotide 4 (Fig 2A, bottom). Lane M contains the mixture of total RNAs of dis3-1, -4, and -9 strains. Arrows indicate the position of 7S pre-RNA and 5.8S rRNA, and arrowheads indicate the long (L), middle (M), and short (S) intermediates. (B) Primer extension analysis of the 5' end of 5.8S rRNA. Total RNA was extracted from gsp1-1907 (lane 1), dis3-1 (lane 2), and DIS3 (lane 3) after incubation at 37° for 8 hr. Primer extension was performed using oligonucleotide 5 (Fig 2A, bottom). A sequencing reaction on the rDNA repeat using the same primer is also shown.

To determine the 5' end of 5.8S rRNA, primer extension analysis of total RNAs extracted from gsp1-1907 was carried out using as a primer the oligonucleotide 5, which hybridizes to all intermediate 5.8S rRNA fragments and to 7S pre-rRNA, but not to mature 5.8S rRNA (Fig 2B, probe 5). dis3-1 and gsp1-1907 accumulate one and three intermediates, respectively, at 37° (Fig 2A and Table 4). In both mutants, the 5' end of 5.8S rRNA terminates near nucleotides 2855(A) and 2864(A), corresponding to the B1(L) and B1(S), respectively, as reported (HENRY et al. 1994 ; Fig 4B). Therefore, the 5' end of 5.8S rRNA was terminated correctly in both gsp1 and dis3, even when the 3' end processing was blocked. Consistent with a previous report (HENRY et al. 1994 ), there was much less 5.8S(L) than 5.8S(S) in both dis3 and gsp1.

These results indicate that loss of RanGTPase activity inhibits 3' end processing, but not 5' end processing, of 5.8S rRNA maturation. It is notable that the number of intermediate fragments depends on the gsp1 allele, as for dis3 mutants.

Overexpression of Dis3p and Mtr4p enhances 3' end processing of 5.8S rRNA:
To further examine the relationship between the RanGTPase and the exosome, a high copy DIS3 plasmid, p195DIS3P, was introduced into the gsp1 strains and 3' processing of 5.8S rRNA was examined in the resulting transformants. Representative results are shown in Fig 5A. Upon overexpression of Dis3p, the ladders of 5.8S rRNA intermediates were shifted toward the shorter species (Fig 5A, a), while all 5.8S rRNA intermediates were removed by overexpression of Gsp1p. In the case of gsp1-640, all 5.8S rRNA intermediates disappeared upon overexpression of Dis3p (Fig 5A, Fig B). The same change occurred upon overexpression of Rrp4p, although Dis3p and Rrp4p did not rescue the ts growth phenotype of gsp1 (data not shown).

View larger version (35K): In this window In a new window Download PPT slide   Figure 5. Overexpression of Dis3p and Mtr4p, but not Ski2p, rescues 3' end processing defects of 5.8S rRNA maturation. (A) Cultures of gsp1-1486 (a) and gsp1-640 (b) containing p195DIS3P, p195GSP1, or YEplac195 vector alone, as indicated, were incubated at 36° for 0, 2, 4, and 8 hr. Cultures of gsp1-640 (c) containing pGM410 (GAL10::MTR4), pYSKI2 (ADH::SKI2), or YEplac195 vector alone, as indicated, which had been grown to OD660 = 0.8 at 26°, were precultured in synthetic medium containing 2% galactose for 1 hr at 26° and then cultured in the same medium at 37° for 0, 2, 4, 8, 12, and 24 hr. Total RNAs extracted at the indicated times were analyzed by Northern hybridization with oligonucleotide 4 (Fig 2A, bottom). Lane M contains the mixture of total RNA of dis3-1, -4, and -9 strains. Arrowheads indicate 7S pre- and 5.8S rRNAs, and the long (L), middle (M), and short (S) intermediates. (B) dis3 and mtr4 are synthetic lethal. The strains ENY42-2 (mtr4-1 dis3-4), ENY46-2 (mtr4-1 dis3-6), ENY47-6 (mtr4-1 dis3-8), ENY48-1 (mtr4-1 dis3-11), ENY49-5 (mtr4-1 dis3-12), ENY50-7 (mtr4-1 dis3-14), and, as controls, dis3 strains, all carrying the plasmid p195DIS3, were streaked on 5'-FOA-containing plates to counterselect p195DIS3 and were incubated at 26° for 3 days.

A putative ATP-dependent RNA helicase, Mtr4p/Dob1p, functions as a cofactor for the exosome (DE LA CRUZ et al. 1998 ). When the MTR4 gene was overexpressed in gsp1-640, growth was still temperature sensitive, but a 5.8S rRNA intermediate disappeared (Fig 5A, Fig C). Moreover, when the mtr4-1 mutation (LIANG et al. 1996 ) was introduced into dis3, strains carrying some alleles of dis3 did not grow even at 26° (Fig 5B). Thus, there is an allele-specific synthetic lethality between mtr4-1 and dis3. Taken together with the previous report that a strain (dob1-1 rrp4-1) grows poorly (DE LA CRUZ et al. 1998 ), these results indicate a functional interaction between Dob1p/Mtr4p and the exosome. Indeed, Mtr4p was reported to be required for the nuclear exosome, which carries out 5.8S rRNA maturation, but not for the cytoplasmic exosome, which performs 3'-to-5' mRNA degradation (VAN HOOF et al. 2000 ). Accordingly, overexpression of Ski2p, another putative ATP-dependent RNA helicase that is required for mRNA degradation (JACOBS ANDERSON and PARKER 1998 ), did not eliminate the 5.8S rRNA intermediates (Fig 5A, Fig C).

Nucleocytoplasmic transport mutants show no defect in exosomal activity:
Several snoRNPs and ribosomal proteins are involved in rRNA processing (WOOLFORD 1991 ; LAFONTAINE and TOLLERVEY 1995 ; MAXWELL and FOURNIR 1995 ; BURGE et al. 1999 ). Those proteins and RNAs are imported into the nucleolus for ribosome genesis. Therefore, inhibition of Ran-dependent nucleocytoplasmic transport could cause defects of 5.8S rRNA maturation. To address this possibility, 5.8S rRNA maturation was investigated in strains defective in the nucleocytoplasmic transport. We chose four mutants defective in a general nuclear import or export pathway: mog1, in which both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports are defective (OKI and NISHIMOTO 1998 ); srp1, which is a ts mutant of the importin- homologue (YANO et al. 1994 ); lrc4, an allele of crm1 that is required for leucine-rich nuclear export signal-dependent nuclear export (STADE et al. 1997 ); and yrb2, which is defective in the Ran-dependent nuclear export (NOGUCHI et al. 1999 ).

After incubation at 37° for 0–8 hr, total RNAs were analyzed by Northern hybridization using oligonucleotide 4. Even after incubation for 8 hr at 37°, no intermediates were seen (data not shown). To confirm that the nucleocytoplasmic transport defects were induced under our experimental conditions, the presence of pre-mRNA was examined in the same RNA extracts by using a CRY1 probe. As controls for pre-mRNA splicing, we used prp8-1 (JAMIESON et al. 1991 ) and three alleles of prp20 (SEKI et al. 1996 ). After incubation at 37° for 8 hr, pre-mRNA, which appeared after 2 hr of incubation at 37° in prp8-1, was seen in NOY612 (srp1) and ENY9-22 (lrc4) strains, as for prp20 alleles (Fig 6A and Fig B).

View larger version (63K): In this window In a new window Download PPT slide   Figure 6. mRNA splicing is affected by dis3 and nucleocytoplasmic transport mutants. Total RNA was prepared as described above from cultures of strains (A) prp8-1 (SPJ8.31), prp20-1 (prp20/2c), srm1-1 (SY1115), and mtr1-2 (T18); (B) moy1, srp1 (NOY612), lrc4 (ENY9-22), yrb2 (ENY38-6), and wt (wild type); and (C) dis3-11, dis3-12, dis3-13, dis3-14, and dis3-6 after incubation at 37° for 0, 2, 4, and 8 hr as indicated and was then analyzed by using a CRY1 probe. Arrows indicate the position of unspliced CRY1 mRNA.

Using the same probe, we then sought to detect pre-mRNA in total RNA extracts from gsp1 and dis3 strains, to learn whether mRNA splicing occurred in our experimental conditions. No pre-mRNA appeared in more than half of dis3 and gsp1 mutant strains that accumulate intermediate 5.8S rRNA fragments upon incubation at 37° (Table 3 and Table 4). Only four dis3 alleles showed inhibition of mRNA splicing, equivalent to prp8-1 (Fig 6C).

Early steps of rRNA processing are defective in gsp1, as in dis3:
The absence of 5.8S rRNA intermediates does not imply that the exosome functions normally. Using oligonucleotide 1, the position of which is shown at the bottom of Fig 2A, we have therefore evaluated accumulation of the 5'-A0 fragment, which is generated by processing of 35S-to-33S rRNA and is degraded by the exosome (DE LA CRUZ et al. 1998 ). As shown in Fig 7A, the 5'-A0 fragment accumulated in most of gsp1 and dis3 mutant strains examined. It is notable that the 5'-A0 fragment accumulated even in those dis3 and gsp1 mutants that show no 5.8S rRNA intermediates (Fig 7A, dis3-6, mtr17-1, and gsp1-882, -1060, -1757, and -1817). Thus, all gsp1 mutants examined are defective in exosomal function. In contrast, the 5'-A0 fragment was not detected in nucleocytoplasmic transport mutants even after 8 hr of incubation at 37° (Fig 7A, srp1 and lrc4).

View larger version (37K): In this window In a new window Download PPT slide   Figure 7. Effects of dis3 and gsp1 mutations on steady-state levels of products of rRNA processing. Cultures (50 ml) were grown at 26° to OD660 = 0.8, centrifuged, and resuspended in the same volume of prewarmed (37°) YPD medium. After incubation at 37° for 0, 2, 4, and 8 hr, 10 ml of cultures was centrifuged, and total RNA was extracted. Equal amounts of total RNA were resolved on 8.3 M urea-5.0% Long Ranger (A) or 1.5% agarose-formaldehyde gels (B) and transferred to a nylon membrane for Northern hybridization. (A) Hybridization with oligonucleotide probe 1 (Fig 2A, bottom). Each lane contains total RNA extracted from indicated strains. Zero- (left) and 8-hr (right) samples were analyzed. Positions of 35S rRNA and 5'-A0 fragments are shown by arrows. (B) Hybridization with oligonucleotide probe 7 (27S) and 2 (18S). Each contains total RNA extracted from indicated strains. Zero-, 4-, and 8-hr incubation samples were analyzed. Positions of 27S and 18S are shown by arrows.

The steady-state level of 35S pre-rRNA increased in both dis3 and gsp1 mutants that accumulated the 5'-A0 fragment (Fig 7A, compare 35S and 5'-A0). This finding may indicate that the pre-rRNA cleavage at the A0 and A1 sites was affected in these mutants, which is consistent with the report that depletion of each of the individual exosome components inhibits the early pre-rRNA cleavage at sites A0, A1, A2, and A3 (ALLMANG et al. 2000 ). In this context, we examined the steady-state level of 27S and 18S rRNA using nucleotide probes 2 and 7. As reported, some dis3 strains showed a strong reduction of 27S rRNA (Fig 7B, dis3-6 and mtr17-1). Similarly, the level of 27S rRNA was reduced in some alleles of gsp1 (Fig 7B, right side). In contrast, nucleocytoplasmic transport mutants did not show any change in the level of 27S (Fig 7B, srp1 and lrc4). We did not detect a significant reduction in the level of 18S rRNA.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We mutagenized the DIS3 gene with error-prone PCR. The lethality of the PCR product was >90%. Such a high lethality may be important for efficient isolation of ts mutants. All 12 new dis3 alleles contain distinct amino acid change(s). Using these mutants, the functional interaction between Dis3p and Gsp1p was investigated.

Almost all dis3 mutants show a ladder(s) of 3' extended fragments intermediate in length between 7S and 5.8S rRNAs at 37°, the nonpermissive temperature. This is consistent with the previous report that Dis3p is a subunit of the exosome (MITCHELL et al. 1997 ). The lengths of 5.8S rRNA intermediates were grouped into three size classes. Although 5.8S rRNA intermediates were previously reported to appear upon loss of exosome function (MITCHELL et al. 1996 , MITCHELL et al. 1997 ; DE LA CRUZ et al. 1998 ; ALLMANG et al. 1999 ), there is no report indicating that 3' processing can be blocked at three distinct points. According to the model of VAN NUES et al. 1995 , the arrest site proximal to the 3' end of 5.8S rRNA seems to be at helical domain II in the ITS2 region of the 35S pre-rRNA unit, since the shortest intermediate fragment hybridizes with nucleotide 5, but not with nucleotide 6. On the other hand, the 3' end of the longest intermediate fragment could be near the 3' end of helical domain IV in the ITS2 region. Pulse-chase experiments revealed that 5.8S rRNA maturation was blocked at these three sites by loss of exosome function.

Since Ran/Gsp1p is required for nucleocytoplasmic transport, 5.8S rRNA maturation could be indirectly inhibited in gsp1 due to defects in nucleocytoplasmic transport. But it is unlikely for the following reasons. First, the majority of gsp1 mutants showing intermediate 5.8S rRNA fragments did not accumulate a pre-mRNA, which does accumulate in nucleocytoplasmic transport mutants. Therefore, any nucleocytoplasmic transport is active enough to carry out pre-mRNA splicing in most of gsp1 strains. Second, the 5' end of 5.8S rRNA intermediates was correctly terminated in gsp1, as in dis3. A large number of trans-acting factors are required for rRNA processing, which have been characterized as nucleases, ribonucleoprotein particles, putative RNA helicases, and ribosome assembly factors (ALLMANG et al. 2000 ). The fact that the 5' end of 5.8S rRNA is correctly terminated therefore indicates that at least some rRNA processing factors function correctly in gsp1 strains. Finally, in both dis3 and gsp1, 5.8S rRNA maturation was blocked at the same three distinct sites. Such an allele-specific phenotype is consistent with there being a direct interaction between Gsp1p and the exosome. In fact, S. cerevisiae Dis3p does bind directly to Gsp1p (NOGUCHI et al. 1996 ). Dis3p binds to either GTP-Gsp1p or GDP-Gsp1p and enhances RCC1-stimulated nucleotide exchange. Recently, Ran was reported to induce spindle assembly by releasing importin- from the TPX2 complex (GRUSS et al. 2001 ) or importin-ß from the NuMA complex (NACHURY et al. 2001 ). Ran was already known to stimulate both assembly and disassembly of protein complexes during nucleocytoplasmic transport of macromolecules (MATTAJ and ENGLMEIER 1998 ; GORLICH and KUTAY 1999 ). In this context, Ran/Gsp1p may regulate assembly/disassembly of the exosome, which comprises 11 components (ALLMANG et al. 1999 ; VAN HOOF and PARKER 1999 ). In this case, overexpression of Dis3p may rescue the exosome when the interaction between Dis3p and mutated Gsp1p is weak, and overexpression of Mtr4p may increase exosomal activity, which is weakened by binding to mutated Gsp1p. It is notable that Ski2p, which is an RNA helicase like Mtr4p, did not rescue a defect of 5.8S rRNA processing. Since Ski2p is required for 3'-to-5' mRNA degradation (JACOBS ANDERSON and PARKER 1998 ), this finding indicates that a defect in Gsp1p specifically inhibits 5.8S rRNA maturation, which is carried out by the nuclear exosome. Consistently, early steps of rRNA processing, which are carried out by the nuclear exosome (VAN NUES et al. 1995 ), are also affected in gsp1 as in dis3. Recently, Ski7p, which belongs to the GTPase family, was reported to be required for 3'-to-5' mRNA degradation (VAN HOOF et al. 2000 ). In this context, Ran/Gsp1p may substitute for Ski7p in 5.8S rRNA processing.

It is notable that dis3 strains, which quickly accumulate pre-mRNA, contain amino acid changes in the region from residue 562 to 566 [G562D (dis3-6), E565K (dis3-14), and V566G (dis3-12)]. V566 is conserved from yeast to humans (SHIOMI et al. 1998 ). These residues may be required for a function of Dis3p in pre-mRNA splicing, although it is not known how the exosome may function in mRNA splicing. Alternatively, Dis3p could function in pre-mRNA splicing by virtue of its being part of distinct complexes other than the exosome.

	ACKNOWLEDGMENTS

We thank Drs. M. Nomura and D. Tollervey for rRNA vector and the strains GAL::RRP44, GAL10::RRP4, and rrp4-1, respectively. This work was supported by Grants-in-Aid for Specially Promoted Research from The Ministry of Education, Science, Sports and Culture of Japan.

Manuscript received August 25, 2000; Accepted for publication March 13, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

AEBI, M., M. W. CLARK, U. VIJAYRAGHAVAN, and J. ABELSON, 1990  A yeast mutant, PRP20, altered in mRNA metabolism and maintenance of the nuclear structure, is defective in a gene homologous to the human gene RCC1 which is involved in the control of chromosome condensation. Mol. Gen. Genet. 224:72-80[Medline].

ALLMANG, C., E. PETFALSKI, A. PODTELEJNIKOV, M. MANN, and D. TOLLERVEY et al., 1999  The yeast exosome and human PM-Scl are related complexes of 3' 5' exonucleases. Genes Dev. 13:2148-2158[Abstract/Free Full Text].

ALLMANG, C., P. MITCHELL, E. PETFALSKI, and D. TOLLERVEY, 2000  Degradation of ribosomal RNA precursors by the exosome. Nucleic Acids Res. 28:1684-1691[Abstract/Free Full Text].

AZUMA, Y. and M. DASSO, 2000  The role of Ran in nuclear function. Curr. Opin. Cell Biol. 12:302-307[Medline].

BECKMAN, R. A., A. S. MILDVAN, and L. A. LOEB, 1985  On the fidelity of DNA replication: manganese mutagenesis in vitro. Biochemistry 24:5810-5817[Medline].

BISCHOFF, F. R., H. KREBBER, T. KEMPF, I. HERMES, and H. PONSTINGL, 1995  Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport. Proc. Natl. Acad. Sci. USA 92:1749-1753[Abstract/Free Full Text].

BOEKE, J. D., F. LACROUTE, and G. R. FINK, 1984  A positive selection for mutants lacking ortidine-5'-phosphate decarboxylase activity in yeast. Mol. Gen. Genet. 197:345-346[Medline].

BURGE, C. B., T. TUSCHL and P. A. SHARP, 1999 Splicing of precursors to mRNAs by the spliceosomes, pp. 525–560 in The RNA World, Ed. 2, edited by R. F. GESTELAND, T. R. CECH and J. F. ATKINS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

CHENG, Y., J. E. DAHLBERG, and E. LUND, 1995  Diverse effects of the guanine nucleotide exchange factor RCC1 on RNA transport. Science 267:1807-1810[Abstract/Free Full Text].

CLARK, K. L. and G. F. SPRAGUE, JR., 1989  Yeast pheromone response pathway: characterization of a suppressor that restores mating to receptorless mutants. Mol. Cell. Biol. 9:2682-2694[Abstract/Free Full Text].

DE LA CRUZ, J., D. KRESSLER, D. TOLLERVEY, and P. LINDER, 1998  Dob1p (Mtr4p) is a putative ATP-dependent RNA helicase required for the 3' end formation of 5.8S rRNA in Saccharomyces cerevisiae. EMBO J. 17:1128-1140[Medline].

GORLICH, D. and U. KUTAY, 1999  Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15:607-660[Medline].

GRUSS, O. J., R. E. CARAZO-SALAS, C. A. SCHATZ, G. GUARGUAGLINI, and J. KAST et al., 2001  Ran induces spindle assembly by reversing the inhibitory effect of importin on TPX2 activity. Cell 104:83-93[Medline].

HILL, J., K. DONALD, and D. E. GRIFFITHS, 1991  DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res. 19:5791[Free Full Text].

HENRY, Y., H. WOOD, J. P. MORRISSEY, E. PETFALSKI, and S. KEARSEY et al., 1994  The 5' end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site. EMBO J. 13:2452-2463[Medline].

HOPPER, A. K., F. BANKS, and V. EVANGELIDIS, 1978  A yeast mutant which accumulates precursor tRNAs. Cell 14:211-219[Medline].

HUTCHISON, H. T., L. H. HARTWELL, and C. S. MCLAUGHLIN, 1969  Temperature-sensitive yeast mutant defective in ribonucleic acid production. J. Bacteriol. 99:807-814[Abstract/Free Full Text].

JACOBS ANDERSON, J. S. and R. PARKER, 1998  The 3' to 5' degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3' to 5' exonucleases of the exosome complex. EMBO J. 17:1497-1506[Medline].

JAMIESON, D. J., B. RAHE, J. PRINGLE, and J. D. BEGGS, 1991  A suppressor of a yeast splicing mutation (prp8–1) encodes a putative ATP-dependent RNA helicase. Nature 349:715-717[Medline].

KADOWAKI, T., D. GOLDFARB, L. M. SPITZ, A. M. TARTAKOFF, and M. OHNO, 1993  Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily. EMBO J. 12:2929-2937[Medline].

KAHANA, J. A. and D. W. CLEVELAND, 1999  Beyond nuclear transport: Ran-GTP as a determinant of spindle assembly. J. Cell Biol. 146:1205-1209[Abstract/Free Full Text].

LAFONTAINE, D. and D. TOLLERVEY, 1995  Trans-acting factors in yeast pre-rRNA and pre-snoRNA processing. Biochem. Cell Biol. 73:803-812[Medline].

LEUNG, D. W., E. CHEN, and D. V. GOEDDEL, 1989  A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11-15.

LIANG, S., M. HITOMI, Y-H. HU, Y. LIU, and A. M. TARTAKOFF, 1996  A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA. Mol. Cell. Biol. 16:5139-5146[Abstract].

MATTAJ, I. W. and L. ENGLMEIER, 1998  Nucleocytoplasmic transport: the soluble phase. Annu. Rev. Biochem. 67:265-306[Medline].

MAXWELL, E. S. and M. J. FOURNIR, 1995  The small nucleolar RNAs. Annu. Rev. Biochem. 35:897-934.

MELCHIOR, F. and L. GERACE, 1998  Two-way trafficking with Ran. Trends Cell Biol. 8:171-179[Medline].

MITCHELL, P., E. PETFALSKI, and D. TOLLERVEY, 1996  The 3' end of yeast 5.8S rRNA is generated by an exonuclease processing mechanism. Genes Dev. 10:502-513[Abstract/Free Full Text].

MITCHELL, P., E. PETFALSKI, A. SHEVCHENKO, M. MANN, and D. TOLLERVEY, 1997  The exosome: a conserved eukaryotic RNA processing complex containing multiple 3' 5' exoribonucleases. Cell 91:457-466[Medline].

NACHURY, M. V., T. J. MARESCA, W. C. SALMON, C. M. WATERMAN-STORER, and R. HEALD et al., 2001  Importin ß is a mitotic target of the small GTPase Ran in spindle assembly. Cell 104:95-106[Medline].

NAKAMURA, M., H. MASUDA, J. HORII, K. KUMA, and N. YOKOYAMA et al., 1998  A novel centrosomal protein, RanBPM, when overexpressed, causes ectopic microtubule nucleation, similar to -tubulin. J. Cell Biol. 143:1041-1052[Abstract/Free Full Text].

NAKIELNY, S. and G. DREYFUSS, 1999  Transport of proteins and RNAs in and out of the nucleus. Cell 99:677-690[Medline].

NISHIMOTO, T., 1999  A new role of Ran GTPase. Biochem. Biophys. Res. Commun. 262:571-574[Medline].

NISHIMOTO, T., 2000  Upstream and downstream of Ran GTPase. Biol. Chem. 381:397-405[Medline].

NOGUCHI, E., N. HAYASHI, Y. AZUMA, T. SEKI, and M. MAKAMURA et al., 1996  Dis3, implicated in mitotic control, binds directly to Ran and enhances the GEF activity of RCC1. EMBO J. 15:5595-5605[Medline].

NOGUCHI, E., N. HAYASHI, N. NAKASHIMA, and T. NISHIMOTO, 1997  Yrb2p, Nup2p-related yeast protein has functional overlap with Rna1p, yeast RanGAP protein. Mol. Cell. Biol. 17:2235-2246[Abstract].

NOGUCHI, E., Y. SAITOH, S. SAZER, and T. NISHIMOTO, 1999  Disruption of YRB2 gene retards nuclear protein export, causing a profound mitotic delay, and can be rescued by overexpression of XPO1/CRM1. J. Biochem. 125:574-585[Abstract/Free Full Text].

OKI, M. and T. NISHIMOTO, 1998  A protein required for nuclear import, Mog1p, directly interacts with GTP-Gsp1p, the Saccharomyces cerevisiae Ran homologue. Proc. Natl. Acad. Sci. USA 95:15388-15393[Abstract/Free Full Text].

OKI, M., E. NOGUCHI, N. HAYASHI, and T. NISHIMOTO, 1998  Nuclear protein import, but not mRNA export, is defective in all of the temperature-sensitive mutants of the Saccharomyces cerevisiae Ran homologue, Gsp1-GTPase. Mol. Gen. Genet. 257:624-634[Medline].

PECULIS, B. A. and J. A. STEITZ, 1993  Disruption of U8 nucleolar snRNA inhibits 5.8S and 28S rRNA processing in the Xenopus oocyte. Cell 73:1233-1245[Medline].

SACHS, A. B. and R. W. DAVIS, 1990  Translation initiation and ribosomal biogenesis: involvement of a putative rRNA helicase and RPL46. Science 247:1077-1079[Abstract/Free Full Text].

SEKI, T., N. HAYASHI, and T. NISHIMOTO, 1996  RCC1 in the Ran pathway. J. Biochem. 120:207-214[Abstract/Free Full Text].

SHIOMI, T., K. FUKUSHIMA, N. SUZUKI, N. NAKASHIMA, and E. NOGUCHI et al., 1998  Human Dis3p, which binds to either GTP- or GDP-Ran, complements Saccharomyces cerevisiae dis3. J. Biochem. 123:883-890[Abstract/Free Full Text].

STADE, K., C. S. FORD, C. GUTHRIE, and K. WEIS, 1997  Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90:1041-1050[Medline].

TOLLERVEY, D., H. LEHTONEN, R. JANSEN, H. KERN, and E. C. HURT, 1993  Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly. Cell 72:443-457[Medline].

VAN HOOF, A. and R. PARKER, 1999  The exosome: a proteasome for RNA. Cell 99:347-350[Medline].

VAN HOOF, A. V., P. LENNERTZ, and R. PARKER, 2000  Yeast exosome mutants accumulate 3'-extended polyadenylated forms of U4 small nuclear RNA and small nucleolar RNAs. Mol. Cell. Biol. 20:441-452[Abstract/Free Full Text].

VAN NUES, R. W., J. VENEMA, J. M. J. RIENTJES, A. DIRKS-MULDER, and H. A. RAUE, 1995  Processing of eukaryotic pre-rRNA: the role of the transcribed spacers. Biochem. Cell Biol. 73:789-801[Medline].

VEGVAR, H. E. N. and J. DAHLBERG, 1990  Nucleocytoplasmic transport and processing of small nuclear RNA precursors. Mol. Cell. Biol. 10:3365-3375[Abstract/Free Full Text].

WOOLFORD, J. L., JR., 1991  The structure and biogenesis of yeast ribosomes. Adv. Genet. 29:63-118[Medline].

WOZNIAK, R. W., M. P. ROUT, and J. P. AITCHISON, 1998  Karyopherins and kissing cousins. Trends Cell Biol. 8:184-188[Medline].

YANO, R., M. L. OAKES, M. M. TABB, and M. NOMURA, 1994  Yeast Srp1p has homology to armadillo/plakoglobin/beta-catenin and participates in apparently multiple nuclear functions including the maintenance of the nucleolar structure. Proc. Natl. Acad. Sci. USA. 91:6880-6884[Abstract/Free Full Text].

YU, Y-T., E. C. SCHARL, C. M. SMITH and J. A. STEITZ, 1999 The growing world of small nuclear ribonucleoproteins, pp. 487–524 in The RNA World, Ed. 2, edited by R. F. GESTELAND, T. R. CECH and J. F. ATKINS. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

This article has been cited by other articles:

				T. Ohba, H. Nishijima, H. Nishitani, and T. Nishimoto Schizosaccharomyces pombe Snf2SR, a novel SNF2 family protein, interacts with Ran GTPase and modulates both RanGEF and RanGAP activities. Genes Cells, June 1, 2008; 13(6): 571 - 582. [Abstract] [Full Text] [PDF]