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• W2085098052 abstract "Rea1, the largest predicted protein in the yeast genome, is a member of the AAA+ family of ATPases and is associated with pre-60 S ribosomes. Here we report that Rea1 is required for maturation and nuclear export of the pre-60 S subunit. Rea1 exhibits a predominantly nucleoplasmic localization and is present in a late pre-60 S particle together with members of the Rix1 complex. To study the role of Rea1 in ribosome biogenesis, we generated a repressible GAL::REA1 strain and temperature-sensitive rea1 alleles. In vivo depletion of Rea1 results in the significant reduction of mature 60 S subunits concomitant with defects in pre-rRNA processing and late pre-60 S ribosome stability following ITS2 cleavage and prior to the generation of mature 5.8 S rRNA. Strains depleted of the components of the Rix1 complex (Rix1, Ipi1, and Ipi3) showed similar defects. Using an in vivo 60 S subunit export assay, a strong accumulation of the large subunit reporter Rpl25-GFP (green fluorescent protein) in the nucleus and at the nuclear periphery was seen in rea1 mutants at restrictive conditions. Rea1, the largest predicted protein in the yeast genome, is a member of the AAA+ family of ATPases and is associated with pre-60 S ribosomes. Here we report that Rea1 is required for maturation and nuclear export of the pre-60 S subunit. Rea1 exhibits a predominantly nucleoplasmic localization and is present in a late pre-60 S particle together with members of the Rix1 complex. To study the role of Rea1 in ribosome biogenesis, we generated a repressible GAL::REA1 strain and temperature-sensitive rea1 alleles. In vivo depletion of Rea1 results in the significant reduction of mature 60 S subunits concomitant with defects in pre-rRNA processing and late pre-60 S ribosome stability following ITS2 cleavage and prior to the generation of mature 5.8 S rRNA. Strains depleted of the components of the Rix1 complex (Rix1, Ipi1, and Ipi3) showed similar defects. Using an in vivo 60 S subunit export assay, a strong accumulation of the large subunit reporter Rpl25-GFP (green fluorescent protein) in the nucleus and at the nuclear periphery was seen in rea1 mutants at restrictive conditions. The synthesis of ribosomes is one of the major and most energy-consuming processes in the cell. In Saccharomyces cerevisiae, ribosome biogenesis begins in the nucleolus with the transcription of two rRNA precursors, the 35 S and the pre-5 S RNA, by RNA polymerases I and III, respectively. The 35 S pre-rRNA contains the sequences for the mature 18 S, 5.8 S, and 25 S rRNAs, two external transcribed spacers (ETS) 1The abbreviations used are: ETS, external transcribed spacer; ITS, internal transcribed spacer; AAA, ATPases associated with different activities; CaM, calmodulin; GFP, green fluorescent protein; MIDAS, metal ion dependant adhesion signal; sl, synthetic lethal; ts, temperature-sensitive strain; td, temperature-sensitive degron mutant; TAP, tandem affinity purification; HA, hemagglutinin. and two internal transcribed spacers (ITS). During the maturation process, the pre-rRNA has to undergo a number of modifications and is subjected to cleavages and trimming events. At least 170 accessory proteins including putative RNA helicases, endo- and exonucleases, and putative GTPases and AAA-ATPases as well as small nucleolar ribonucleoprotein particles are involved in the maturation of rRNA and its assembly into ribosomal subunits (1Kressler D. Linder P. De La Cruz J. Mol. Cell. Biol. 1999; 19: 7897-7912Crossref PubMed Scopus (309) Google Scholar, 2Tschochner H. Hurt E. Trends Cell Biol. 2003; 13: 255-263Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Concomitant with rRNA processing, ribosomal and non-ribosomal proteins are assembled on the pre-35 S rRNA, giving rise to a large 90 S pre-ribosomal particle (see Fig. 6B) (3Dragon F. Gallagher J.E. Compagnone-Post P.A. Mitchell B.M. Porwancher K.A. Wehner K.A. Wormsley S. Settlage R.E. Shabanowitz J. Osheim Y. Beyer A.L. Hunt D.F. Baserga S.J. Nature. 2002; 417: 967-970Crossref PubMed Scopus (555) Google Scholar, 4Grandi P. Rybin V. Bassler J. Petfalski E. Strauss D. Marzioch M. Schäfer T. Kuster B. Tschochner H. Tollervey D. Gavin A.C. Hurt E. Mol. Cell. 2002; 10: 105-115Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). The initial cleavages at sites A0–A2 separate the two subunits. The pre-40 S subunit is exported relatively rapidly to the cytoplasm, where it undergoes further processing. In contrast, the maturation of the large subunit continues in the nucleoplasm with recruitment of 60 S-specific biogenesis factors and further processing of the 27 S pre-rRNA. This includes the late cleavage and processing in the ITS2 region, which generates mature 5.8 S and 25 S rRNA. In the last few years, the maturation of 40 S and 60 S pre-ribosomes has been extensively analyzed by purification of pre-ribosomal particles (5Nissan T.A. Bassler J. Petfalski E. Tollervey D. Hurt E.C. EMBO J. 2002; 21: 5539-5547Crossref PubMed Scopus (291) Google Scholar, 6Saveanu C. Bienvenu D. Namane A. Gleizes P.E. Gas N. Jacquier A. Fromont-Racine M. EMBO J. 2001; 20: 6475-6484Crossref PubMed Scopus (156) Google Scholar, 7Harnpicharnchai P. Jakovljevic J. Horsey E. Miles T. Roman J. Rout M. Meagher D. Imai B. Guo Y. Brame C.J. Shabanowitz J. Hunt D.F. Woolford J.L. Mol. Cell. 2001; 8: 505-515Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 8Fatica A. Cronshaw A.D.M., D. Tollervey D. Mol. Cell. 2002; 9: 341-351Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9Schäfer T. Strauss D. Petfalski E. Tollervey D. Hurt E.C. EMBO J. 2003; 22: 1370-1380Crossref PubMed Scopus (248) Google Scholar, 10Bassler J. Grandi P. Gadal O. Lessmann T. Tollervey D. Lechner J. Hurt E.C. Mol. Cell. 2001; 8: 517-529Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Interestingly, a large number of non-ribosomal proteins were identified in pre-60 S particles without an assigned function in RNA metabolism. In contrast to the pre-40 S particles, the nascent 60 S particles contain several putative GTPases and AAA-type ATPases (2Tschochner H. Hurt E. Trends Cell Biol. 2003; 13: 255-263Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar, 11Gadal O. Strauss D. Braspenning J. Hoepfner D. Petfalski E. Philippsen P. Tollervey D. Hurt E.C. EMBO J. 2001; 20: 3695-3704Crossref PubMed Scopus (78) Google Scholar). To understand the events of ribosome biogenesis, we previously purified pre-ribosomal 60 S particles, which represent different maturation states from early nucleolar through cytoplasmic export (5Nissan T.A. Bassler J. Petfalski E. Tollervey D. Hurt E.C. EMBO J. 2002; 21: 5539-5547Crossref PubMed Scopus (291) Google Scholar). One of these, the late nucleoplasmic Rix1 particle, was selected for further study. The Rix1 particle is specifically enriched in the products of three uncharacterized open reading frames, YHR085w (Ipi1), YNL182c (Ipi3), and Rea1. Ipi1 has a single ARM repeat motif (12Mulder N.J. Apweiler R. Attwood T.K. Bairoch A. Barrell D. Bateman A. Binns D. Biswas M. Bradley P. Bork P. Bucher P. Copley R.R. Courcelle E. Das U. Durbin R. Falquet L. Fleischmann W. Griffiths-Jones S. Haft D. Harte N. Hulo N. Kahn D. Kanapin A. Krestyaninova M. Lopez R. Letunic I. Lonsdale D. Silventoinen V. Orchard S.E. Pagni M. Peyruc D. Ponting C.P. Selengut J.D. Servant F. Sigrist C.J. Vaughan R. Zdobnov E.M. Nucleic Acids Res. 2003; 31: 315-318Crossref PubMed Scopus (604) Google Scholar), whereas Ipi3 contains two WD40 domains. In contrast, Rea1, which at 560 kDa is the largest protein identified in the yeast genome, contains several interesting domains and homologies. The N terminus of Rea1 possesses six AAA ATPase protomers and a C-terminal region containing the MIDAS (metal ion-dependant adhesion site) motif. Furthermore, sequence analysis has indicated relatedness to dynein (13Garbarino J.E. Gibbons I.R. BMC Genomics. 2002; 3: 18-28Crossref PubMed Scopus (84) Google Scholar). In this study, we characterize Rea1 and the Rix1 complex-containing pre-60 S particle. Our results show that Rea1 and the Rix1 complex exhibit similar subcellular localization and a similar late ITS2 rRNA processing defect. Although the Rix1 complex mutants accumulate the Rpl25-GFP reporter throughout the nucleoplasm, rea1 temperature-sensitive (ts) mutants also show a later defect, with accumulation around the nuclear periphery. Our data demonstrate that Rea1 and the Rix1 complex play an essential role in maturation and nuclear export of nascent 60 S subunits from the nucleoplasm to the cytoplasm. Yeast Strains and Plasmids—Genomic integration of GFP (HIS3MX6 marker) as a C-terminal tag into yeast strains to create fusion proteins of Ipi1 (strain DS1-2b, MATa, ura3, trp1, his3, leu2) and Ipi3 (strain JBa, MATa trp1 ura3 ade2 ade3 leu2 his3) was performed as described (14Longtine M.S. McKenzie A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 10: 953-961Crossref Scopus (4172) Google Scholar). For construction of GAL1::GFP-REA1 and GAL1::HA-REA1 strains, GAL1::GFP and GAL1::HA cassettes containing the TRP1 marker were integrated 5′ upstream of the ATG start codon of REA1 (strain RS453). Integration of the GFP and HA tags was confirmed by Western blot. The REA1 shuffle strain was obtained by transforming plasmid pYCG-YLR106c (REA1) into the heterozygous strain BY4743 (rea1::kanMX4/REA1; derived from EUROSCARF) and selection of kanrURA+ haploid progeny after tetrad dissection. The degron yeast strains ipi3-td, ipi1-td, and rix1-td as well as the isogenic wild type (wt-td) (15Kanemaki M. Sanchez-Diaz A. Gambus A. Labib K. Nature. 2003; 423: 720-724Crossref PubMed Scopus (215) Google Scholar), strains expressing Rix1-TAP and Rix1-GFP (5Nissan T.A. Bassler J. Petfalski E. Tollervey D. Hurt E.C. EMBO J. 2002; 21: 5539-5547Crossref PubMed Scopus (291) Google Scholar), and Ipi3-TAP (16Nissan T.A. Galani K. Maco B. Tollervey D. Aebi U. Hurt E. Mol. Cell. 2004; 15: 295-301Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) were described previously. The degron strains were grown at 23 °C in selective raffinose-glucose medium containing CuSO4 (permissive condition) before shift to galactose medium lacking CuSO4, first for 40 min at 23 °C for induction of Ubr1 expression and then to 37 °C to induce protein degradation of the mutant proteins (15Kanemaki M. Sanchez-Diaz A. Gambus A. Labib K. Nature. 2003; 423: 720-724Crossref PubMed Scopus (215) Google Scholar, 17Dohmen R.J. Wu P. Varshavsky A. Science. 1994; 263: 1273-1276Crossref PubMed Scopus (294) Google Scholar). For RNA analysis, cells were grown similarly except that the raffinose medium contained no glucose and that cells were washed twice with prewarmed galactose medium without CuSO4. The following described plasmids were used in this study: pFA6a-TRP1-PGAL1-GFP, pFA6a-TRP1-PGAL1-3HA (14Longtine M.S. McKenzie A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 10: 953-961Crossref Scopus (4172) Google Scholar), pRS316-RPL25-GFP (18Gadal O. Strauss D. Kessl J. Trumpower B. Tollervey D. Hurt E. Mol. Cell. Biol. 2001; 21: 3405-3415Crossref PubMed Scopus (257) Google Scholar), pRS314-RPS2-GFP (19Milkereit P. Strauss D. Bassler J. Gadal O. Kühn H. Schütz S. Gas N. Lechner J. Hurt E. Tschochner H J. Biol. Chem. 2002; 278: 4072-4081Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Plasmid pYCG-YLR106c was obtained from EUROSCARF. For construction of plasmid pLEU2-REA1, the LEU2 gene was amplified by PCR from plasmid pRS315 and used to replace the URA3 gene from plasmid pYCG-YLR106c. Generation of Rea1 ts Alleles—20 μg of pLEU2-REA1 plasmid DNA was incubated in 500 μl of 2 m hydroxylamine buffer for 20 h at 55 °C (20Amberg D.C. Fleischmann M. Stagljar I. Cole C.N. Aebi M. EMBO J. 1993; 12: 233-241Crossref PubMed Scopus (119) Google Scholar). The REA1 shuffle strain was transformed with the mutagenized plasmid, and cells were streaked on plates containing selection medium lacking leucine for 5 days at 23 °C in the dark. About 2000 single colonies were picked and streaked twice on 5-fluoroorotic acid-containing plates at 23 °C before it was streaked on YPD plates at 23 and 37 °C. Two ts rea1 mutants (rea1-7 and rea1-21) were derived from this screen from which the mutagenized pLEU2-REA1 plasmids were recovered and reintroduced into the REA1 shuffle strain to verify the ts phenotype. The ts mutant rea1-21 was used for further characterization. Affinity Purification and MgCl2 Treatments—Affinity purification of TAP-tagged proteins was performed as described previously using 2–6 liters of yeast culture (21Gavin A-C. Bösche M. Krause R. Grandi P. Marzioch M. Bauer M. Schultz J. Rick J.M. Michon A.M. Cruciat C.-M. Remor M. Höfert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. 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. Superti-Furga G Nature. 2002; 415: 141-147Crossref PubMed Scopus (3998) Google Scholar). Normal buffer composition for purification was 0.1 m NaCl, 50 mm Tris, pH 7.5, 1.5 mm MgCl2, 0.75% Nonidet P-40. For MgCl2 salt elution, the Rix1-TAP purification was as described until after the beads were bound to calmodulin (CaM)-Sepharose. The beads were then incubated for 10 min at 30 °C with LB-buffer plus 100 mm MgCl2, the eluate was collected, and the column was washed with 5 ml of LB + 100 mm MgCl2. After repeating the procedure with LB + 200 mm MgCl2, the sample was eluted from the beads by boiling for 10 min in SDS sample buffer (0.5% SDS, 50 mm NaCl, and 10 mm Tris, pH 7.5). RNA Analysis—Northern hybridization and primer extension were performed as described on whole cell extracts (22Beltrame M. Tollervey D. EMBO J. 1992; 11: 1531-1542Crossref PubMed Scopus (216) Google Scholar, 23Tollervey D. Lehtonen H. Jansen R.P. Kern H. Hurt E.C. Cell. 1993; 72: 443-457Abstract Full Text PDF PubMed Scopus (411) Google Scholar). Oligonucleotides used were: 003, 5′-TGT TAC CTC TGG GCC C-3′; 004, 5′-CGG TTT TAA TTG TCC TA-3′; 006, 5′-AGA TTA GCC GCA GTT GG-3′; 007, 5′-CTC CGC TTA TTG ATA TGC-3′; 008, 5′-CAT GGC TTA ATC TTT GAG AC-3′; 017, 5′-GCG TTG TTC ATC GAT GC-3′; 020, 5′-TGA GAA GGA AAT GAC GCT-3′; 041, 5′-CTA CTC GGT CAG GCT C-3′; 250, 5′-ATCCCGGCCGCCTCCATCAC-3′; 306, 5′-GCA TCT TAC GAT ACC TG-3′. Miscellaneous—Western blot analyses were performed according to Ref. 24Siniossoglou S. Wimmer C. Rieger M. Doye V. Tekotte H. Weise C. Emig S. Segref A. Hurt E.C. Cell. 1996; 84: 265-275Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar. Fluorescence microscopy was done as described (11Gadal O. Strauss D. Braspenning J. Hoepfner D. Petfalski E. Philippsen P. Tollervey D. Hurt E.C. EMBO J. 2001; 20: 3695-3704Crossref PubMed Scopus (78) Google Scholar). The fluorescence-based visual assay to analyze the nuclear export of large and small ribosomal subunits using the Rpl25-GFP and Rps2-GFP reporters, respectively, in living cells was performed according to Refs. 18Gadal O. Strauss D. Kessl J. Trumpower B. Tollervey D. Hurt E. Mol. Cell. Biol. 2001; 21: 3405-3415Crossref PubMed Scopus (257) Google Scholar, 19Milkereit P. Strauss D. Bassler J. Gadal O. Kühn H. Schütz S. Gas N. Lechner J. Hurt E. Tschochner H J. Biol. Chem. 2002; 278: 4072-4081Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, and 25Gadal O. Strauss D. Petfalski E. Gleizes P.E. Gas N. Tollervey D. Hurt E. J. Cell Biol. 2002; 157: 941-951Crossref PubMed Scopus (65) Google Scholar. Sedimentation analysis of ribosomes under low salt conditions by sucrose gradient centrifugation was performed as described (10Bassler J. Grandi P. Gadal O. Lessmann T. Tollervey D. Lechner J. Hurt E.C. Mol. Cell. 2001; 8: 517-529Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Mass spectrometry using tryptic digests from Coomassie Blue-stained bands derived from SDS-PAGE was performed as described (10Bassler J. Grandi P. Gadal O. Lessmann T. Tollervey D. Lechner J. Hurt E.C. Mol. Cell. 2001; 8: 517-529Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Synthetic lethality was determined by tetrad dissection of either the rix1-1 (10Bassler J. Grandi P. Gadal O. Lessmann T. Tollervey D. Lechner J. Hurt E.C. Mol. Cell. 2001; 8: 517-529Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) or the rix7-1 (11Gadal O. Strauss D. Braspenning J. Hoepfner D. Petfalski E. Philippsen P. Tollervey D. Hurt E.C. EMBO J. 2001; 20: 3695-3704Crossref PubMed Scopus (78) Google Scholar) strain mated to the rea1 deletion strain complemented with the pRS316-REA1 plasmid. Haploid progeny containing either the rix1-1 or the rix7-1 mutation and the rea1 deletion were transformed with pRS315 plasmids containing no insert or rea1-7 or rea1-21 mutant alleles. Strains were considered synthetic lethal if they could not grow on 5-fluoroorotic acid, i.e. if they could not lose the wild-type pRS316-REA1 plasmid. High Salt Dissociates Rea1 and 60 S Subunits from the Rix1 Complex—We have previously reported the isolation and identification of 60 S pre-ribosomal particles, which are characterized by the presence of different ribosomal precursors depending on the maturation state of each of the particles (5Nissan T.A. Bassler J. Petfalski E. Tollervey D. Hurt E.C. EMBO J. 2002; 21: 5539-5547Crossref PubMed Scopus (291) Google Scholar, 10Bassler J. Grandi P. Gadal O. Lessmann T. Tollervey D. Lechner J. Hurt E.C. Mol. Cell. 2001; 8: 517-529Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). The Rix1 particle represents a late intermediate in 60 S biogenesis and is highly enriched in the AAA-ATPase Rea1/Mdn1, as well as in two other non-ribosomal proteins, Yhr085/Ipi1 and Ynl182/Ipi3 (Fig. 1A; see also Ref. 5Nissan T.A. Bassler J. Petfalski E. Tollervey D. Hurt E.C. EMBO J. 2002; 21: 5539-5547Crossref PubMed Scopus (291) Google Scholar). All three proteins are specifically associated with the Rix1 particle since they are largely absent both from earlier and from later pre-60 S ribosomes. The observation that Rix1, Ipi1, Ipi3, and Rea1 co-enrich during biochemical purification indicates that these four proteins could be organized in a complex attached to nascent 60 S subunits. To test this possibility, we affinity-purified Rix1-TAP by the tandem affinity purification method. However, instead of eluting the purified complexes from the final CaM-Sepharose with EGTA (as in Fig. 1A), we treated the beads with increasing concentrations of MgCl2 (Fig. 1B). The additional salt should release the interacting proteins that are not tightly bound with the affinity-purified bait proteins (26Simos G. Segref A. Fasiolo F. Hellmuth K. Shevchenko A. Mann M. Hurt E.C. EMBO J. 1996; 15: 5437-5448Crossref PubMed Scopus (211) Google Scholar). When Rix1-TAP, which is immobilized on CaM beads, was incubated with a MgCl2 step gradient, Rea1 and 60 S subunit proteins were released with 100 mm salt (Fig. 1B, lane 1). However, Rix1, Ipi1, and Ipi3 remained bound under these conditions and were also resistant to 200 mm MgCl2, but they eluted by SDS-sample buffer (Fig. 1B, lane 3). Ipi1 reproducibly appeared substoichiometric after salt washing. It remains to be shown whether Ipi1 is present in lower amounts in the Rix1 complex or is not stained effectively by Coomassie Blue. Similar results were obtained for salt-treated Ipi3-TAP purification (data not shown). We conclude that Rix1, Ipi1, and Ipi3 form a salt-stable complex, to which Rea1 and the nascent 60 S subunit are less tightly attached. Recently, Krogan et al. (27Krogan N.J. Peng W.T. Cagney G. Robinson M.D. Haw R. Zhong G. Guo X. Zhang X. Canadien V. Richards D.P. Beattie B.K. Lalev A. Zhang W. Davierwala A.P. Mnaimneh S. Starostine A. Tikuisis A.P. Grigull J. Datta N. Bray J.E. Hughes T.R. Emili A. Greenblatt J.F. Mol. Cell. 2004; 13: 225-239Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar), in a large scale effort to isolate RNA processing complexes by TAP purification following a ultracentrifugation step to remove ribosomes, have reported a similar complex. Finally, since Rea1 was observed to be specifically enriched in the Rix1-TAP purification, we examined whether a synthetic lethal relationship exists between the rea1 ts mutants and the rix1-1 mutant (10Bassler J. Grandi P. Gadal O. Lessmann T. Tollervey D. Lechner J. Hurt E.C. Mol. Cell. 2001; 8: 517-529Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). However, we did not observe any sl phenotype (data not shown). Rea1 and the Rix1 Complex Members Exhibit a Nucleoplasmic Location—Since Rea1 and the Rix1 complex are co-enriched, we wanted to know whether their subcellular distribution is also similar. Previously, we showed that Rix1 is localized in the nucleus (5Nissan T.A. Bassler J. Petfalski E. Tollervey D. Hurt E.C. EMBO J. 2002; 21: 5539-5547Crossref PubMed Scopus (291) Google Scholar). Ipi1 and Ipi3 were genomically tagged with the GFP epitope at their C terminus, which ensures that protein expression remains under the control of the native promoter. Expression of the genomically N-terminally tagged GFP-Rea1 is under the GAL promoter due to difficulties in obtaining a fully functional C-terminal GFP fusion protein. 2K. Galani, unpublished data. After confirming that the GFP tagging had no effect on the growth rate of the strains (Supplemental Fig. 1), we examined the yeast cells under the fluorescence microscope. Similar to Rix1, Rea1, Ipi1, and Ipi3 were localized throughout the nucleoplasm (Fig. 2). We also expressed the DsRed-tagged nucleolar protein Nop1 in the GFP-tagged strains, and we did not observe nucleolar concentration for any of the proteins as this can be judged by the lack of colocalization between the GFP and the DsRed signals (Supplemental Fig. 2). These results are in agreement with the previously reported localization of Ipi1 and Rea1 proteins (28Huh W.K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Nature. 2003; 425: 686-691Crossref PubMed Scopus (3306) Google Scholar) and Rix1 (5Nissan T.A. Bassler J. Petfalski E. Tollervey D. Hurt E.C. EMBO J. 2002; 21: 5539-5547Crossref PubMed Scopus (291) Google Scholar). Thus, Rea1 and the Rix1 complex could function in late nucleoplasmic maturation of pre-60 S subunits and/or their export to the cytoplasm. Taken together, the identified Rix1 complex is present together with the AAA-type ATPase Rea1 in a late pre-60 S particle that is located in the nucleoplasm. Rea1 and the Rix1 Complex Are Required for Nuclear Export of the 60 S Subunit—We wanted to determine whether Rea1 and the components of the Rix1 complex are involved in late nucleoplasmic steps during 60 S subunit biogenesis. Previously, we showed that the rix1-1 ts mutant is strongly impaired in 60 S subunit export, but rRNA processing was not significantly affected (10Bassler J. Grandi P. Gadal O. Lessmann T. Tollervey D. Lechner J. Hurt E.C. Mol. Cell. 2001; 8: 517-529Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). We followed different strategies to obtain conditional-lethal mutants of the Rea1 protein and Rix1 complex members. For REA1, both a repressible GAL1::REA1 construct and ts rea1 alleles were generated (see “Experimental Procedures”). For the essential Rix1, Ipi1, and Ipi3, conditional-lethal degron (td) mutants were used. These degron mutants target the proteins for rapid degradation in vivo upon shift to 37 °C (15Kanemaki M. Sanchez-Diaz A. Gambus A. Labib K. Nature. 2003; 423: 720-724Crossref PubMed Scopus (215) Google Scholar). Furthermore, we have confirmed that rea1-7 and rea1-21 ts mutants could be rescued by the presence of a plasmid carrying the REA1 wild-type gene (Supplemental Fig. 3A). In the case of the td mutants, we used the available RIX1 wild-type plasmid (10Bassler J. Grandi P. Gadal O. Lessmann T. Tollervey D. Lechner J. Hurt E.C. Mol. Cell. 2001; 8: 517-529Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) to verify that the expression of the wild-type gene is sufficient to rescue the lethal phenotype of rix1-td mutant (Supplemental Fig. 3B). The Rpl25-GFP reporter assay was developed to monitor 60 S export in vivo (18Gadal O. Strauss D. Kessl J. Trumpower B. Tollervey D. Hurt E. Mol. Cell. Biol. 2001; 21: 3405-3415Crossref PubMed Scopus (257) Google Scholar, 29Hurt E. Hannus S. Schmelzl B. Lau D. Tollervey D. Simos G. J. Cell Biol. 1999; 144: 389-401Crossref PubMed Scopus (145) Google Scholar). In wild-type cells, Rpl25-GFP is incorporated into nascent pre-60 S ribosomes and is rapidly exported to the cytoplasm. In all ts mutants generated (rea1-21, rix1, ipi1, and ipi3), the reporter protein was cytoplasmic at steady state (Fig. 3). Similar results were obtained with the rea1-7 mutant (data not shown). At early time points upon shift to the restrictive condition, the td mutants exhibited a mixed phenotype with cells accumulating the Rpl25-GFP protein either at their nucleolus or throughout their nucleoplasm (Fig. 3A, 1-h shift). At a later time point, all td mutants exhibited strong nuclear accumulation, whereas the nucleolus was either similar to nucleoplasmic staining or else devoid of any signal (Fig. 3A, 2-h shift; Supplemental Fig. 4). A similar assay for nuclear export of the small subunit showed cytoplasmic localization of Rps2-GFP comparable with wild-type in all of the mutants (Fig. 3). Both the rea1-21 ts and the repressible GAL::REA1 mutants accumulated the Rpl25-GFP in their nucleoplasm upon shift to restrictive conditions (Fig. 3, B and C). Interestingly, in the case of the rea1-21 ts mutant, ∼20% of the cells accumulated the Rpl25-GFP protein at their nuclear periphery after 9 h of shift. The same phenotype was also observed with the rea1-7 ts mutant, albeit to a lesser extent (15%; data not shown). The fact that pre-60 S ribosomes accumulated in the entire nucleoplasm or at the nuclear periphery (Fig. 3, rea1-21 insert; Supplemental Fig. 5) suggests that a late step in nuclear export from the nucleoplasm to the cytoplasm is blocked in rea1 and rix1 complex mutants. Rea1 and the Rix1 Complex Are Required for Normal Levels of the 60 S Subunit—As the export is impaired for Rea1 and the Rix1 complex mutants, we wished to see whether the overall production of 60 S subunits is likewise reduced. For this reason, we analyzed the ribosomal and polysomal profiles in rea1 and rix1 complex mutants by sucrose density gradient centrifugation. This analysis revealed a significant reduction of 60 S subunits as compared with 40 S in the mutants. Moreover, the appearance of half-mer polysomes was observed in mutant strains (Fig. 4), indicating a lack of mature 60 S to bind to the 43 S preinitiation complex. These data are consistent with the export defect, demonstrating that it is specific to the 60 S pathway. Depletion of Rea1 or the Rix1 Complex Inhibits Synthesis of the 5.8 S rRNA—To determine whether the rea1 and rix1 complex member mutants are impaired in pre-rRNA processing, we performed Northern hybridization and primer extension analyses. The locations of oligonucleotide probes are indicated in Fig. 6A. Analyses of low molecular weight RNAs (Fig. 5A, upper panel) revealed that genetic depletion of Rea1 resulted in strong inhibition of processing from 7 S pre-rRNA to 5.8 S + 30 and 6 S pre-rRNA, as shown by their substantial loss 16 h after transfer of the GAL::REA1 strain to glucose medium. Since the 7 S pre-rRNA was mildly accumulated, whereas the normal products of its processing were drastically reduced, it is likely that much of the 7 S pre-rRNA is degraded, presumably reflecting degradation of the entire pre-60 S particle. It is, however, difficult to directly assess this experimentally since 7 S is only faintly visible in pulse-chase labeling of wild-type strains, and the 5.8 S + 30 and 6 S pre-rRNAs are not observed (data not shown). Mature 5.8 S was reduced relative to the tRNA Leu3 loading control, as was the 5 S rRNA component of the 60 S subunit. Analyses of high molecular weight RNA by Northern hybridization (Fig. 5A, middle panel) and primer extension (Fig. 5A, lower panel) showed the accumulation of the 35 S pre-rRNA and 23 S RNA, accompanied by a mild reduction in the 20 S and 27 SA2 pre-rRNA. Very similar processing defects were observed in the rea1-21 ts strain 4 h after transfer to the non-permissive temperature (data not shown). These phenotypes indicate a delay in early pre-rRNA processing at sites A0, A1, and A2 (Fig. 6B), which are very frequently seen in strains defective in 60 S subunit synthesis (30Venema J. Tollervey D. Annu. Rev. Genet. 1999; 33: 261-311Crossref PubMed Scopus (652) Google Scholar). The 27 SB pre-rRNAs were mildly accumulated, with little alteration in the levels of processing at the alternative B1S and B1L sites, indicating that the early steps in 60 S synthesis continue in the Rea1-depleted strain. Related defects in 5.8 S synthesis were seen in strains depleted of Rix1, Ipi1, or Ipi3 (Fig. 5B, upper panel). In each case, the 7 S pre-rRNA was accumulated relative to the wild-type control following transfer to non-permissive conditions, accompanied by reduced 6 S pre-rRNA. The level of the 5.8 S + 30 was reduced in the wild-type following transfer to 37 °C but was further reduced in the mutant strains. Some reduction in the levels of the mature 5.8 S was seen after 4 h in non-permissive conditions. The mature rRNAs are not generally turned over and are therefore depleted only by growth under non-permissive conditions. Greater depletion would not therefore have been expected over this perio" @default.
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• W2085098052 title "Rea1, a Dynein-related Nuclear AAA-ATPase, Is Involved in Late rRNA Processing and Nuclear Export of 60 S Subunits" @default.
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