Coupled GTPase and remodelling ATPase activities form a checkpoint for ribosome export

Eukaryotic ribosomes are assembled by a complex pathway that extends from the nucleolus to the cytoplasm and is powered by many energy-consuming enzymes 1-3. Nuclear export is a key, irreversible step in pre-ribosome maturation4-8, but mechanisms underlying the timely acquisition of export competence remain poorly understood. Here we show that a conserved GTPase Nug2/ Nog2 (called NGP-1, Gnl2 or nucleostemin 2 in human9) plays a key role in the timing of export competence. Nug2 binds the inter-subunit face of maturing, nucleoplasmic pre-60S particles, and the location clashes with the position of Nmd3, a key pre-60S export adaptor10. Nug2 and Nmd3 are not present on the same pre-60S particles, with Nug2 binding prior to Nmd3. Depletion of Nug2 causes premature Nmd3 binding to the pre-60S particles, whereas mutations in the Gdomain of Nug2 block Nmd3 recruitment, resulting in severe 60S export defects. Two pre-60S remodeling factors, the Rea1 ATPase and its co-substrate Rsa4, are present on Nug2-associated particles, and both show synthetic lethal interactions with nug2 mutants. Release of Nug2 from pre-60S particles requires both its K+-dependent GTPase activity and the remodeling ATPase activity of Rea1. We conclude that Nug2 is a regulatory GTPase that monitors pre-60S maturation, with release from its placeholder site linked to recruitment of the nuclear export machinery. The conserved GTPase Nug2/Nog2 (Extended Data Fig. 1) is associated with a number of pre-60S particles located in the nucleoplasm, but was not detected on particles with a known cytoplasmic location (see also Extended Data Fig. 2). The bacterial homolog of Nug2, YlqF, binds directly to rRNA11, and we therefore used the CRAC UV cross-linking method to localize the binding site for yeast Nug2 within the pre-60S particle12. Direct contacts for Nug2 were identified only with the 25S rRNA, at sites in helices H38, H69, H71, H80, H81-83, H84-86, H89, H91-92 and H93 (Fig. 1a, c). Yeast 3-hybrid analyses confirmed Users may view, print, copy, download and text and datamine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence and requests for materials should be addressed to E.H. ([email protected]).. Author Contributions Experiments were designed and the data were interpreted by Y.M. and E.H.; all experiments excepting CRAC analysis were performed by Y.M.; CRAC experiments and data analyses were performed by S.G. in collaboration with D.T.; M.T. constructed rea1 mutants and performed the in vitro release assay of rea1 mutants, ctNug2 complementation assay and immunodepletion assay; G.M. developed the methods of in vitro assay for nucleotide binding and GTPase activity measurement; the manuscript was written by Y.M. and E.H.; all authors discussed the results and commented on the manuscript. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Europe PMC Funders Group Author Manuscript Nature. Author manuscript; available in PMC 2014 July 02. Published in final edited form as: Nature. 2014 January 2; 505(7481): 112–116. doi:10.1038/nature12731. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts interactions between Nug2 and these rRNA helices (Fig. 1b). Mapping the major rRNA crosslink sites of Nug2 onto the 60S subunit structure (Fig. 1d) showed a distinct cluster on the inter-subunit joining surface13. Nug2 binding sites overlap with regions occupied by the export factor Nmd3 in cryo-EM10. CRAC was therefore applied to Nmd3 to more precisely identify its binding sites, which were found to lie in H38, H69 and H89 of 25S rRNA (Fig. 1a, c, e). Strikingly, the Nug2 and Nmd3 binding sites overlapped in H38, H69 and H89 (Fig. 1c, f), suggesting that binding of these two proteins is mutually exclusive. To test this, pre-60S particles were purified with tagged Nug2 and shown to lack detectable Nmd3, and vice versa (Extended Data Fig. 2). Nmd3 is an essential nuclear export factor that recruits the export receptor Crm1 to the nascent 60S subunits14,15. These observations suggested that Nug2 acts as a “placeholder” to prevent premature recruitment of Nmd3 to earlier, exportincompetent pre-60S particles. Like other GTP-binding proteins, Nug2 has characteristic G1, G3 and G4 motifs in its Gdomain (Fig. 2a, Extended Data Fig. 1), suggesting that GTP-binding or hydrolysis16 might regulate dynamic interactions between Nug2 and the pre-ribosome. Dominant-negative mutations were previously described in two GTPases involved in ribosome biogenesis, the G1-motif of Lsg1 (K349N/R/T)17 and G3-motif of Nog1 (G224A)18. Orthologous G1and G3-motif mutants, nug2K328R and nug2G369A, respectively (Fig. 2a, Extended Data Fig. 1), each showed severe growth defect phenotypes (Fig. 2b), and were also dominantnegative when overexpressed in the presence of chromosomal NUG2 (Fig. 2c). Preribosome analysis by sucrose gradient centrifugation showed that the Nug2K328R and Nug2G369A proteins were efficiently assembled into pre-60S subunits, but induced a ‘halfmer’ polysome phenotype (in particular for Nug2K328R), characteristic of reduced 60S subunit synthesis (Fig. 2d). The reduced 60S levels were more apparent under low Mg2+ conditions that cause 80S ribosomes to dissociate into 60S and 40S subunits (Fig. 2d). The nug2K328R and nug2G369A strains showed nuclear accumulation of a RpL25-GFP reporter, but not RpS3-GFP, revealing a specific block in pre-60S nuclear export (Fig. 2e). We conclude that mutations in the GTPase domain of Nug2 allow recruitment to the preribosomes, but block nuclear export. To determine the basis of the defects associated with Nug2K328R and Nug2G369A, we assayed in vitro guanine nucleotide-binding activity and GTP hydrolysis. Nug2 from Saccharomyces cerevisiae was unstable when expressed in E.coli (data not shown). In contrast, good yields were obtained for wild-type and mutant Nug2 from the eukaryotic thermophile Chaetomium thermophilum (ctNug2, ctNug2K339R and ctNug2G380A, respectively; Fig. 2f), whose thermostable proteins have superior biochemical properties19. ctNug2 is highly homologous to yeast Nug2 (74% identity; Extended Data Fig. 1), and can complement albeit not perfectly a yeast nug2Δ mutant (Extended Data Fig. 3). As Nug2 may act as a potassium-dependent GTPase20, we tested the cation requirement for GTP hydrolysis. The GTPase activity of ctNug2 was low in NaCl-containing buffer, but was substantially stimulated by KCl (Fig. 2f). In contrast, ctNug2K339R and ctNug2G380A exhibited only background GTPase activity (Fig. 2f). In binding assays, wild-type ctNug2 and ctNug2G380A readily bound fluorescent MANT-GTP or MANT-GDP, whereas ctNug2K339R did not (Fig. 2g). We conclude that ctNug2K339R is defective in GTP binding, whereas ctNug2G380A binds but cannot hydrolyse GTP. This K+-stimulated GTPase activity might regulate interaction of Nug2 with nascent 60S particles. Nug2 is associated with nucleoplasmic pre-60S particles that also carry the Rix1-Ipi1-Ipi3 heterotrimer, the dynein-related AAA-ATPase Rea1 and its co-substrate Rsa4 (Extended Data Fig. 2; see also below and21). The enzymatic activity of Rea1 is required for the release of Ytm122 and Rsa421 and a genetic screen revealed synthetic lethality between the G1-motif mutant nug2K328R and the mutant alleles rea1-DTS and rsa4-121 (Fig. 3a). We Matsuo et al. Page 2 Nature. Author manuscript; available in PMC 2014 July 02. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts therefore investigated whether ATP-dependent remodeling of the Rix1-particle by the AAAATPase activity of Rea121 is altered in particles containing Nug2K328R or Nug2G369A. Pre-60S particles carrying Flag-tagged RpL3 were affinity-purified with Rix1-TAP via IgG binding and TEV elution. The pre-60S particles were incubated in vitro to allow factor release, and then re-isolated on Flag-beads via RpL3-Flag (Fig. 3b). Consistent with previous data21, incubation of the pre-60S particles with ATP in Na+-containing buffer resulted in release of Rsa4 and Rea1, but not Nug2 (Fig. 3c). In contrast, incubation in K+containing buffer caused the ATP-dependent release of Nug2, in addition to Rsa4 and Rea1 (Fig. 3c). Incubation with GTP in Na+ or K+-containing buffer did not induce the release of biogenesis factors (Fig. 3d). However, neither Nug2K328R nor Nug2G369A could be dissociated from pre-60S particles upon ATP treatment in K+ buffer (Fig. 3e). In the case of Nug2K328R (defective in GTP-binding), incubation with ATP in K+ buffer failed to release Rsa4, whereas pre-60S particles carrying Nug2G369A (defective in GTP-hydrolysis) still showed Rsa4 release upon ATP-treatment (Fig. 3e). Mutation of one of the six ATP-binding protomers of Rea1 (AAA2; rea1 K659A) inhibited remodeling, including Nug2 release (Extended Data Fig. 4). These findings indicate that the GTP-binding activity of Nug2 influences the remodeling activity of the Rea1 ATPase, whereas GTP hydrolysis is necessary for the final Nug2 release from the pre-60S subunit. In vitro, Rea1-dependent release of Rsa4 and Nug2 required only ATP and K+ without addition of GTP, whereas the mutational analyses suggested that GTPase activity is necessary for Nug2 release. These findings suggest that Nug2 on the Rix1-particle might have retained bound GTP during purification (which is possible due to its low intrinsic GTPase activity). Alternatively, ribosome-associated nucleotide diphosphate kinases can transfer the γ-phosphate from ATP to GDP to generate GTP-loaded GTPases23,24. The pre-60S particles co-purified with Rix1 also contained small amounts of Ytm1 and Erb1 (Fig. 3c), which were previously described as nucleolar co-substrates for Rea122, and both were released by incubation with ATP in Na+ or K+ buffer (Fig. 3c). To determine the step in 60S subunit biogenesis at which dissociation of Nug2 is disturbed in vivo, we affinity-purified different pre-60S particles from Nug2 wild-type and mutant cells using bait proteins that specifically enrich nucleolar, nucleoplasmic or cytoplasmic intermediates (Fig. 4a). The nug2K328R mutation did not markedly alter the biochemical composition of most pre-60S particles tested. The exception was Arx1-associated particles, which showed a marked depletion of the export adapter Nmd3 and the cytoplasmic factor Rei1 that stimulates recycling of Arx125 (Fig. 4a). Nmd3 was also largely absent from Arx1particles purified from nug2G369A cells (Fig. 4b). To test the model that Nug2 depletion allows premature recruitment of Nmd3 we employed an auxin-inducible degron system28. Nug2 was expressed as a fusion protein (sAid-Nug2sAid) with two copies of the sAid tag (small Auxin-inducible degron), which is targeted by the F-box E3 ubiquitin ligase TIR1 in the presence of auxin, inducing fast proteasomal degradation28 (Extended Data Fig. 5). Nmd3 is normally not detected on Rix1-associated particles, but was prematurely recruited to this pre-60S particle upon Nug2 depletion (Fig. 4c). Concomitant with Nmd3 association, the recovery of Rea1 and Rsa4 decreased during Nug2 depletion (Fig. 4c). We conclude that Nug2 promotes the stable association of Rsa4 and Rea1 with the Rix1-particles, while blocking premature recruitment of Nmd3. To address the timing of Nug2 recruitment to pre-60S particles in comparison to Rea1, Rsa4 and the Rix1-Ipi1-Ipi3 complex, we employed a combination of affinity-purification and immunodepletion. Affinity-purification of Nug2-TAP yielded a mixture of different pre-60S particles including Rix1/Rea1-particles. Rix1-FLAG immunoprecipitation was used to Matsuo et al. Page 3 Nature. Author manuscript; available in PMC 2014 July 02. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts deplete Rix1/Rea1-particles from this mixture, leaving Nug2 particles that contained Rsa4 and a number of intermediate pre-60S factors including Nog1, Arx1, Nug1, Nop53, Nsa3, Rpf2, Rlp7 and Nsa2, (Fig. 4d). However, this Nug2-particle lacked other (further upstream) pre-60S factors such as Ytm1, Erb1 and Has1, suggesting that it corresponds to the precursor particle to which Nug2 was recruited. These data complement previous findings that Nog2/Nug2 is the last “B-factor” to associate with pre-ribosomes after dissociation of Has129. As outlined in Fig. 4e, we propose that a previously uncharacterized step in the reorganization of the evolving pre-60S subunit primes it for nuclear export. This involves a regulatory GTPase Nug2 that overlaps with the binding site for the essential nuclear export adaptor Nmd3. As long as intranuclear maturation is incomplete, the pre-60S subunit cannot be exported, since recruitment of this essential export factor is not possible. However, a late nucleoplasmic remodeling step, catalyzed by the AAA-ATPase Rea1 and its co-factor Rsa4, restructures the pre-60S particle, which could lead to both an rRNA and assembly factor rearrangement. This conformational change could also stimulate Nug2’s K+-dependent GTPase activity, thereby triggering its release from the matured pre-60S particles. We suggest that the Nug2 GTPase acts as molecular switch to proofread pre-ribosome maturation and regulate the acquisition of export competence. After this reorganization step, the binding site for Nmd3 becomes accessible on the pre-60S subunit, which further triggers Crm1 and RanGTP recruitment to generate nuclear export competence. Thus, our data indicate coordination between a remodeling AAA-ATPase and a conformation-sensing GTPase. The human Nug2 orthologue Gnl2 is highly expressed in proliferating cells including cancer cells and involved in the control of cell cycle progression30. The discovery of the role of Nug2 during surveillance of ribosome biogenesis may help reveal the molecular mechanisms by which nucleostemin family members interconnect the elementary cellular processes of ribosome biogenesis and cell proliferation.