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• W4309593280 abstract "•Cms1 is a suppressor of nop14 mutants impaired in Rrp12-Enp1 recruitment to the 90S•Cms1 associates with early 90S to restrict premature Rrp12-Enp1 recruitment•Cms1 allows timely pseudouridylation by snR83 at the exposed 3′ major domain Ribosome synthesis begins in the nucleolus with 90S pre-ribosome construction, but little is known about how the many different snoRNAs that modify the pre-rRNA are timely guided to their target sites. Here, we report a role for Cms1 in such a process. Initially, we discovered CMS1 as a null suppressor of a nop14 mutant impaired in Rrp12-Enp1 factor recruitment to the 90S. Further investigations detected Cms1 at the 18S rRNA 3′ major domain of an early 90S that carried H/ACA snR83, which is known to guide pseudouridylation at two target sites within the same subdomain. Cms1 co-precipitates with many 90S factors, but Rrp12-Enp1 encircling the 3′ major domain in the mature 90S is decreased. We suggest that Cms1 associates with the 3′ major domain during early 90S biogenesis to restrict premature Rrp12-Enp1 binding but allows snR83 to timely perform its modification role before the next 90S assembly steps coupled with Cms1 release take place. Ribosome synthesis begins in the nucleolus with 90S pre-ribosome construction, but little is known about how the many different snoRNAs that modify the pre-rRNA are timely guided to their target sites. Here, we report a role for Cms1 in such a process. Initially, we discovered CMS1 as a null suppressor of a nop14 mutant impaired in Rrp12-Enp1 factor recruitment to the 90S. Further investigations detected Cms1 at the 18S rRNA 3′ major domain of an early 90S that carried H/ACA snR83, which is known to guide pseudouridylation at two target sites within the same subdomain. Cms1 co-precipitates with many 90S factors, but Rrp12-Enp1 encircling the 3′ major domain in the mature 90S is decreased. We suggest that Cms1 associates with the 3′ major domain during early 90S biogenesis to restrict premature Rrp12-Enp1 binding but allows snR83 to timely perform its modification role before the next 90S assembly steps coupled with Cms1 release take place. IntroductionEukaryotic ribosome synthesis is a highly energy-consuming process that occurs along a cascade of numerous assembly, modification, and maturation steps from the nucleolus to the cytoplasm. There, the small 40S and large 60S ribosomal subunits are supplied for protein synthesis. In yeast, four ribosomal RNAs (18S, 5.8S, 25S, and 5S rRNA), 79 ribosomal proteins, about 70 different small nucleolar RNAs (snoRNAs), and approximately 200 assembly factors hierarchically enter and dynamically participate in this enormous assembly line (Ben-Shem et al., 2011Ben-Shem A. Garreau de Loubresse N. Melnikov S. Jenner L. Yusupova G. Yusupov M. The structure of the eukaryotic ribosome at 3.0 A resolution.Science. 2011; 334: 1524-1529Crossref PubMed Scopus (790) Google Scholar; Klinge et al., 2012Klinge S. Voigts-Hoffmann F. Leibundgut M. Ban N. Atomic structures of the eukaryotic ribosome.Trends Biochem. Sci. 2012; 37: 189-198Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar; Kressler et al., 2010Kressler D. Hurt E. Bassler J. Driving ribosome assembly.Biochim. Biophys. Acta. 2010; 1803: 673-683Crossref PubMed Scopus (363) Google Scholar).Following transcription by RNA polymerase I, a large rRNA precursor, called 35S pre-rRNA in yeast, is produced. This is composed of two external transcribed spacers (5′-ETS and 3′-ETS) and two internal transcribed spacers (ITS1 and ITS2) separating the mature 18S, 5.8S, and 25S rRNAs (Granneman and Baserga, 2004Granneman S. Baserga S.J. Ribosome biogenesis: of knobs and RNA processing.Exp. Cell Res. 2004; 296: 43-50Crossref PubMed Scopus (184) Google Scholar; Henras et al., 2015Henras A.K. Plisson-Chastang C. O'Donohue M.F. Chakraborty A. Gleizes P.E. An overview of pre-ribosomal RNA processing in eukaryotes.Wiley Interdiscip. Rev. RNA. 2015; 6: 225-242Crossref PubMed Scopus (337) Google Scholar). The 5S rRNA is synthesized separately by RNA polymerase III before assembling into pre-ribosomal particles. The first steps of ribosome assembly are initiated while pre-rRNA synthesis is taking place. These include modification of pre-rRNA base and ribose moieties, RNA folding and processing, and recruitment of early ribosomal proteins and assembly factors (Venema and Tollervey, 1999Venema J. Tollervey D. Ribosome synthesis in Saccharomyces cerevisiae.Annu. Rev. Genet. 1999; 33: 261-311Crossref PubMed Scopus (649) Google Scholar; Zhang et al., 2016Zhang L. Wu C. Cai G. Chen S. Ye K. Stepwise and dynamic assembly of the earliest precursors of small ribosomal subunits in yeast.Genes Dev. 2016; 30: 718-732Crossref PubMed Google Scholar). This eventually yields the 90S pre-ribosome (also known as the small subunit processome), the first stable intermediate amenable to high-resolution structural analysis (Cheng et al., 2019Cheng J. Baßler J. Fischer P. Lau B. Kellner N. Kunze R. Griesel S. Kallas M. Berninghausen O. Strauss D. et al.Thermophile 90S pre-ribosome structures reveal the reverse order of Co-transcriptional 18S rRNA subdomain integration.Mol. Cell. 2019; 75: 1256-1269.e7Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar; Kornprobst et al., 2016Kornprobst M. Turk M. Kellner N. Cheng J. Flemming D. Koš-Braun I. Koš M. Thoms M. Berninghausen O. Beckmann R. Hurt E. Architecture of the 90S pre-ribosome: a structural view on the birth of the eukaryotic ribosome.Cell. 2016; 166: 380-393Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar; Singh et al., 2021Singh S. Vanden Broeck A. Miller L. Chaker-Margot M. Klinge S. Nucleolar maturation of the human small subunit processome.Science. 2021; 373: eabj5338Crossref Scopus (12) Google Scholar). During this nucleolar phase, pre-60S assembly also begins on the nascent 35S pre-rRNA. Cleavages within ITS1, at either site A2 (occurring co-transcriptionally) or site A3 (occurring post-transcriptionally), separate the 40S and 60S subunit biogenesis routes until the small and large subunits have reached their maturity in the cytoplasm (Henry et al., 1994Henry Y. Wood H. Morrissey J.P. Petfalski E. Kearsey S. Tollervey D. The 5' end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site.EMBO J. 1994; 13: 2452-2463Crossref PubMed Google Scholar; Lebaron et al., 2013Lebaron S. Segerstolpe A. French S.L. Dudnakova T. de Lima Alves F. Granneman S. Rappsilber J. Beyer A.L. Wieslander L. Tollervey D. Rrp5 binding at multiple sites coordinates pre-rRNA processing and assembly.Mol. Cell. 2013; 52: 707-719Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar; Lygerou et al., 1996Lygerou Z. Allmang C. Tollervey D. Séraphin B. Accurate processing of a eukaryotic precursor ribosomal RNA by ribonuclease MRP in vitro.Science. 1996; 272: 268-270Crossref PubMed Google Scholar).When 90S biogenesis begins co-transcriptionally in the nucleolus, the emerging 5′-ETS already binds several 90S modules, such as UTP-A, UTP-B, and the U3 snoRNP. As the pre-rRNA continues to grow in the 18S rRNA region, more 90S factors (e.g., Utp20) and modules (e.g., Noc4-Nop14-Emg1-Enp1-Rrp12, UTP-C, Bms1-Rcl1, Mpp10-Sas10-Imp3-Imp4, Utp7-Sof1-Utp14) become successively integrated before the archetypal 90S has formed (Chaker-Margot et al., 2015Chaker-Margot M. Hunziker M. Barandun J. Dill B.D. Klinge S. Stage-specific assembly events of the 6-MDa small-subunit processome initiate eukaryotic ribosome biogenesis.Nat. Struct. Mol. Biol. 2015; 22: 920-923Crossref PubMed Scopus (77) Google Scholar; Cheng et al., 2019Cheng J. Baßler J. Fischer P. Lau B. Kellner N. Kunze R. Griesel S. Kallas M. Berninghausen O. Strauss D. et al.Thermophile 90S pre-ribosome structures reveal the reverse order of Co-transcriptional 18S rRNA subdomain integration.Mol. Cell. 2019; 75: 1256-1269.e7Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar; Hunziker et al., 2019Hunziker M. Barandun J. Buzovetsky O. Steckler C. Molina H. Klinge S. Conformational switches control early maturation of the eukaryotic small ribosomal subunit.Elife. 2019; 8: e45185Crossref PubMed Google Scholar; Zhang et al., 2016Zhang L. Wu C. Cai G. Chen S. Ye K. Stepwise and dynamic assembly of the earliest precursors of small ribosomal subunits in yeast.Genes Dev. 2016; 30: 718-732Crossref PubMed Google Scholar). At a certain point, the 90S undergoes a dramatic transition, which involves shedding of most of the 90S factors until the primordial pre-40S (Dis-C) emerges (Cheng et al., 2020Cheng J. Lau B. La Venuta G. Ameismeier M. Berninghausen O. Hurt E. Beckmann R. 90S pre-ribosome transformation into the primordial 40S subunit.Science. 2020; 369: 1470-1476Crossref PubMed Google Scholar; Du et al., 2020Du Y. An W. Zhu X. Sun Q. Qi J. Ye K. Cryo-EM structure of 90S small ribosomal subunit precursors in transition states.Science. 2020; 369: 1477-1481Crossref PubMed Google Scholar). However, Dis-C still carries the U3 snoRNA and a few residual 90S factors, including the helicase Dhr1. Their final removal and recruitment of the next set of pre-40S factors occur in the pre-40S maturation steps that follow (Cheng et al., 2020Cheng J. Lau B. La Venuta G. Ameismeier M. Berninghausen O. Hurt E. Beckmann R. 90S pre-ribosome transformation into the primordial 40S subunit.Science. 2020; 369: 1470-1476Crossref PubMed Google Scholar; Sardana et al., 2015Sardana R. Liu X. Granneman S. Zhu J. Gill M. Papoulas O. Marcotte E.M. Tollervey D. Correll C.C. Johnson A.W. The DEAH-box helicase Dhr1 dissociates U3 from the pre-rRNA to promote formation of the central pseudoknot.PLoS Biol. 2015; 13: e1002083Crossref PubMed Google Scholar).The 90S carries only a single snoRNA, the U3 snoRNA, which, together with Nop56, Nop58, Snu13, and Nop1, constitutes a typical C/D box small nucleolar ribonucleoprotein (snoRNP) (Chaker-Margot et al., 2017Chaker-Margot M. Barandun J. Hunziker M. Klinge S. Architecture of the yeast small subunit processome.Science. 2017; 355: eaal1880Crossref PubMed Scopus (88) Google Scholar; Hunziker et al., 2016Hunziker M. Barandun J. Petfalski E. Tan D. Delan-Forino C. Molloy K.R. Kim K.H. Dunn-Davies H. Shi Y. Chaker-Margot M. et al.UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly.Nat. Commun. 2016; 7: 12090Crossref PubMed Scopus (47) Google Scholar; Kornprobst et al., 2016Kornprobst M. Turk M. Kellner N. Cheng J. Flemming D. Koš-Braun I. Koš M. Thoms M. Berninghausen O. Beckmann R. Hurt E. Architecture of the 90S pre-ribosome: a structural view on the birth of the eukaryotic ribosome.Cell. 2016; 166: 380-393Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, U3 is not involved in pre-rRNA methylation (see below) but has a structural role in the 90S. In this case, the 5′ single-stranded region of the U3 snoRNA anneals at distinct regions in the 5′-ETS and 18S rRNA. These contacts help to organize the nascent pre-RNA, and also keep the 18S rRNA immature at distinct sites (Beltrame and Tollervey, 1992Beltrame M. Tollervey D. Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA.EMBO J. 1992; 11: 1531-1542Crossref PubMed Scopus (0) Google Scholar; Hughes, 1996Hughes J.M. Functional base-pairing interaction between highly conserved elements of U3 small nucleolar RNA and the small ribosomal subunit RNA.J. Mol. Biol. 1996; 259: 645-654Crossref PubMed Scopus (125) Google Scholar; Sharma and Tollervey, 1999Sharma K. Tollervey D. Base pairing between U3 small nucleolar RNA and the 5' end of 18S rRNA is required for pre-rRNA processing.Mol. Cell Biol. 1999; 19: 6012-6019Crossref PubMed Scopus (0) Google Scholar).However, there exist many other snoRNAs in eukaryotic cells, which guide numerous covalent pre-rRNA modifications during ribosome synthesis (Dunbar and Baserga, 1998Dunbar D.A. Baserga S.J. The U14 snoRNA is required for 2'-O-methylation of the pre-18S rRNA in Xenopus oocytes.RNA. 1998; 4: 195-204PubMed Google Scholar; Jaafar et al., 2021Jaafar M. Contreras J. Dominique C. Martín-Villanueva S. Capeyrou R. Vitali P. Rodríguez-Galán O. Velasco C. Humbert O. Watkins N.J. et al.Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast.Nat. Commun. 2021; 12: 6153Crossref PubMed Scopus (0) Google Scholar; Venema and Tollervey, 1999Venema J. Tollervey D. Ribosome synthesis in Saccharomyces cerevisiae.Annu. Rev. Genet. 1999; 33: 261-311Crossref PubMed Scopus (649) Google Scholar). These snoRNAs form snoRNPs that belong to either the C/D box (such as U3) or the H/ACA box particles (Kiss et al., 2006Kiss T. Fayet E. Jády B.E. Richard P. Weber M. Biogenesis and intranuclear trafficking of human box C/D and H/ACA RNPs.Cold Spring Harb. Symp. Quant. Biol. 2006; 71: 407-417Crossref PubMed Scopus (124) Google Scholar; Watkins and Bohnsack, 2012Watkins N.J. Bohnsack M.T. The box C/D and H/ACA snoRNPs: key players in the modification, processing and the dynamic folding of ribosomal RNA.Wiley Interdiscip. Rev. RNA. 2012; 3: 397-414Crossref PubMed Scopus (309) Google Scholar). The C/D box snoRNPs catalyze 2′-O-ribose methylation, with Nop1/fibrillarin as the methyltransferase and Nop58, Nop56, and Snu13 as further core factors. The H/ACA snoRNPs mediate pseudouridylation, with Cbf5 acting as a pseudouridine synthase and Gar1, Nhp2, and Nop10 as its core factors (Duan et al., 2009Duan J. Li L. Lu J. Wang W. Ye K. Structural mechanism of substrate RNA recruitment in H/ACA RNA-guided pseudouridine synthase.Mol. Cell. 2009; 34: 427-439Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar; Lapinaite et al., 2013Lapinaite A. Simon B. Skjaerven L. Rakwalska-Bange M. Gabel F. Carlomagno T. The structure of the box C/D enzyme reveals regulation of RNA methylation.Nature. 2013; 502: 519-523Crossref PubMed Scopus (122) Google Scholar; Li and Ye, 2006Li L. Ye K. Crystal structure of an H/ACA box ribonucleoprotein particle.Nature. 2006; 443: 302-307Crossref PubMed Scopus (0) Google Scholar; Lin et al., 2011Lin J. Lai S. Jia R. Xu A. Zhang L. Lu J. Ye K. Structural basis for site-specific ribose methylation by box C/D RNA protein complexes.Nature. 2011; 469: 559-563Crossref PubMed Scopus (92) Google Scholar). Many of the known rRNA modifications are thought to occur quite early during ribosome assembly, when the pre-rRNA is not yet compactly folded or target sites are masked by assembly factors. Although the mechanistic details of these processes remain largely unknown, it is conceivable that a stepwise assembly of 90S might grant the many different snoRNPs access to their modification sites, which number approximately 100 in yeast rRNA and 200 in human, with many conserved in evolution (Sharma and Lafontaine, 2015Sharma S. Lafontaine D.L.J. 'View from A bricge': a new perspective on eukaryotic rRNA base modification.Trends Biochem. Sci. 2015; 40: 560-575Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar; Sloan et al., 2017Sloan K.E. Warda A.S. Sharma S. Entian K.D. Lafontaine D.L.J. Bohnsack M.T. Tuning the ribosome: the influence of rRNA modification on eukaryotic ribosome biogenesis and function.RNA Biol. 2017; 14: 1138-1152Crossref PubMed Scopus (301) Google Scholar).Since little is known about snoRNA-mediated modification during early ribosome formation and its coupling with downstream assembly steps, we sought to investigate this mechanism, which is assumed to be coordinated. We observed such a coupling with our initial discovery that Cms1, a poorly characterized ribosome biogenesis factor, suppresses a specific nop14 mutant defective in Noc4 module assembly. Further characterization of Cms1 showed that it is associated with a specific box H/ACA snoRNA, snR83, which guides pseudouridylation at two specific uridines in the 3′ major domain of the 18S rRNA. Based on these insights, we propose a model in which Cms1 could coordinate snR83-guided modification within the 3′ major domain, coupled with the stepwise assembly of the Noc4 module in the same region of the 90S pre-ribosome.ResultsThe Nop14-N motif connects the Noc4 module and Rcl1-Bms1 in the 90S pre-ribosomeAnalysis of 90S pre-ribosome structures from evolutionarily distant organisms such as yeast, Chaetomium thermophilum, and human revealed a conserved interaction between the Noc4 module (Noc4-Nop14-Emg1-Enp1-Rrp12) and the Rcl1-Bms1 heterodimer, mediated by a short β strand in the Nop14 N-terminal extension (residues 110–114 in yeast) that contacts another β strand in Rcl1 (Figures 1A and 1B ). This observation prompted us to investigate the role of the extended Nop14 N terminus by performing a deletion analysis in yeast. Whereas Nop14 N-terminal residues 1–104 (nop14ΔN1) could be removed without impairing growth, deleting farther into the β-strand (residues 1–125, nop14ΔN2) generated a slow-growth phenotype that was not exacerbated in the nop14ΔN3 mutant lacking residues 1–159 (Figure 1C). To demonstrate that the strong inhibition of growth observed for the two longer nop14 N-terminal deletions was caused by the removal of the interacting β-strand residues 110–114, five “alanine scan” mutations (nop14-5Ala) were introduced into this part of Nop14 (Figures 1A and 1D). As anticipated, the nop14-5Ala mutant showed a strong growth defect, albeit slightly less pronounced than the nop14ΔN2 deletion (Figure 1D).CMS1 gene disruption rescues nop14 N-terminal deletion mutantsWe observed that the two slow-growing nop14 N-terminal deletion mutants, when plated at high cell density, occasionally produced a few faster-growing colonies (Figure 1C and data not shown). To ascertain whether these are extragenic suppressors, we performed genetic tests (Figures S1A–S1C). Notably, when one of these suppressors (called sup1-1) was backcrossed to an isogenic nop14ΔN3 initial strain, the resulting diploid exhibited a loss of suppressor activity, but after sporulation and tetrad analysis, a 2:2 segregation of fast- versus slow-growing colonies was observed (Figures S1A and S1B). These genetic data suggest that sup1-1 carries a single recessive mutation responsible for the extragenic suppressor activity that restores growth of the nop14 N-terminal mutants.To identify the suppressor gene locus, the genome of the sup1-1 yeast strain was sequenced. This uncovered a meaningful T nucleotide insertion in the nonessential CMS1 gene, causing a Ser68Phe substitution in the Cms1 protein (full-length Cms1 has 291 amino acids), with a subsequent frameshift into a short open reading frame (ORF) before a stop codon (Figure S1D). We assumed that this C-terminally truncated Cms1 in the sup1-1 strain is nonfunctional, and hence a cms1-null-like phenotype might be responsible for the suppression of the nop14 N-deletion mutants. To obtain experimental evidence for this, the CMS1 gene was disrupted in the nop14ΔN3 and nop14-5Ala strains, which in both cases restored cell growth to a large, but not full, extent (Figure 1D). However, another slow-growing mutant, nop14ΔC80, lacking the long C-terminal α helix that pokes into the 90S at another site (see Figure 1B), was not suppressed by cms1Δ (Figure 1E). Together, these data show that the CMS1 gene disruption specifically suppresses nop14 N-terminal mutations.CMS1 encodes a poorly characterized but conserved protein (human homolog CMSS1; Figure S2A) that was initially found as a high-copy suppressor of MCM10 (Wang and Wu, 2001Wang J.W. Wu J.R. Overexpression of a novel gene, Cms1, can rescue the growth arrest of a Saccharomyces cerevisiae mcm10 suppressor.Cell Res. 2001; 11: 285-291Crossref Scopus (0) Google Scholar). However, based on other analyses, it was implicated in 90S ribosome biogenesis (Grandi et al., 2002Grandi P. Rybin V. Bassler J. Petfalski E. Strauss D. Marzioch M. Schäfer T. Kuster B. Tschochner H. Tollervey D. et al.90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors.Mol. Cell. 2002; 10: 105-115Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar; Hunziker et al., 2019Hunziker M. Barandun J. Buzovetsky O. Steckler C. Molina H. Klinge S. Conformational switches control early maturation of the eukaryotic small ribosomal subunit.Elife. 2019; 8: e45185Crossref PubMed Google Scholar; van Leeuwen et al., 2020van Leeuwen J. Pons C. Tan G. Wang J.Z. Hou J. Weile J. Gebbia M. Liang W. Shuteriqi E. Li Z. et al.Systematic analysis of bypass suppression of essential genes.Mol. Syst. Biol. 2020; 16: e9828Google Scholar; Gavin et al., 2002Gavin A.-C. Bösche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. et al.Functional organization of the yeast proteome by systematic analysis of protein complexes.Nature. 2002; 415: 141-147Crossref PubMed Scopus (3978) Google Scholar; Sturm et al., 2017Sturm M. Cheng J. Baßler J. Beckmann R. Hurt E. Interdependent action of KH domain proteins Krr1 and Dim2 drive the 40S platform assembly.Nat. Commun. 2017; 8: 2213Crossref PubMed Scopus (18) Google Scholar). Cms1 exhibits a helicase fold, but it is likely inactive as a helicase owing to the absence of classical ATPase/helicase motifs (e.g., Walker A, DEAD box) and a second RecA-like domain (Figures S2B and S2C).Based on our genetic and structural data, we wondered if a mutation in the pairing Rcl1 β strand could be suppressed by cms1Δ as well. To examine this, we replaced a charged loop emerging from this Rcl1 β strand with a neutral glycine-serine (GGGGS) loop. Notably, this Rcl1 GGGGS loop mutant exhibited a slow-growing phenotype that could be suppressed by cms1Δ (Figure S3A).Prompted by these findings, we tested whether a complete chromosomal deletion of either NOP14 or RCL1, both of which are essential genes in yeast, could be suppressed by cms1Δ. Whereas the cms1-null allele did not rescue nop14Δ (data not shown), the rcl1Δ cms1Δ double-gene-disruption strain was viable, although cells grew very slowly (Figure 2A ). This observation is consistent with a recent systematic bypass suppression analysis of essential yeast genes, including RCL1, which yielded a similar result (van Leeuwen et al., 2020van Leeuwen J. Pons C. Tan G. Wang J.Z. Hou J. Weile J. Gebbia M. Liang W. Shuteriqi E. Li Z. et al.Systematic analysis of bypass suppression of essential genes.Mol. Syst. Biol. 2020; 16: e9828Google Scholar).Figure 2Biochemical and structural analysis of 90S particles isolated from the viable rcl1Δ cms1Δ and nop14-5Ala cms1 suppressor strainsShow full caption(A) Complete chromosomal deletion of RCL1, which is lethal in yeast, can be rescued by cms1Δ. Dot-spot growth analysis of wild type (RCL1 CMS1), single cms1Δ and cms1Δ mutants, and the double-disruption rcl1Δ cms1Δ suppressor strains. The shuffle strains were grown on 5-FOA plates at 30°C for 4 days.(B) Affinity purification of FTpA-Utp18 from wild type (RCL1 CMS1), single cms1Δ mutant, and double-disruption strain rcl1Δ cms1Δ.(C) Cryo-EM analysis of 90S pre-ribosomal particles, isolated from the double-disruption rcl1Δ cms1Δ strain via the 90S bait protein FTpA-Utp18. Note that there is no electron density for Rcl1 (shown in red in the wild-type 90S) in the mutant 90S particles, but Bms1 (yellow) could be well discerned at its authentic position, together with other typical 90S substructures. For comparison, the cryo-EM structure of an intact yeast 90S particle in state B2 (EMD-11358) is shown in a related orientation. The overall resolution of the 90S from the rcl1Δ cms1Δ strain is 6.7 Å (using a loose mask from cryoSPARC).(D) Cryo-EM analysis of 90S pre-ribosomal particles isolated from the nop14-5Ala cms1Δ suppressor strain via the 90S bait protein FTpA-Utp18. In total, two 90S particles, state B2 (middle) and state b (right), could be classified from the dataset, which are highly similar to published 90S particles state B2 (EMD-11358) and b (EMD-8859), respectively. Notably, Utp20 is missing in the 90S state b. For comparison, the cryo-EM structure of an intact yeast 90S particle in state B2 (EMD-11358) is shown in a related orientation (left). All structures are color coded to indicate the different 90S modules.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cryo-EM structure of the 90S particles isolated from cms1Δ suppressorsSince the cms1Δ gene disruption not only rescued the nop14-5Ala mutant, but also allowed yeast cells to live without the essential RCL1, we sought to gain insight into the structure of 90S particles from these different suppressors. Affinity purification of Utp18 (UTP-B factor) from the rcl1Δ cms1Δ suppressor revealed co-enrichment of many 90S factors, but Rcl1, Bms1, and a few other factors such as Utp20, Utp22, and Utp14 were absent or present in low abundance (Figure 2B). Moreover, the free pool of the UTP-B module was diminished. Subsequent cryoelectron microscopy (cryo-EM) analysis of these suppressor-derived 90S particles showed a cavity at the expected position of Rcl1. However, adjacent to this hole, Rcl1’s partner Bms1 was still present at its authentic position (Figures 2C and S3B–S3E). In addition, several other typical 90S substructures, such as the 5′-ETS with its attached UTP-A, UTP-B, and U3 snoRNP modules, were readily identified on these unusual 90S particles. In contrast, another area corresponding to the 5′ rRNA domain, including Utp20, was either not as clearly visible or not resolved at all, suggesting that this subpart of the 90S is in a more immature, and therefore still more dynamic, state, which is not yet correctly folded and/or rigidly integrated into the 90S structure (Figures 2C and S3B–S3E). Thus, quite unexpectedly, ribosome biogenesis can take place without the essential Rcl1 under cms1Δ-suppressing conditions. However, such 90S pre-ribosomes are biochemically less stable and structurally immature or not correctly assembled in distinct regions, providing an explanation for the slow-growth phenotype of the rcl1Δ cms1Δ strain.Cryo-EM analysis of the 90S particles purified from the nop14-5Ala cms1Δ suppressor revealed two major populations; one was similar to the 90S wild-type B2 state (Cheng et al., 2020Cheng J. Lau B. La Venuta G. Ameismeier M. Berninghausen O. Hurt E. Beckmann R. 90S pre-ribosome transformation into the primordial 40S subunit.Science. 2020; 369: 1470-1476Crossref PubMed Google Scholar), whereas the other exhibited a more immature appearance, with flexible central and 5′ domains (Figures 2D and S3F). Since the nop14-5Ala cms1Δ suppressor does not exhibit optimal cell growth compared with wild-type yeast (see Figure 1D), this could explain the existence of these two different 90S classes.90S pre-ribosomes from the nop14-5Ala mutant retain Cms1 instead of Rrp12-Enp1To investigate whether the nop14-5Ala mutation causes disruption to the assembly of the Noc4 module, we isolated 90S particles from the mutant strain via FTpA-Utp18 and identified the co-precipitated factors. Although the overall pattern of co-enriched 90S factors was similar compared with that of wild-type particles, the Rrp12 band, together with its direct binding partner Enp1, was markedly decreased in the nop14-5Ala strain, which could be confirmed by western blotting using α-Rrp12 antibodies (Figure 3A ). However, this defect was corrected in the nop14-5Ala cms1Δ suppressor strain, where co-enrichment of Rrp12-Enp1 was restored in the FTpA-Utp18 preparation (Figures 3A and S4 and Table S1). Thus, disrupting CMS1 in nop14-5Ala cells not only improved cell growth, but also induced recovery from arrested pre-ribosome maturation with reappearance of Rrp12-Enp1 in the suppressor-derived 90S particles.Figure 3Characterization of 90S particles from the nop14-5Ala mutant and its cms1Δ suppressorShow full caption(A) FTpA-Utp18 was affinity purified from the wild type (lane 1), the single mutants cms1Δ (lane 2) and nop14-5Ala (lane 3), and the nop14-5Ala cms1Δ double mutant (suppressor, lane 4). Equivalent amounts of the final eluates were analyzed on a 4%–12% gradient SDS-polyacrylamide gel stained with Coomassie blue (top) and analyzed by western blotting using anti-Rrp12 and anti-Sof1 (90S marker) antibodies (bottom). An independent series of FTpA-Utp18 affinity purifications from wild type and the same mutant strains is shown in Figure S4, from which the final eluates were further analyzed by semiquantitative mass spectrometry (Table S1), but also used for RNA analysis (see also Figure 6). The experiment was repeated four times, yielding in all cases similar outcomes.(B) Dhr1-Dim1 split-tag affinity purification of particles in the 90S → pre-40S transition from the wild type (NOP14, lane 1), the nop14-5Ala mutant (lane 2), and the nop14ΔC80 mutant (lane 3). The final eluates were analyzed on a 4%–12% gradient SDS-polyacrylamide gel and stained with Coomassie blue. Bands numbered 1–11 were excised from the gel and identified by MALDI-TOF mass spectrometry. Shown on the right are Mascot score histograms (probability-based MOWSE algorithm; scores greater than 50 were regarded as statistically significant) for the individual excised bands 1–3; 90S factors are identified above the red bars. The Mascot score is a statistical score for how well the experimental data match the database sequences.(C) Cryo-EM structure comparison of 90S pre-ribosomes in the post-A1 state purified from wild-type NOP14 (left) and nop14-5Ala cells (right), both isolated via Dhr1-Dim1 split tags. Rcl1 and Nop14 are colored in yellow and pink, respectively. The Rcl1-Nop14 interaction region from both structures is shown in detail as insets in the middle. The Nop" @default.
• W4309593280 created "2022-11-28" @default.
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• W4309593280 date "2022-11-01" @default.
• W4309593280 modified "2023-10-01" @default.
• W4309593280 title "Cms1 coordinates stepwise local 90S pre-ribosome assembly with timely snR83 release" @default.
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• W4309593280 doi "https://doi.org/10.1016/j.celrep.2022.111684" @default.
• W4309593280 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/36417864" @default.
• W4309593280 hasPublicationYear "2022" @default.
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