Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast

ARTICLE Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast Mariam Jaafar1,2,9, Julia Contreras3,4, Carine Dominique1, Sara Martín-Villanueva 3, Régine Capeyrou1, Patrice Vitali1, Olga Rodríguez-Galán3,4, Carmen Velasco3,5, Odile Humbert1, Nicholas J. Watkins6, Eduardo Villalobo 3,5, Katherine E. Bohnsack 7, Markus T. Bohnsack 7,8, Yves Henry1, Raghida Abou Merhi2, Jesús de la Cruz 3,4 & Anthony K. Henras 1✉ Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit. https://doi.org/10.1038/s41467-021-26207-w OPEN 1Molecular, Cellular and Developmental Biology Unit (MCD), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, 31062 Toulouse, France. 2Genomic Stability and Biotherapy (GSBT) Laboratory, Faculty of Sciences, Rafik Hariri Campus, Lebanese University, Beirut, Lebanon. 3Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41013 Seville, Spain. 4Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41012 Seville, Spain. 5Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla, 41012 Seville, Spain. 6Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE1 7RU, UK. 7Department of Molecular Biology, University Medical Centre Göttingen, 37073 Göttingen, Germany. 8Göttingen Center for Molecular Biosciences, Georg- August University Göttingen, 37077 Göttingen, Germany. 9 Present address: Cancer Research Center of Lyon (CRCL), 69 008 Lyon, France. ✉email: [email protected] NATURE COMMUNICATIONS | (2021) 12:6153 | https://doi.org/10.1038/s41467-021-26207-w | www.nature.com/naturecommunications 1 1234567890():,; Ribosomes are universal molecular machines responsible for protein synthesis. The first crystal structures of ribosomal subunits (r-subunits)1,2revealed that the ribosome is a ribozyme3, namely an RNA-based enzyme. This implies that an essential aspect of ribosome biogenesis is the accurate folding of the rRNAs to generate their functional structure. In yeast, rRNAs contained in the 40S (18S) and 60S (5S, 5.8S, and 25S) r-subunits are generated by transcription of rDNA genes by RNA polymerases I and III (Pol I and Pol III). Pol I-mediated transcription generates a primary transcript containing the sequences of 18S, 5.8S, and 25S rRNAs. Maturation of this pre-rRNA occurs within a series of preribosomal particles containing also RPs and numerous AFs. This process comprises pre-rRNA nucleoside modifications, cleavage and folding events and the incorporation and proper positioning of RPs that gradually lead to the formation of mature r-subunits4–6. Assembly of the earliest precursors to the large (60S) r-subunit is the least understood step of eukaryotic ribosome biogenesis. These particles contain numerous small nucleolar ribonucleoprotein particles (snoRNPs)7, which belong to the C/D- and H/ ACA-type families. C/D-type snoRNPs typically catalyze the 2’- O-methylation of selected nucleosides. They contain a box C/D small nucleolar RNA (snoRNA) associated with a protein core including the methyltransferase Nop1. H/ACA-type snoRNPs are composed of a box H/ACA snoRNA and several proteins including the pseudouridine synthase Cbf5, which isomerizes uridines into pseudouridines. In both cases, the snoRNAs contain antisense elements that base-pair to target sequences on the prerRNA, and function as a guide for specific nucleoside modifications8,9. A subset of snoRNAs from both families do not function as modification guides but as RNA chaperones, assisting folding of the pre-rRNA to promote proper processing. In yeast, four snoRNP chaperones, containing U3, U14, snR30, and snR10 snoRNAs, are involved in the biogenesis of the small r-subunit10–13. An important issue that remains to be addressed is when and how snoRNA base-pairings with the pre-rRNA are disrupted, given that some of these interactions are thermodynamically highly stable. NTP-dependent RNA helicases have the potential to dissociate snoRNA-pre-rRNA interactions. Twelve RNA helicases (Dbp2, Dbp3, Dbp6, Dbp7, Dbp9, Dbp10, Drs1, Mak5, Mtr4, Spb4, Prp43, and Has1) participate in the synthesis of the large r-subunit14,15 and interestingly, eight of them (Dbp3, Dbp6, Dbp7, Dbp9, Drs1, Has1, Mak5, and Prp43) are present in early pre-60S r-particles7. These proteins are believed to fulfill various functions in the remodeling events occurring within pre-60S r-particles, such as promoting formation, stability or dissociation of RNA secondary structures or regulating the binding or dissociation of proteins16,17. Among these enzymes, only the RNA helicase Prp43 and Dbp3 have been shown to function in the release of snoRNAs18,19. The structural organization of the RNA components of the earliest pre-60S r-particles remains ill-defined. In the mature 60S r-subunit, the 25S and 5.8S rRNAs are organized in six structural domains (I to VI), each beginning with short stems referred to as root helices (Supplementary Fig. 1). These root helices are clustered in the mature particles and their clustering and compaction has been proposed to occur early during assembly of pre-60S rparticles20. In the earliest pre-60S r-particles, root helix I and base-pairing interactions between the 25S and 5.8S rRNAs, key determinants of domain I folding, are already established21. In the earliest pre-60S particle cryo-electron microscopy structure reported to date (State A22), the root helix of domain I is formed, and domains I, II and part of domain VI have acquired their final secondary structure. These structured domains form a rigid platform, corresponding to the solvent-exposed side of the mature subunit, onto which takes place assembly and compaction of the other domains22. Failure to correctly complete these early folding events is believed to lead to turnover of pre-60S rparticles. Formation and/or stability of the earliest pre-60S r-particles in yeast require the Npa1 complex, a protein module composed of 5 AFs: Npa1, Npa2, Nop8, Rsa3, and the RNA helicase Dbp67,23,24. Mapping of RNA interaction sites of Npa1 using the crosslinking and analysis of cDNA approach (CRAC) revealed that Npa1 interacts with both the 5’(domain I) and 3’(domains V and VI) regions of the 25S rRNA (Supplementary Fig. 1), and has therefore the potential to act as a long-range, physical link between domains I, V, and VI25. The Npa1 complex may therefore promote or stabilize circularization of the 25S rRNA in the earliest stages of pre-60S r-particle assembly, by tethering the 5’and 3’ domains. Npa1 also crosslinks to a subset of C/D- and H/ACA- type snoRNAs involved in the chemical modification of nucleosides of the decoding center and PTC of the large r-subunit. Interestingly, the snR190 box C/D snoRNA is particularly efficiently crosslinked to Npa1. Its antisense sequence upstream of box D’(box A, Supplementary Fig. 2a) is complementary to a sequence embedded within helix H73 in domain V of the 25S rRNA and it is predicted to guide ribose 2’-O-methylation at position G2395 (Supplementary Fig. 1). However, this methylation has not been detected in recent large scale studies26–28. snR190 also contains another 15 nucleoside sequence (box B, Supplementary Fig. 2a) that is perfectly complementary to a sequence in domain I of 25S rRNA near the Npa1 binding site (Supplementary Fig. 1). Like the Npa1 complex, snR190 therefore also has the potential to make a physical link between the 5’and 3’domains of the 25S rRNA. Here, we provide evidence that yeast snR190 does not function as a methylation guide snoRNP, but rather as a placeholder preventing specific 25S rRNA sequences from establishing inappropriate interactions during pre-rRNA folding. Moreover, our data strongly support the model that Dbp7 regulates the dynamics of base pairing between snR190 and the 25S rRNA within early pre-60S r-particles. Results Genetic link between Dbp7 and snR190 base-pairing site on 25S rRNA. The absence of Dbp7 impairs production of the 27S and 7S pre-rRNA intermediates during early pre-60S r-particle maturation, and compromises synthesis of mature 25S and 5.8S rRNAs and the 60S subunit29,30. The precise molecular function of Dbp7 in this process remains elusive. DBP7 displays genetic interactions with most genes encoding members of the Npa1 complex24, suggesting that Dbp7 function may be related to these AFs25. To gain further insight into the function of Dbp7, we exploited the severe growth defect of the dbp7Δstrain to isolate suppressors bearing mutations in rRNAs. We made use of the yeast strain NOY89131, in which all the chromosomal rDNA genes are deleted and the 35S pre-rRNA is expressed from a plasmid (pNOY353, 2 µ, TRP1) driven by the galactoseinducible GAL7 promoter31. We further engineered this strain to express a HA-tagged version of Dbp7 under control of another galactose-inducible promoter (GAL1::HA-DBP7,strain EMY65). To identify rRNA suppressors of Dbp7 loss-of-func- tion, a mutant library of a second plasmid, (pNOY373, 2 µ, LEU2) expressing the 35S pre-rRNA from its natural Pol I-dependent promoter, was transformed into strain EMY65. Transformants were screened on selective SD medium for faster-growing clones, condition where neither pNOY353 nor the GAL1::HA-DBP7 construct are expressed (Supplementary Fig. 3). Only two plasmids recovered from candidate clones, referred to as Sup. #2 and Sup. #10, reproducibly improved the ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26207-w 2NATURE COMMUNICATIONS | (2021) 12:6153 | https://doi.org/10.1038/s41467-021-26207-w | www.nature.com/naturecommunications growth of strain EMY65 when retransformed (Fig. 1a). Their rDNA inserts were found to carry different mutations (Supplementary Table 1). Strikingly, both featured, among others, the same rRNA mutation, a C-to-U transition at position C2392 in helix H73 of the 25S rRNA sequence. To confirm that C2392U was itself responsible for the suppression effect, we specifically introduced the C2392T mutation in the wild-type pNOY373 plasmid. In parallel, we also introduced the T2392C reversion in the Sup. #2 and Sup. #10 plasmids. Growth of the resulting strains at 30 °C on selective SGal and SD media showed that the C2392U substitution was both necessary and sufficient to rescue the growth defect of the dbp7Δstrain (Fig. 1a). This suppression effect was not complete, as growth of the EMY65 strain transformed with the Sup. #10 plasmid on SD c a Pol I::35S Pol I::35S Sup. #10 Pol I::35S C2392T Pol I::35S Sup. #10 T2392C Pol I::35S Sup. #2 SGal-His-Leu SD-His-Leu Pol I::35S Sup. #2 T2392C GAL1::HA-DBP7 GAL7::35S b Pol I::35S Sup. #10 DBP7 SGal-His-Leu SD-His-Leu GAL::HA-DBP7 Pol I::35S Pol I::35S Fig. 1 rRNA suppressors of the Dbp7 depletion. a Serial dilution growth test of control (Pol I::35S) and suppressor (Pol I::35S Sup. #2 and Pol I::35S Sup. #10) strains isolated in the genetic screen for rRNA suppressors of Dbp7 depletion. Growth of strain EMY65 transformed with plasmid bearing the specific C2392T mutation (Pol I::35S C2392T), or with plasmids Sup. #2 and Sup. #10 in which mutation T2392 has been reverted to the original C2392 (Pol I::35S Sup. #2 T2392C and Pol I::35S Sup. #10 T2392C) is also tested. All strains grow similarly on selective SGal medium, which allows expression of Dbp7. On selective SD medium, the control strain shows a strong growth defect resulting from Dbp7 depletion, which is alleviated in all the strains expressing the mutant 25S rRNA with the C2392U substitution. Plates were incubated at 30 °C for 4 days. bSerial dilution growth test of strain NOY891 (original Nomura’s lab strain, DBP7) and strain EMY65 (GAL::HA-DBP7) transformed with either the control plasmid (Pol I::35S) or the suppressor Sup. #10 plasmid (Pol I:: 35S Sup. #10). All strains grow similarly on selective SGal medium. On selective SD medium, strain EMY65 transformed with the control plasmid showed a strong growth defect, which is only partially rescued, with respect to the reference NOY891 strain, by the Sup. #10 plasmid. Plates were incubated at 30°C for 4 days. cSchematic representation of the secondary structure of the 25S rRNA in the PTC region (domain V). The sequence complementary to snR190 box A is highlighted in pink. The predicted methylation site of snR190 (G2395) and the nucleoside found mutated in the genetic screen (C2392U) are indicated in red. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26207-w ARTICLE NATURE COMMUNICATIONS | (2021) 12:6153 | https://doi.org/10.1038/s41467-021-26207-w | www.nature.com /naturecommunications 3 medium was slower than that of the original NOY891 strain expressing Dbp7 (Fig. 1b). Helix H73 corresponds to the root helix of domain V of the 25S rRNA, which forms the PTC of the mature 60S r-subunit (Fig. 1c and Supplementary Fig. 1). Interestingly, the C2392U substitution lies in the immediate vicinity of G2395, predicted to undergo ribose 2’-O-methylation guided by the snR190 box C/D snoRNA (Supplementary Fig. 1), and is expected to weaken the base pairing between snR190 and its complementary sequence on the pre-rRNA. snR190 is the snoRNA most efficiently crosslinked to Npa1 in CRAC experiments25. This intimate biochemical relationship between Npa1 and snR190 along with (i) the genetic interaction between Dbp7 and Npa1, and (ii) the suppression of the growth defect of the loss-of-function of Dbp7 by a mutation in the 25S rRNA predicted to weaken the base pairing with snR190, led us to hypothesize that the function of Dbp7 might be linked to that of snR190. snR190 is required for optimal 60S r-subunit biogenesis.In yeast, snR190 is expressed from a dicistronic precursor transcript also supporting expression of U14, an essential box C/D snoRNA that functions as an RNA chaperone in the maturation of the 18S rRNA32. This particular context precluded using conventional genomic knockout approaches to inactivate snR190 expression. To interfere with snR190 expression post-transcriptionally, we mutagenized the genomic sequences encoding box C and the terminal stem of the snoRNA (snr190-[mut.C], Supplementary Fig. 2b) using the CRISPR-Cas9 approach33 (see “Materials and Methods”section for details). We selected two independent snr190-[mut.C] clones in two different yeast genetic backgrounds, W303 and BY4741, and confirmed snR190 depletion by northern blotting (Fig. 2a and Supplementary Fig. 4a). Strains having undergone the mutagenesis procedure, but which lacked mutations in SNR190 were used as controls (referred to as “WT”on the figures). To assess the impact of the loss-of-function of snR190 on cell fitness, we carried out growth tests in liquid YPD medium at 30 °C. We observed that the snr190-[mut.C] strains showed a moderate but reproducible decrease in growth rate (Supplementary Table 2 and Supplementary Fig. 15). In order to verify that this defect was not due to off-target mutations introduced by the CRISPR-Cas9 approach, we rescued snR190 expression in these strains. The SNR190-U14 gene was PCR amplified from yeast genomic DNA and inserted into a centromeric vector. The resulting plasmid, as well as an empty vector (E.V.), as a negative control, were transformed into the snr190-[mut.C] strains. We confirmed by northern blotting re-expression of snR190 in the snr190-[mut.C] strain in the W303 background (Fig. 2b). Ectopic expression of snR190 in the snr190-[mut.C] strains (BY4741 and W303 backgrounds) fully restored wild-type growth rates (Supplementary Table 2 and Supplementary Fig. 15). These results indicated that, in contrast to numerous box C/D snoRNAs functioning as methylation guides, snR190 is required for optimal yeast proliferation. In yeast, the few known examples of snoRNAs required for yeastproliferation(U3,U14,snR10,andsnR30)functionas chaperones in the 40S r-subunit biogenesis10–13. We next tested whether snR190 might function as a snoRNA chaperone in the maturation of pre-60S r-particles by analyzing pre-rRNA processing in the absence of snR190. Total RNA was extracted from snr190-[mut.C] strains and steady-state levels of prerRNA species were analyzed using northern blotting. Loss-of- function of snR190 resulted in a strong increase in the steadystate levels of the 35S and 33S/32S pre-rRNAs in the snr190- [mut.C] mutant compared to the wild-type strains (Fig. 2a and Supplementary Fig. 5a for the W303 background; Supplementary Fig. 4a and Supplementary Fig. 5b for the BY4741 background), indicating that the maturation of early 90S r-particles was delayed. We further observed a slight increase in the 27SA 2 precursor levels and reduced accumulation of the 27SB pre-rRNAs. The 27SB/27SA 2 ratios therefore substantially decreased in the absence of snR190 in both genetic backgrounds (Supplementary Fig. 5c, d). We conclude that snR190 loss-of-function impairs conversion of 27SA 2 to 27SB pre-rRNA within pre-60S r-particles. Low-molecular-weight rRNAs were also studied to determine whether the absence of snR190 also influences production of the downstream intermediates. The levels of the 7S pre-rRNAs did not show any apparent change (Supplementary Fig. 6a, b) nor did the levels of the mature 5.8S rRNAs (Supplementary Fig. 7a, b). Furthermore, no change in the level of 20S pre-rRNA was observed in absence of snR190, suggesting that snR190 is not involved in pre-40S r-particle maturation. Importantly, loss-of-function of snR190 slightly affected production of the mature 25S rRNA (Fig. 2a and Supplementary Fig. 4a). These phenotypes are not due to off-target mutations introduced by the CRISPR-Cas9 approach, as ectopic re-expression of snR190 in the snr190- [mut.C] strain partially rescued the pre-rRNA processing defects as shown by the reduced levels of the 35S and 27SA 2 intermediates (Fig. 2b). Although these levels did not drop to those observed in the wild-type W303 strain, accumulation of the 27SB intermediate appeared fully restored to wild-type levels (Fig. 2b and Supplementary Fig. 8a). Similar observations were made in the BY4741 background (Supplementary Fig. 8b and Supplementary Fig. 8c), indicating that although these phenotypes are relatively weak, they are highly reproducible. To determine whether the pre-rRNA processing defects observed in the absence of snR190 impact 60S r-subunit biogenesis, we analyzed polysome profiles from cell extracts of the snr190-[mut.C] strains in both genetic backgrounds. We observed that loss-of-function of snR190 resulted in a minor deficit in 60S r-subunits, as judged from the slight reduction in the levels of free 60S relative to 40S r-subunits, and the appearance of half-mer polysomes indicative of a shortage in 60S subunit and/or translation defects34,35 (Fig. 2c, W303 background). Re-expression of snR190 restored a close to wildtype polysome profile, clearly reducing the presence of half-mer peaks (Fig. 2d). Similar results were obtained in a strain derived from BY4741 (Supplementary Fig. 4b). These results indicate that loss-of-function of snR190 induces minor but significant prerRNA processing defects in the maturation pathway of the 25S rRNA, thus affecting production of 60S r-subunits and possibly also translation. snR190 does not function as a ribose 2’-O-methylation guide. snR190 contains an antisense element upstream of its box D’that is complementary to a sequence embedded within the root helix (helix H73) of domain V of the 25S rRNA (box A, Supplementary Fig. 2a). This antisense sequence is predicted to guide methylation of G2395 in the 25S rRNA36,37. Several large scale studies have been undertaken to exhaustively detect and quantify rRNA methylations in yeast26–28, but none of them detected a ribose 2′- O-methylation at position G2395. To verify these data with a specific emphasis on G2395 methylation, we mapped methylations in this region of the 25S rRNA using primer extensions at low dNTP concentration38. Two different primers, proximal and distal, were used in these experiments, located 19 and 40 nucleosides upstream of G2395 respectively (Supplementary ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26207-w 4NATURE COMMUNICATIONS | (2021) 12:6153 | https://doi.org/10.1038/s41467-021-26207-w | www.nature.com/naturecommunications Fig. 9). For this experiment, we used the snr190-[mut.C] strain (BY4741 background) transformed with the SNR190-U14 expression plasmid (WT) or the empty vector (E.V.) as a control. Primer extension using the distal primer revealed two expected signals corresponding to methylations of nucleosides U2417 and U2421 (Fig. 3, right panel). No clear signal located further upstream at G2395 was observed. Primer extension with the proximal primer (Fig. 3, left panel) confirmed the absence of a strong, snR190-dependent signal at position G2395. Analysis of snR190 putative secondary structure revealed that a potential internal stem can be formed between part of the antisense sequence and a region of the snoRNA including box C’and the downstream sequence (Supplementary Fig. 2a), possibly inactivating the guide element. To test whether this internal stem prevents methylation of G2395, we introduced mutations in the SNR190-U14 expression plasmid to destabilize the stem (snr190- [mut.S], Supplementary Fig. 2b). These mutations did not provoke any change in the signal obtained at position G2395 (Fig. 3). c 40S 60S 80S Polysomes Absorbance (254 nm) Fractions W303 WT 40S 60S 80S Polysomes Absorbance (254 nm) Fractions snr190-[mut.C] d 40S 60S 80S Polysomes Absorbance (254 nm) Fractions snr190-[mut.C] + E.V. 40S 60S 80S Polysomes Absorbance (254 nm) Fractions snr190-[mut.C] + SNR190-U14 b W303 #1 #1#2 #2 E.V. snr190-[mut.C] SNR190-U14 27SA2 20S U14 snR190 35S 33S/32S 27SB 27SA2 a W303 #1 #1#2 #2 WT snr190-[mut.C] 27SA2 20S U14 snR190 35S 33S/32S 27SB 27SA2 18S 25S Fig. 2 snR190 functions in pre-60S r-particle maturation. a Accumulation levels of rRNA precursors in W303 wild-type (WT) and snr190-[mut.C] strains. Total RNA extracted from two independent clones (#1 and #2) for each strain was analyzed by northern blotting. The RNA molecules detected using radiolabeled oligonucleotide probes (Supplementary Table 4) are indicated on the left. bAccumulation levels of rRNA precursors in W303 snr190-[mut.C] strain and upon ectopic re-expression of snR190. Total RNA extracted from two independent clones (#1 and #2) of strain W303 snr190-[mut.C] transformed with a vector rescuing snR190 expression (SNR190-U14) or the corresponding empty vector as control (E.V.) was analyzed by northern blotting. The same probes as in awere used. cPolysome profiles on sucrose gradients of wild-type (green) and snr190-[mut.C] (red) strains in the W303 background. Total cellular extracts prepared from these strains were centrifuged through 10–50% sucrose gradients. A 254 was measured during gradient fractionation. The identity of the different peaks is indicated and half-mers are marked with arrows. dPolysome profiles on sucrose gradients of W303 snr190-[mut.C] strain transformed with an empty vector (E.V., red) or upon ectopic re-expression of snR190 (SNR190-U14, purple). Total cellular extract preparation and analysis were carried out as in c. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26207-w ARTICLE NATURE COMMUNICATIONS | (2021) 12:6153 | https://doi.org/10.1038/s41467-021-26207-w | www.nature.com /naturecommunications 5 We conclude that snR190 does not direct 25S rRNA modification at position G2395. snR190 functions as a chaperone in pre-60S r-particle maturation. In addition to its antisense box A, snR190 contains another 15 nucleoside-long potential antisense element showing perfect complementarity to a sequence within domain I of the 25S rRNA, located in the vicinity of one of the Npa1 crosslinking sites25 (box B, Supplementary Figs. 1 and 2a). In the mature 60S r-subunit, this 25S rRNA sequence is base paired to the 5.8S rRNA. Part of snR190 box B has been described previously to reinforce the base pairing of box A in the vicinity of G239539.In any case, snR190 contains two long antisense elements with the potential to base-pair to two different regions of the 25S rRNA in domains I and V. In order to test whether boxes A and B are required for snR190 function in the maturation of pre-60S rparticles, we introduced drastic mutations in boxes A and/or B on the SNR190-U14 expression vector described above (snr190- [mut.A],snr190-[mut.B],orsnr190-[mut.AB], Supplementary Fig. 2c). The resulting plasmids were used to transform the snr190-[mut.C] strain in the BY4741 or W303 genetic background. Mutation of box A or box B did not affect yeast cell proliferation as both mutant strains displayed a doubling time very similar to that of the snr190-[mut.C] strain rescued by the plasmid expressing wild-type snR190 (Supplementary Table 2 and Supplementary Fig. 15). In contrast, simultaneous mutation of boxes A and B induced a reproducible growth defect in both genetic backgrounds, with a doubling time intermediate between those of the wild-type and snr190-[mut.C] strains (Supplementary Table 2 and Supplementary Fig. 15). In no case was this the result of reduced steady-state levels of the mutated snoRNAs (Fig. 4b). We next analyzed r-particle profiles in these mutants after fractionation of total cell extracts on density gradients. Mutation of boxes A or B alone showed polysome profiles very similar to that of the wild-type W303 strain (Fig. 4a). In contrast, mutation of both boxes A and B induced a minor imbalance of free 40S and 60S r-subunits, comparable to that observed in the snr190- [mut.C] strain, and the appearance of half-mer polysomes. PrerRNA processing analyses in the different strains gave results consistent with the polysome profiles (Fig. 4b, W303 background). The 27SB/27SA 2 ratio dropped to approximately half that of the wild-type in the absence of snR190 (Supplementary Fig. 8a), reflecting the accumulation of the 27SA 2 and depletion of the 27SB species. Ectopic expression of wild-type snR190, or the mutations of box A or box B alone, restored a close to wild-type 27SB/27SA 2 ratio. In contrast, mutation of both boxes resulted in a 27SB/27SA 2 ratio close to that of the snr190-[mut.C] strain (Fig. 4b and Supplementary Fig. 8a). These results were also reproduced in the BY4741 background (Supplementary Fig. 8b, c). Altogether, these data show that individual mutation of box A or B does not negatively affect the function of snR190 whereas simultaneous mutation of both boxes induces pre-60S r-particle maturation defects. snR190 is retained in preribosomal particles in absence of Dbp7. The genetic screen described in Fig. 1suggests that Dbp7 may contribute to the dissociation of snR190 from its 25S rRNA target(s) within pre-60S r-particles. To further test this hypothesis, we used a strain expressing an HTP-tagged version of Nop7, G2395 ? U2417 U2421 TGCA E.V. WT [mut.S] Primer #1 Strain Plasmid snr190-[mut.C] Primer #2 TGCA E.V. WT [mut.S] G2395 ? U2347 U2337 snr190-[mut.C] Strain Plasmid Fig. 3 snR190 does not function as a methylation guide. Reverse transcription experiment at normal or low dNTP concentration (black triangles) using total RNA extracted from strain BY4741 snr190-[mut.C] transformed with plasmids expressing the wild-type (WT)orsnr190-[mut.S] versions of snR190 or the empty vector (E.V.) as control. Two different primers were used to initiate reverse transcription: primer #2 (proximal, green) and primer #1 (distal, blue). A sequencing ladder was loaded in parallel to identify the nucleosides inducing reverse transcription stops. Note: a weak reverse transcription stop was observed at position G2395 with the proximal primer, but this signal was detected both in the presence or absence of snR190, suggesting that it may correspond to a nonspecific signal rather than a bona fide methylation-induced reverse transcription stop. The question mark (?) indicates a strong reverse transcription stop that is not associated to currently known nucleoside methylation. These experiments were repeated two times independently with each primer and similar results were obtained. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26207-w 6NATURE COMMUNICATIONS | (2021) 12:6153 | https://doi.org/10.1038/s41467-021-26207-w | www.nature.com/naturecommunications #1 #1#2 #2 E.V. snr190-[mut.C] + plasmid... WT #1 #1#2 #2 #1 #2 [mut.A] [mut.B] [mut.AB] 35S 27SA2 27SB 33S/32S 27SA2 W303 20S snR190 U14 b aWT 40S 60S 80S Polysomes Absorbance (254 nm) Fractions E.V. 80S 40S 60S Polysomes Absorbance (254 nm) Fractions snr190-[mut.A] 40S 60S 80S Polysomes Absorbance (254 nm) Fractions snr190-[mut.B] 40S 60S 80S Polysomes Absorbance (254 nm) Fractions snr190-[mut.AB] 40S60S 80S Polysomes Absorbance (254 nm) Fractions Fig. 4 snR190 boxes A and B are required for its function in LSU synthesis. a Polysome profiles on sucrose gradients of W303 snr190-[mut.C] strain transformed with vectors supporting expression of wild-type snR190 (WT, green) or bearing mutations in its antisense elements box A (snr190-[mut.A], dark blue), box B (snr190-[mut.B], grey) or both (snr190-[mut.AB], pink), or with the empty vector as control (E.V., red). Cell extracts were prepared and analyzed as in Fig. 2c. bSteady-state levels of distinct pre-rRNAs and U14 and snR190 snoRNAs in isogenic wild-type and snr190-[mut.C] strains in the W303 background; cells were transformed with plasmids supporting expression of wild-type snR190 (WT) or bearing mutations in its antisense elements box A ([mut.A]), box B ([mut.B]) or both ([mut.AB]), or with the empty vector as control (E.V.). Experiments were performed as explained in the legend of Fig. 2b. Note: the first five lanes of this figure are identical to those presented in Fig. 2b, where they were used to support the conclusion that ectopic reexpression of snR190 restores pre-rRNA processing in the snr190-[mut.C] strain. This experiment was performed once with each independent clone for the W303 background and twice with each independent clone for the BY4741 background (see also Supplementary Fig. 8). NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26207-w ARTICLE NATURE COMMUNICATIONS | (2021) 12:6153 | https://doi.org/10.1038/s41467-021-26207-w | www.nature.com /naturecommunications 7 a component of 90S and pre-60S r-particles40,41 to purify these particles in both a dbp7Δstrain and its isogenic wild-type counterpart (Fig. 5a). Then, we compared by northern blotting the association of snR190 and other snoRNAs previously found efficiently crosslinked to Npa1 by CRAC25 with these particles. As shown in Fig. 5a, b, snR190 clearly co-immunoprecipitated with Nop7-HTP in both tagged strains, but the efficiency of coimmunoprecipitation was much higher in the dbp7Δstrain. In contrast, the box H/ACA snoRNAs snR5, snR42, and snR10 were weakly co-immunoprecipitated with Nop7-HTP in both conditions, although snR42 and snR10 base-pair to domain V of the 25S rRNA, close to one of the base-pairing sites of snR190. Strikingly, however, the box H/ACA snoRNA snR37, and the box C/D snoRNAs snR38 and snR71 were also preferentially retained cWT dbp7∆ snR190 snR37 40S 60S 80S Polysomes Absorbance (254 nm) Fractions 40S 60S 80S Polysomes Absorbance (254 nm) Fractions snR42 snR5 1 2 3 4 5 6 7 8 9 101112131415161718Fractions 1 2 3 4 5 6 7 8 9 101112131415161718 27SA2 35S 33S/32S snR190 snR37 snR10 snR38 snR71 snR42 snR5 NOP7::HTP DBP7 WTWT ΔΔ ++++ _ WT WT _ Inputs IPs ab snR37 snR42 snR10 snR5 snR190 snR38 snR71 IP efficiencies dbp7Δ 35S 27SA 2 DBP7 0 0,5 1 1,5 2 2,5 3 3,5 4 Fig. 5 snR190 is retained into preribosomal particles in the absence of Dbp7. a Immunoprecipitation of Nop7-HTP-containing preribosomal particles in the presence or absence of Dbp7. Particles were immunoprecipitated from total cell extracts prepared from wild-type or dbp7Δstrains expressing an HTP- tagged version of Nop7. As a background control, the wild-type strain expressing untagged Nop7 was similarly processed. Some rRNA precursors and snoRNAs (indicated on the left) present in the total extracts (Inputs) or in the immuno-precipitates (IPs) were analyzed by northern blotting using selected radiolabeled probes (Supplementary Table 4). bQuantification of the northern blot signals obtained for the hybridizations shown in ausing PhosphorImager data and the MultiGauge software. The histogram represents the co-immunoprecipitation efficiencies (IPs over Inputs) of the indicated RNA species with Nop7-HTP in the presence (DBP7, black) or absence (dbp7Δ, red) of Dbp7. cSedimentation profile of snR190, snR37, snR42, and snR5 snoRNAs in the presence or absence of Dbp7. Total cellular extracts prepared from the wild-type (WT, green) or dbp7Δ(dbp7Δ, red) strains were centrifuged through 10–50% sucrose gradients. A 254 was measured during gradient fractionation and the profiles are represented. RNAs extracted from the first 18 fractions were analyzed by northern blotting to detect the indicated snoRNAs. Note: the RNA samples corresponding to each gradient were processed separately (electrophoresis, transfer, hybridization, exposure, data acquisition). Therefore, the exposures presented for each snoRNA in the two strains have been chosen arbitrarily. Please refer to the intraseries quantifications (preribosome-bound versus free ratios) presented in Supplementary Fig. 10 for the interpretation of these data. This experiment has been performed once with the dbp7Δstrain and once with the strain expressing the Dbp7 K197A catalytic mutant (Fig. 6c) with similar results for the snoRNAs tested. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26207-w 8NATURE COMMUNICATIONS | (2021) 12:6153 | https://doi.org/10.1038/s41467-021-26207-w | www.nature.com/naturecommunications within the preribosomal particles containing Nop7-HTP in the dbp7Δstrain (Fig. 5a, b). These results indicate that the absence of Dbp7 alters the steady-state association of snR190 with early preribosomes, as well as that of some, but not all, snoRNAs interacting with domain V of the 25S rRNA. To further confirm snR190 retention in 90S and pre-60S rparticles in the absence of Dbp7, the sedimentation profiles of snR190 on density gradients were compared in dbp7Δand wildtype cells. Polysome profile analyses revealed that lack of Dbp7 caused a robust decrease in the amount of free 60S r-subunits (Fig. 5c) and formation of prominent half-mer polysomes, in agreement with the previously reported function of Dbp7 in 60S r-subunit production29,30. RNA was isolated from gradient fractions and the sedimentation profile of snR190 analyzed by northern blotting (Fig. 5c). In wild-type cells, snR190 sedimented both with free snoRNPs in the top fractions of the gradient and with preribosome-bound snoRNPs in denser fractions containing 90S and pre-60S r-particles. Deletion of DBP7 induced a clear increase in the proportion of preribosomebound snR190. Similarly, the sedimentation profile of snR37 also appeared affected to the same extent, whereas that of snR5 and snR42 did not show the same preribosome enrichment (Fig. 5c). Quantification of the signals detected in the top (1–5) or preribosome-bound (7–18) fractions of the gradients for each snoRNA confirmed an increase in the bound/free ratios for snR190 and snR37, but not for snR42 and snR5 (Supplementary Fig. 10a). These data were further supported using sedimentations through low magnesium (low-Mg2+) sucrose gradients, in which polysomes are dissociated into free 40S and 60S r-subunits. Compared to the wild-type control, lack of Dbp7 resulted in an increase in the proportion of the snR190, snR71, snR37, and snR38 snoRNAs co-sedimenting with pre-60S and 90S r-particles (Supplementary Fig. 11a). In contrast, the sedimentation profile of other snoRNAs such as snR42, snR5, snR10, snR61, and the 5 S rRNA, were less affected. Quantification of the radioactive signals detected in the different fractions of the gradient confirmed the increased sedimentation in fractions 9–13 of the gradient of snR190, snR71, snR37, and snR38 snoRNAs in absence of Dbp7 (Supplementary Fig. 11b). As a member of the DExD-box family of putative ATP- dependent RNA helicases, Dbp7 is expected to possess ATPase activity, which may contribute to release of snR190 from the prerRNA. We tested in vitro the ATPase activity of recombinant wild-type Dbp7 protein (Dbp7 WT ) and a mutant version carrying a lysine to alanine amino acid substitution in the conserved motif I (also referred to as Walker motif A) of the RecA1 domain (Dbp7 K197A ). Recombinant His-ZZ tagged Dbp7 WT and Dbp7 K197A proteins were expressed and purified to apparent homogeneity from Escherichia coli (Fig. 6a) and their in vitro ATPase activity was tested using a NADH-coupled ATPase assay in the presence or absence of model RNA (Fig. 6b). We observed that Dbp7 on its own displayed a very weak ATPase activity, which was strongly stimulated by RNA. In contrast the Dbp7 K197A mutant displayed a greatly reduced activity in the same conditions (Fig. 6b). To determine whether the ATPase activity of Dbp7 is required in vivo for release of snR190 from preribosomes, a yeast strain expressing Dbp7 K197A was generated. This strain showed a sedimentation profile of r-particles very similar to that of the dbp7Δstrain (Fig. 6c) but, compared to a wild-type strain, a milder growth defect was observed for the dbp7Δstrain (Supplementary Fig. 12). Analysis of the levels of free versus preribosome-bound snoRNAs showed a strong accumulation of snR190 into preribosomes in the strain expressing Dbp7 K197A , similar to that previously observed in the dbp7Δstrain. In contrast, the sedimentation profile of two other snoRNAs, snR10, and snR42, were not drastically changed upon expression of Dbp7 K197A . Quantification of the signals detected in the top or preribosome-bound fractions of the gradients clearly revealed an increase in the bound/free ratio for snR190 but not for snR10 and snR42 (Supplementary Fig. 10b). We conclude that the enzymatic activity of Dbp7 is required for efficient release of snR190 from preribosomal particles. snR190 mutations alleviate the growth defect of the dbp7Δ strain. We hypothesized that the growth and processing defects observed in the dbp7Δstrain may be due to the aberrant retention of snR190 and other snoRNAs in early preribosomal particles. In order to test this, we eliminated snR190 in the dbp7Δstrain and analyzed the phenotypes of the resulting double mutant. The absence of snR190 slightly alleviated the growth defect of a dbp7Δ strain at 30 °C (Fig. 7a and Supplementary Table 2 and Supplementary Fig. 15), and the recovery was even more evident at 37 °C. Loss of snR190 also improved the growth rate of the strain expressing Dbp7 K197A at 22, 25, and 30 °C (Supplementary Fig. 12). This result indicates that the absence of snR190 alleviates the phenotypes resulting from the loss of the ATPase activity of Dbp7, in line with the model that Dbp7 contributes to the release of snR190 from its base-pairing sites with the pre-rRNA. We next determined whether weakening of the base pairing between snR190 and the 25S rRNA was responsible for this phenotypic rescue. We expressed in the dbp7Δsnr190-[mut.C] strain, different mutant versions of snR190 bearing either multiple mutations in box A (snr190-[mut.A]) or a single substitution of snR190 guanosine 34 into a cytidine (snr190- [mut.G34C]), to mimic the mutation on the 25S rRNA (C2392U) isolated in our suppressor screen (Fig. 1). In both cases, the growth defect of the dbp7Δstrain was partially alleviated (Supplementary Table 2 and Supplementary Fig. 15). Analysis of pre-60S r-particle maturation in these strains showed rescue effects consistent with the growth data. As previously described30, the dbp7Δstrain showed an increase in the steady-state levels of the 35S and the 27SA 2 precursors and a marked decrease of the 27SB pre-rRNAs (Fig. 7b, c). We observed a partial restoration of 27SB precursor levels in the double dbp7Δ snr190-[mut.C] mutant and upon mutagenesis of box A. In particular, the single G34C substitution in snR190 (snr190- [mut.G34C]) partially restored production of the 27SB intermediates when introduced in the dbp7Δstrain (Fig. 7b, c). These data indicated that the loss of snR190 directly alleviates the 27SB pre-rRNA production defects arising in the absence of Dbp7. Interestingly, however, 35S pre-rRNA accumulation was not restored to the wild-type level in these strains, which could reflect additional snR190-independent functions of Dbp7 (see Discussion). Altogether, these data show that the lack of snR190, or mutations weakening the base pairing between its box A and prerRNA, partially suppress the growth and pre-rRNA processing defects of a dbp7Δstrain. Thus, the function of Dbp7 becomes less limiting in these conditions, strongly suggesting that the snR190-pre-rRNA duplex is a cellular substrate of Dbp7. Stable association of the Npa1 complex with preribosomes requires snR190. snR190 is the snoRNA most efficiently crosslinked to Npa125. To understand further this relationship, we next studied the impact of the lack of snR190 and Dbp7 on the association of the Npa1 complex with nascent preribosomes. To specifically purify early 90S and pre-60S r-particles containing the Npa1 complex, we fused Noc1, another known component of these early r-particles42, to the Tandem-Affinity Purification NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26207-w ARTICLE NATURE COMMUNICATIONS | (2021) 12:6153 | https://doi.org/10.1038/s41467-021-26207-w | www.nature.com /naturecommunications 9 Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 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Acknowledgements Our work has benefited from fruitful technical and scientific contacts with the neighboring groups of Jérôme Cavaillé (J.C., Jade Hebras), Olivier Gadal (O.G., Christophe Dez, Frédéric Beckouët), Pierre-Emmanuel Gleizes (Simon Lebaron), and Tamás Kiss at the LBME/ CBI and Alain Kamgoué. We thank Jade Hebras for technical help with the detection of ribose-methylated nucleosides. We thank Hussein Hamze for experiments related to the study not presented in this manuscript. We thank all members of the Henry/Henras team for helpful discussions. The Henry/Henras group is supported by grants from ANR (ANR- 20-CE12-0026) and funding from CNRS and University of Toulouse. R.A.M. is supported by grants from the Rectorat of Lebanese University. M.J. is supported by a Ph.D. fellowship from the Lebanese University and CIOES Organization. The group of J.d.l.C. is supported by the Spanish Ministry of Science and Innovation [PID2019-103859-GB-I00 AEI/ 10.13039/501100011033], and the Andalusian Regional Government (JA; BIO-271). J.C. was supported by a Ph.D. fellowship (PIF) from the University of Seville, and S.M.-V. is an academic research staff of the JA (PAIDI2020). M.T.B. and K.E.B. are supported by funding from the Deutsche Forschungsgemeinschaft (SFB860) and the University Medical Centre Göttingen. We thank F. Espinar-Marchena and H. Domínguez-Martín for assistance during the initial design of the rRNA suppressor screen. Author contributions M.J., J.C., P.V., O.H., N.J.W., E.V., M.T.B., K.E.B., Y.H., R.A.M., J.d.l.C. and A.H. designed the experiments. Experiments were performed by M.J., J.C., C.D., S.M.-V., R.C., P.V., O.R.- G., C.V., Y.H., E.V., M.T.B., K.E.B., J.d.l.C. and A.H. All authors interpreted the data. M.J., J.d.l.C. and A.H. wrote the manuscript with strong contributions from Y.H. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-26207-w. Correspondence and requests for materials should be addressed to Anthony K. Henras. Peer review information Nature Communications thanks Sebastian Klinge and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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