Author response: Mechanism of completion of peptidyltransferase centre assembly in eukaryotes

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies. https://doi.org/10.7554/eLife.44904.001 eLife digest Biological machines called ribosomes make proteins in the cells of our body. Mammalian cells build roughly 7,500 new ribosomes every minute, each one containing 80 proteins and four RNA molecules. Problems that prevent ribosomes from assembling correctly have been linked to cancers such as leukemia, and a class of disorders called ribosomopathies that increase the likelihood of someone developing cancer. Understanding how ribosomes assemble could therefore help to develop new treatments for these diseases. Ribosomes are mostly constructed in the cell nucleus, but the final stages of assembly occur in the cytoplasm of the cell. A protein called Nmd3 binds to the partly constructed ribosome to export it out of the nucleus. Then, the final ribosomal proteins integrate into the structure to form a key site called the peptidyltransferase centre (PTC), which is where the ribosome joins together amino acids when making new proteins for the cell. Questions remained about how these final assembly steps occur, and how Nmd3 is removed from the ribosome. Kargas et al. have now examined how the PTC forms by using a method known as cryo-electron microscopy to determine the structures that the ribosome forms at different stages of assembly. This revealed that when the last two ribosomal proteins integrate into the ribosome, the ribosomal RNA goes through large shape changes that evict Nmd3 from the PTC. Quality control factors then check the structure of the newly formed ribosome and, if it passes their checks that it works correctly, license it to start making cell proteins. This stage of ribosome assembly is likely to occur in the same way in all plant, animal and other eukaryotic species. The results presented by Kargas et al. will also help researchers to better understand the consequences of the mutations that affect ribosomal proteins in cancer cells. Ultimately, this knowledge may help to uncover new ways to treat cancer and ribosomopathies. https://doi.org/10.7554/eLife.44904.002 Introduction The assembly of eukaryotic ribosomes involves the concerted action of over 200 trans-acting assembly factors. Following their assembly in the nucleus, pre-60S ribosomal subunits are exported to the cytoplasm where they are converted to translation-competent particles. The pre-60S particle attains export competence following release of the GTPase Nog2 by binding of the adaptor protein Nmd3 that recruits the nuclear export receptor Crm1 (Xpo1) through a C-terminal leucine-rich nuclear export signal sequence (Ho et al., 2000; Thomas and Kutay, 2003; Trotta et al., 2003). Crm1-independent adaptors, including Arx1 (Hung and Johnson, 2006; Bradatsch et al., 2007), Bud20 (Bassler et al., 2012), Mex67 (Yao et al., 2007) and Ecm1 (Yao et al., 2010) facilitate pre-60S nuclear export. Once the ribosomal precursor enters the cytoplasm, the final assembly factors are removed and the last remaining ribosomal proteins integrated. The AAA-ATPase Drg1 initiates the final cascade of cytoplasmic events by recycling the assembly factors Rlp24 and Nog1 (Kappel et al., 2012; Pertschy et al., 2007; Lo et al., 2010). Downstream cytoplasmic maturation events include the release and recycling of additional shuttling proteins including the export factors Arx1, Mex67 and Nmd3, removal of the GTPase Lsg1 as well as incorporation of late joining ribosomal proteins. The Arx1-Alb1 heterodimer is bound at the end of the peptide exit tunnel (Bradatsch et al., 2012; Leidig et al., 2014; Wu et al., 2016), from where it is released by the concerted action of the zinc-finger protein Rei1 and the cytosolic J protein Jjj1 (human DNAJC21) that stimulates the ATPase activity of the Hsp70 chaperone protein Ssa (Hung and Johnson, 2006; Lebreton et al., 2006; Demoinet et al., 2007; Meyer et al., 2007; Meyer et al., 2010; Lo et al., 2010). Incorporation of Rei1 requires prior release of Nog1, whose C-terminal tail seals the exit tunnel as the particle transitions from the nucleolus to the cytoplasm (Wu et al., 2016). Interestingly, Rei1 also inserts into the exit tunnel (Greber et al., 2012; Greber et al., 2016) from where it is displaced during later cytoplasmic maturation steps by Reh1 (Ma et al., 2017). However, it is unclear when Reh1 is exchanged for Rei1 and how long Reh1 persists on the particle. A parallel branch of the cytoplasmic maturation pathway involves Yvh1-dependent exchange of uL10 for the placeholder protein Mrt4 to form the P-stalk (Kemmler et al., 2009; Lo et al., 2009; Rodríguez-Mateos et al., 2009). The subsequent cytoplasmic maturation steps are crucial to correctly shape the peptidyltransferase centre (PTC), evolutionarily the oldest part of the ribosome, in a strictly controlled sequence of events. Single-particle cryo-electron microscopy (cryo-EM) has identified the binding sites for Nmd3 and Lsg1 on the intersubunit face of the 60S subunit (Ma et al., 2017; Malyutin et al., 2017). Nmd3 spans the tRNA corridor from the uL1 protein at the L1 stalk through the tRNA exit site and the PTC to interact with the anti-association factor eIF6 (yeast Tif6; herein called eIF6) at the sarcin-ricin loop (SRL), while Lsg1 embraces H69. The timing and mechanism of Nmd3 release remains unclear, but is dependent on the GTPase Lsg1 (Hedges et al., 2005) and the integration of ribosomal proteins eL40 (Fernández-Pevida et al., 2012) and uL16 (Hedges et al., 2005). The eIF6 protein prevents premature joining of the ribosomal 60S and 40S subunits by binding to the SRL and ribosomal proteins uL14 and eL24 (Gartmann et al., 2010). Release of eIF6 in the cytoplasm is catalysed by EFL1 (elongation factor like GTPase 1, an EF-2 homolog) and its cofactor SBDS (Shwachman-Bodian-Diamond syndrome, yeast Sdo1) (Bécam et al., 2001; Senger et al., 2001; Menne et al., 2007; Finch et al., 2011; Wong et al., 2011; Weis et al., 2015). The recruitment of SBDS to the 60S subunit depends on the prior integration of uL16 into the PTC in vivo (Weis et al., 2015). Although recent structural studies suggest that eIF6 is released after Nmd3 (Weis et al., 2015; Ma et al., 2017), an alternate model posits that the binding of uL16 breaks the interaction of the Nmd3 N-terminus with eIF6, allowing the recruitment of SBDS to promote eIF6 removal prior to the eviction of Nmd3 (Lo et al., 2010; Malyutin et al., 2017; Patchett et al., 2017). Maintaining the fidelity of late cytoplasmic 60S subunit maturation is crucial for all eukaryotic cells as defects in this process cause human developmental defects and cancer predisposition. Multiple mutations associated with human bone marrow failure and leukaemia target this pathway. For example, recurrent somatic mutations in the RPL10 gene (encoding the ribosomal protein uL16) have been identified in 10% of cases of paediatric T-cell acute lymphoblastic leukaemia (T-ALL) (De Keersmaecker et al., 2013). The T-ALL associated uL16-R98S missense variant impairs the release of both Nmd3 and eIF6 (De Keersmaecker et al., 2013). However, the underlying mechanism is unknown. Inherited mutations in the SBDS gene have been identified in 90% of individuals with Shwachman-Diamond syndrome (SDS), an autosomal recessive disorder characterized by poor growth, exocrine pancreatic insufficiency, skeletal abnormalities and bone marrow failure with an increased risk of progression to myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) (Boocock et al., 2003; Warren, 2018). SDS is also associated with mutations in DNAJC21, the human homologue of yeast JJJ1 (Dhanraj et al., 2017; Tummala et al., 2016; D'Amours et al., 2018) and EFL1 (Stepensky et al., 2017). To elucidate the mechanism of PTC completion and understand how this process in corrupted by leukaemia-associated mutations, we used single-particle cryo-EM and cross-linking mass spectrometry to delineate the sequential steps that lead to assembly of the key functional site of the ribosome. We show how integration of the final ribosomal proteins eL40 and uL16 initiates a hierarchical sequence of RNA and protein rearrangements that result in release of the essential nuclear export adaptor Nmd3, a key conserved step in PTC formation. Defective completion of the PTC causes developmental disorders associated with an increased propensity for malignant transformation. Hence, our atomic models not only illuminate the mechanism of PTC assembly but also suggest how mutations found in leukaemia disrupt this process. Results Overview of the pre-60S Lsg1 particles We set out to determine the mechanism of cytoplasmic 60S subunit maturation and completion of PTC assembly by affinity purifying native pre-60S particles from S. cerevisiae using tandem affinity purification (TAP)-tagged Lsg1 as bait (Figure 1—figure supplement 1A) and subjecting them to immunoblotting (Figure 1—figure supplement 1B), cryo-EM analysis (Figure 1A, Figure 1—figure supplement 1C–F, Figure 1—figure supplements 2–4, Supplementary file 1A, B) and crosslinking mass spectrometry (XL-MX) (Figure 1—figure supplement 5A,B, Supplementary file 2). Immunoblotting revealed enrichment of the assembly factors Lsg1, Nmd3, Arx1 and eIF6 in the purified particles, but under-representation of ribosomal proteins uL16 and uL10 (P0) (Figure 1—figure supplement 1B). Analysis by single-particle cryo-EM with extensive 3D classification yielded a series of structures (hereafter termed states I-VI) that likely reflect sequential snapshots of final cytoplasmic pre-60S maturation (Figure 1A). The ability to capture state VI (lacking both Nmd3 and Lsg1) likely reflects ongoing maturation of the particles or possibly the dissociation of Lsg1 and Nmd3 during immunopurification. We refined all six pre-60S cryo-EM reconstructions to average resolutions of 3.1–3.9 Å with the local resolution extending to 2.3 Å in the core of the particles in states I and III (Figure 1—figure supplement 1E,F). The maps allowed us to fit and refine atomic models for all six assembly states, including the biogenesis factors Arx1, Lsg1, Nmd3, Rei1, Reh1, eIF6, ribosomal proteins eL40, uL16 and uL11 together with the 5S, 5.8S and 25S rRNA (Figure 1B, Figure 1—figure supplements 2–4, Supplementary file 1A, B). In addition, we identified density corresponding to the phosphatase (Baßler et al., 2017) and zinc finger (Zhou et al., 2019) domains of Yvh1 situated between uL11 in the P-stalk and eIF6 (Figure 1A, states I-II). Figure 1 with 5 supplements see all Download asset Open asset Sequential steps in late cytoplasmic 60S subunit maturation. (A) Cryo-EM reconstructions of six cytoplasmic maturation states (I–VI). States, overall resolution, changes in protein composition and rRNA conformation are indicated. The phosphatase (P) and zinc finger (Zn) domains of Yvh1 are indicated in state II. (B) Atomic models of pre-60S states I-VI with rRNA and biogenesis factors highlighted. https://doi.org/10.7554/eLife.44904.003 Sequential maturation of the cytoplasmic pre-60S subunit couples dramatic conformational rearrangements of two long flexible helices (the L1 stalk and H38) to recruitment of the final ribosomal proteins (eL40 and uL16) and assembly factor displacement (Figure 1A). In states I-III, the L1 stalk is displaced inwards with H38 in the ‘closed’ position. Rearrangement of H38 to the mature position (state IV) is followed by partial (state V) then full (state VI) retraction of the L1 stalk. Focused classification of the local density around the P-stalk (Figure 1—figure supplement 2) revealed that binding of ribosomal protein eL40 does not occur until state III, prior to the recruitment of uL16 in state IV. Although there is clear density for uL11 (state I) at the base of the P-stalk, uL10 was poorly defined in our maps, consistent with the paucity of uL10 protein in the Lsg1-TAP particles as detected by immunoblotting (Figure 1—figure supplement 1B). Focused classification around the P-stalk revealed that Yvh1 is predominantly bound in state I, is present at low occupancy in state II but is absent in state III. Thus, Yvh1 and eL40 do not appear to bind simultaneously to the same particle. The homologous C-termini of Reh1 and Rei1 occupy the PET sequentially (Figure 1A). While the departure of Rei1 seems to be concurrent with Arx1 release, the C-terminus of Reh1 unexpectedly persists in the exit tunnel even after the departure of Nmd3 and Lsg1. The timing of the exchange of Rei1 for Reh1 (between states I-II) is consistent with XL-MS analysis that yielded crosslinks between Rei1, Arx1 and eL24, while Reh1 yielded crosslinks to eL24, but not to Rei1 or Arx1 (Figure 1—figure supplement 5A,B and Supplementary file 2). These data are also consistent with a previous structural snapshot (Ma et al., 2017) and with co-immunoprecipitation analysis (Parnell and Bass, 2009). We conclude that the exchange of Reh1 for Rei1 occurs at the time of Arx1 release, but that surveillance of the PET by Reh1 continues throughout the entire process of cytoplasmic pre-60S maturation. While the overall structures of Rei1 and Reh1 are consistent with previous reports (Greber et al., 2016; Ma et al., 2017), our maps reveal additional density that corresponds to the N-termini of both Rei1 and Reh1 extending up across the surface of eL24 to directly interact with eIF6 subdomains C and D and with a loop that extends out from the α-helical domain of Lsg1 (residues G438-T456) (Figure 1A, state I). The Lsg1 α-helical domain (residues G474-D479) also interacts with uL14. We unambiguously distinguished the helical C-termini of Reh1 and Rei1 within the PET based on specific side chain densities (Ma et al., 2017). Interestingly, the position of the extreme C-terminal leucine and glutamine residues of Rei1 and Reh1 differs compared with previous reports (Ma et al., 2017; Greber et al., 2016): the side chains of Q393 (Rei1) and Q432 (Reh1) form an electrostatic interaction with the base of U2875 (H89), while the backbone interacts with the base of U2978 (H93). The coexistence of eIF6 and Reh1 on the same particle (Figure 1B) suggests that state VI is not simply a product of eIF6 rebinding but is a bona fide late pre-60S subunit maturation intermediate that lies downstream of Nmd3 and Lsg1 release. These data support the hypothesis that eIF6 is evicted after Nmd3 (Weis et al., 2015). Nmd3 is assembled from existing ribosomal machinery We set out to understand the mechanism of Nmd3 release, a key event in the completion of PTC assembly. Nmd3 extends across the entire tRNA binding cleft on the intersubunit face of the 60S subunit from uL1 at the L1 stalk through the E, P and A sites to contact Lsg1 and eIF6 at the SRL (Figure 1A,B). The cryo-EM density allowed us to build a complete atomic model for Nmd3 (residues T16-R404) including backbone atoms and side chains (Figure 2A), revealing a multi-domain architecture that includes two treble clef zinc fingers (that superimpose with an RMSD of 1 Å over 13 Cα atoms; Figure 2A, inset), two alpha-beta domains and two beta-barrel domains. The N-terminal treble clef zinc finger (residues 16–42, C19-C22-C35-C38) is grafted into an alpha-beta domain (residues 43–154) that is structurally related to ribosomal protein eL31. The second alpha-beta domain (residues 155–250) is structurally related to ribosomal protein eL22, while the two C-terminal beta-barrel domains have SH3 (251–310) and OB (311-400) folds, respectively. Although the combination of the SH3 and OB domains is similar in sequence and structure to eIF-5A-1, the domains are oriented differently with respect to each other. Thus, Nmd3 comprises a modular assembly of existing structural blocks associated with the ribosomal machinery combined together to form a functionally distinct protein. Figure 2 Download asset Open asset Nmd3 maintains the closed conformation of helix 38. (A) The modular atomic structure of Nmd3 with the individual domains (OB, SH3, eL22-like, eL31-like and the two treble clef zinc fingers) indicated by colour coding. Inset shows the superposition of the two treble clef zinc fingers (RMSD ~1 Å). (B) Interaction of the Nmd3 OB, SH3 and eL22-like domains with the tip of H38. (C) Nmd3 stabilises an A1025 base-flip. Atomic model, derived from the state III map (grey mesh), shows the interaction between Nmd3 and the flipped-out base A1025. https://doi.org/10.7554/eLife.44904.009 Nmd3 promotes H38 closure by stabilising a base flip at A2971 We sought to understand how Nmd3 maintains the ‘closed’ orientation of the L1 stalk (states I-IV) and H38 (states I-III) (Figure 1A). The Nmd3 OB domain holds the L1 stalk in the closed position, while the eL22-like, SH3 and OB domains, together with an extended C-terminal loop, encircle and distort the tip of H38 (Figure 2B,C). Specifically, two loops (β1–2 and β3–4) from the OB domain stabilize a base-flip of A1025. The side chain and backbone atoms of N332 contact the base and backbone phosphate of A1025. H364 contacts the backbone phosphate of A1027, while the side chains of Y402 and R404 contact the backbone phosphates of A1026-G1029. Within the SH3 domain, the side chain of K253 contacts the phosphate backbone of C1023. Helix α1 from the eL22-like domain (particularly the side chains of R169 and Q176) packs against the bases and backbone of H38 (G1020-C1023). Nmd3 contacts Lsg1 and eIF6 throughout cytoplasmic maturation Throughout the steps of pre-60S subunit maturation visualised herein, a conformation that differs from the mature ribosome et al., but that persists even in the of Nmd3 and Lsg1 (state VI) (Figure Lsg1 stabilises a base flip at (Figure while the side chain of Lsg1 against the base of at the tip of (Figure The conformation of is also by a in the SH3 domain of Nmd3 that stabilises a base flip in at the between and (Figure The Nmd3 SH3 domain additional with the 25S rRNA using two and (Figure the conformation of is even in the of both Nmd3 and Lsg1 (state we suggest that may only adopt the mature conformation after joining of the 60S and 40S subunits. Figure with 1 supplement see all Download asset Open asset Nmd3 interacts with Lsg1 and eIF6 throughout 60S maturation. (A) of (states I-VI) differs from the mature 60S subunit et al., (B) Interaction of Lsg1 with the tip of H69. (C) between Nmd3 and base of Nmd3 SH3 domain with and Interaction between Lsg1 and the Nmd3 eL31-like Interaction of the Nmd3 N-terminal domain with the D and domains of with the particle et al., 2017), the Nmd3 eL31-like domain is from the long of the Lsg1 GTPase domain in each of the native Lsg1 and Nmd3 (states (Figure 1A). The interaction involves a interaction between the side chain of Nmd3 and the side chain of Lsg1 (Figure Although classification of state III identified a of particles in which the Nmd3 eL31-like domain is this class Lsg1 and Arx1, with Reh1 present in the We suggest that the conformation of the Nmd3 eL31-like domain in this of state III particles is a of Lsg1 dissociation during a We the of the N-terminus of Nmd3 in using analysis of the Nmd3 (residues that the N-terminal zinc finger (residues has a of the domain on the (Figure supplement 1A,B). residues the of a flexible between the two domains that is These data that the Nmd3 N-terminal zinc finger has that allows it to a of in However, the cryo-EM data that the Nmd3 zinc finger remains bound to eIF6 subdomains D and surface of throughout states (Figure a that the Nmd3 N-terminus in position throughout pre-60S maturation. our data how the modular architecture of Nmd3 it with the to its conformation on the specific stage of pre-60S maturation. of eL40 stabilises to facilitate uL16 recruitment In the late nuclear pre-60S particle purified using the N-terminal domain of Nog1 into two an position (Wu et al., 2016). We therefore sought to understand how into its mature conformation to form one face of the site. Focused classification around the density revealed that in the of eL40 (states a of even with Yvh1 present (Figure However, following the departure of eL40 stabilises in its conformation by two contacts with the including a interaction of with and base between and (Figure contact between Yvh1 and the side chain of with the binding site for the N-terminus of eL40 in state III, the that the recruitment of eL40 may Yvh1 to promote its departure from the pre-60S particle. In turn, the of uL16 to the surface of promotes a dramatic of H38 from Nmd3 (Figure uL16 in a cleft between H38 and (Figure Figure Download asset Open asset of eL40 stabilises to form the uL16 binding site. (A) is flexible in states Cryo-EM density map of Yvh1 is in uL11 in The mature conformation of is in for (B) Atomic model of the interaction between eL40 and the tip of and (state (C) of uL16 a of H38. The of H38 in state III compared with state of with eL40 and uL16 bound (state A conformational switch in Nmd3 PTC completion In ribosomes with tRNA the loop (residues of uL16 extends into the PTC et al., 2016). However, in states the eL22-like domain of Nmd3 extends into the PTC where it is by helices and (Figure on the surface of the eL22-like domain make extensive with the backbone of (Figure stabilises a base interaction between and (Figure the loop of Nmd3 stabilises the ‘closed’ conformation of base A2971 through specific with the side chains of and the backbone of (Figure a the uL16 P-site loop is to into the PTC in state of a with the Nmd3 Figure 5 Download asset Open asset Nmd3 base A2971 in the peptidyltransferase (A) of Nmd3 with the PTC. (B) of the Nmd3 eL22-like domain with (C) Nmd3 stabilises the between bases and The loop of the Nmd3 eL22-like domain maintains base A2971 in the ‘closed’ conformation. The of uL16 is fully with of Nmd3 on the pre-60S particle (Figure 1A, Figure However, uL16 H38 from the C-terminus of Nmd3 partial (state V) and full retraction (state VI) of the L1 stalk (Figure retraction of the L1 stalk (state V) is by a conformational switch in Nmd3 in which the OB, SH3 and eL22-like domains are displaced and from the P and sites by Å through a between the flexible helical the and eL31-like domains, while the N-terminus remains to eIF6 and Lsg1 (Figure of the eL22-like domain breaks the interaction of with and (Figure allowing helix to form a new contact with (Figure supplement of the loop from the to the PTC (state V) the uL16 P-site loop to into the PTC where the essential A2971 in an conformation (Figure Figure with supplements see all Download asset Open asset of Nmd3 release. (A) The Nmd3 loop base A2971 (state shows base A2971 in the ‘closed’ position. H38 is the L1 stalk is ‘closed’ and uL16 is The P and tRNA binding sites are indicated. (B) The uL16 P-site loop competes with the Nmd3 loop for binding to base A2971 (state The L1 stalk is by to the fully closed L1 the Nmd3 loop is displaced from the PTC. The uL16 P-site loop is bound to base A2971 in the flipped-out conformation. (C) The PTC (state The L1 stalk is fully by Nmd3 and Lsg1 have and the uL16 P-site loop is bound to base A2971 in the flipped-out conformation. L1 stalk retraction promotes a conformational switch in the Nmd3 eL22-like Atomic models for the L1 stalk and Nmd3 in states and are The between the and eL22-like domains is indicated. and analysis are consistent with an essential for the uL16 P-site loop in Nmd3 release et al., 2007; et al., 2012; et al., 2014; Weis et al., 2015; Patchett et al., 2017). an the of yeast cells the uL16-R98S as the of uL16 (Figure supplement These data support a functional for the interaction between Nmd3 and helices and (Figure in stabilising the binding of Nmd3 to the 60S We conclude that the uL16 P-site loop the conformational in of Nmd3 release by with Nmd3 for an overlapping binding site within the PTC. Discussion of completion of PTC assembly The cryo-EM structures us to a mechanism for the completion of assembly of a functional PTC (Figure The sequential incorporation of eL40 and uL16 couples rRNA rearrangements to a conformational switch in Nmd3 that allows the P-site loop of uL16 to fully accommodate into the PTC to the conformational Nmd3 dissociation (Figure Figure supplement The incorporation of eL40 (state stabilises to form one face of the and may also facilitate Yvh1 release by its interaction with While the of uL16 to the surface of is with the of Nmd3 on the it promotes a of H38 from Nmd3 to adopt its mature uL16 in the cleft formed by H38 and of the stabilising between Nmd3 and the tip of H38 promotes partial retraction of the L1 stalk (state This is coupled to a conformational switch in the C-terminus of Nmd3 that the SH3 and OB domains