Structural dynamics of the yeast Shwachman-Diamond syndrome protein (Sdo1) on the ribosome and its implication in the 60S subunit maturation

The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.     

The nucle(ol)ar Tif6p and Efl1p are required for a late cytoplasmic step of ribosome synthesis.

Deletion of elongation factor-like 1 (Efl1p), a cytoplasmic GTPase homologous to the ribosomal translocases EF-G/EF-2, results in nucle(ol)ar pre-rRNA processing and pre-60S subunits export defects. Efl1p interacts genetically with Tif6p, a nucle(ol)ar protein stably associated with pre-60S subunits and required for their synthesis and nuclear exit. In the absence of Efl1p, 50% of Tif6p is relocated to the cytoplasm. In vitro, the GTPase activity of Efl1p is stimulated by 60S, and Efl1p promotes the dissociation of Tif6p-60S complexes. We propose that Tif6p binds to the pre-60S subunits in the nucle(ol)us and escorts them to the cytoplasm where the GTPase activity of Efl1p triggers a late structural rearrangement, which facilitates the release of Tif6p and its recycling to the nucle(ol)us.     

Deletion of EFL1 results in heterogeneity of the 60 S GTPase-associated rRNA conformation

Previous work suggested that the release of the nucleolar Tif6 from nascent 60 S subunits occurs in the cytoplasm and requires the cytoplasmic EF-2-like GTPase, Efl1. To check whether this release involves an rRNA structural rearrangement mediated by Efl1, we analyzed the rRNA conformation of the GTPase center of 80 S ribosomes in three contexts: wild-type, Deltaefl1 and a dominant suppressor R1 of Deltaefl1. This analysis was restricted to domain II and VI of 25 S rRNA. The rRNA analysis of R1 ribosomes allows us to distinguish the effects due to depletion of Efl1 from the resulting nucleolar deficit of Tif6. Efl1 inhibits the EF-2 GTPase activity, suggesting that the two proteins share a similar ribosome-binding site. The 80 S ribosomes from either type failed to show any difference of conformation in the two rRNA domains analyzed. However, the same analysis performed on the pool of free 60 S subunits reveals several rRNA conformational differences between wild-type and Deltaefl1 subunits, whereas that from the suppressor strain is similar to wild-type. This suggests that the nucleolar deficit of Tif6 during assembly of the 60 S preribosomes is responsible for the changes in rRNA conformation observed in Deltaefl1 60 S subunits. We also purified 60 S preribosomes from the three genetic contexts by TAP-tagging Tif6. The protein content of 60 S preribosomes associated with Tif6p in a Deltaefl1 strain are obtained at a lower yield but have, surprisingly, a protein composition that is a priori similar to that of wild-type and the suppressor strain.     

Nmd3 is a structural mimic of eIF5A,and activates the cpGTPase Lsg1 during 60S ribosome biogenesis

During ribosome biogenesis in eukaryotes, nascent subunits are exported to the cytoplasm in a functionally inactive state. 60S subunits are activated through a series of cytoplasmic maturation events. The last known events in the cytoplasm are the release of Tif6 by Efl1 and Sdo1 and the release of the export adapter, Nmd3, by the GTPase Lsg1. Here, we have used cryo-electron microscopy to determine the structure of the 60S subunit bound by Nmd3, Lsg1, and Tif6. We find that a central domain of Nmd3 mimics the translation elongation factor eIF5A, inserting into the E site of the ribosome and pulling the L1 stalk into a closed position. Additional domains occupy the P site and extend toward the sarcin–ricin loop to interact with Tif6. Nmd3 and Lsg1 together embrace helix 69 of the B2a intersubunit bridge, inducing base flipping that we suggest may activate the GTPase activity of Lsg1.     

Involvement of MEK-ERK signaling pathway in the inhibition of cell growth by troglitazone in human pancreatic cancer cells

Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes, partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Asc1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation.     

A functional network involved in the recycling of nucleocytoplasmic pre-60S factors

Eukaryotic pre-ribosomes go through cytoplasmic maturation steps before entering translation. The nucleocytoplasmic proteins participating in these late stages of maturation are reimported to the nucleus. In this study, we describe a functional network focused on Rei1/Ybr267w, a strictly cytoplasmic pre-60S factor indirectly involved in nuclear 27S pre-ribosomal RNA processing. In the absence of Rei1, the nuclear import of at least three other pre-60S factors is impaired. The accumulation in the cytoplasm of a small complex formed by the association of Arx1 with a novel factor, Alb1/Yjl122w, inhibits the release of the putative antiassociation factor Tif6 from the premature large ribosomal subunits and its recycling to the nucleus. We propose a model in which Rei1 is a key factor for the coordinated dissociation and recycling of the last pre-60S factors before newly synthesized large ribosomal subunits enter translation.     

Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1

Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.     

The Saccharomyces cerevisiae 60 S ribosome biogenesis factor Tif6p is regulated by Hrr25p-mediated phosphorylation

The biosynthesis of 60 S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eIF6. This protein is necessary for the formation of 60 S ribosomal subunits because it is essential for the processing of 35 S pre-rRNA to the mature 25 S and 5.8 S rRNAs. In the present work, using molecular genetic and biochemical analyses, we show that Hrr25p, an isoform of yeast casein kinase I, phosphorylates Tif6p both in vitro and in vivo. Tryptic phosphopeptide mapping of in vitro phosphorylated Tif6p by Hrr25p and (32)P-labeled Tif6p isolated from yeast cells followed by mass spectrometric analysis revealed that phosphorylation occurred on a single tryptic peptide at Ser-174. Sucrose gradient fractionation and coimmunoprecipitation experiments demonstrate that a small but significant fraction of Hrr25p is bound to 66 S preribosomal particles that also contain bound Tif6p. Depletion of Hrr25p from a conditional yeast mutant that fails to phosphorylate Tif6p was unable to process pre-rRNAs efficiently, resulting in significant reduction in the formation of 25 S rRNA. These results along with our previous observations that phosphorylatable Ser-174 is required for yeast cell growth and viability, suggest that Hrr25p-mediated phosphorylation of Tif6p plays a critical role in the biogenesis of 60 S ribosomal subunits in yeast cells.     

Alteration in location of a conserved GTPase-associated center of the ribosome induced by mutagenesis influences the structure of peptidyltransferase center and activity of elongation factor G

Translocation catalyzed by elongation factor G occurs after the peptidyltransferase reaction on the large ribosomal subunit. Deacylated tRNA in the P-site stimulates multiple turnover GTPase activity of EF-G. We suggest that the allosteric signal from the peptidyltransferase center that activates EF-G may involve the alteration in the conformation of elongation factor binding center of the ribosome. The latter consists of the moveable GTPase-associated center and the sarcin-ricin loop that keeps its position on the ribosome during translation elongation. The position of the GTPase-associated center was altered by mutagenesis. An insertion of additional base pair at positions C1030/G1124 was lethal and affected function of EF-G, but not that of EF-Tu. Structure probing revealed a putative allosteric signal pathway connecting the P-site with the binding site of the elongation factors. The results are consistent with the different structural requirements for EF-G and EF-Tu function, where the integrity of the path between the peptidyltransferase center and both GTPase-associated center and sarcin-ricin loop is important for EF-G binding.     

A flexible loop in yeast ribosomal protein L11 coordinates P-site tRNA binding

High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 ‘P-site loop’. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site.     

Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p: evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth

The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6). In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6. Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I. The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site). The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells. Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability. When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished. Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein. These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.     

Direct interaction between EFL1 and SBDS is mediated by an intrinsically disordered insertion domain

Removal of anti-association factor, Tif6 (eIF6), by elongation factor-like 1 (EFL1) and Shwachman–Bodian–Diamond syndrome (SBDS) protein is a critical step in the late stage of ribosome maturation. Although EFL1 is known to have GTPase activity that is stimulated by SBDS, how they cooperatively trigger dissociation of Tif6 from the ribosome remains to be elucidated. In the present study, the interaction between EFL1 and SBDS was analyzed by size exclusion chromatography, gel shift assay, and isothermal titration calorimetry (ITC). The results showed that EFL1 interacted directly with SBDS. ITC experiments using domain-truncated mutants showed that the interaction between EFL1 and SBDS is governed by the insertion domain of EFL1 and domains II–III of SBDS. Circular dichroism spectroscopy showed that the insertion domain of EFL1 has a random structure in the absence of SBDS, whereas the disadvantageous entropy change observed on ITC suggested a fixed conformation coupled with complex formation with SBDS. Based on these observations together with those reported previously, we propose roles of EFL1 and SBDS in ribosomal maturation.     

Final touches and quality control on the assembly of the eukaryotic ribosome

One of the most fundamental processes of life is protein synthesis by the ribosome. Although much is known about the function and structure of this macromolecular complex, our understanding on its assembly is still vague. In this issue of The EMBO Journal, Malyutin et al ( 2017 ) provide a detailed picture of one of the latest assembly stages of the yeast 60S ribosomal subunit. The cryo-EM map of the 60S-Nmd3-Lsg1-Tif6 complex sheds new light on the function of Nmd3, Lsg1 and Tif6—and their release mechanisms—right before the 60S subunit joins the pool of actively translating ribosomes.     

GTP-independent tRNA Delivery to the Ribosomal P-site by a Novel Eukaryotic Translation Factor

During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNA_i^Met occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.     

Assessing the components of the eIF3 complex and their phosphorylation status

The eukaryotic initiation factor 3 (eIF3) is an essential, highly conserved multiprotein complex that is a key component in the recruitment and assembly of the translation initiation machinery. To better understand the molecular function of eIF3, we examined its composition and phosphorylation status in Saccharomyces cerevisiae. The yeast eIF3 complex contains five core components: Rpg1, Nip1, Prt1, Tif34, and Tif35. 2-D LC-MS/MS analysis of affinity purified eIF3 complexes showed that several other initiation factors (Fun12, Tif5, Sui3, Pab1, Hcr1, and Sui1) and the casein kinase 2 complex (CK2) copurify. In Vivo metabolic labeling of proteins with (32)P revealed that Nip1 is phosphorylated. Using 2-D LC-MS/MS analysis of eIF3 complexes, we identified Prt1 phosphopeptides indicating phosphorylation at S22 and T707 and a Tif5 phosphopeptide with phosphorylation at T191. Additionally, we used immobilized metal affinity chromatography (IMAC) to enrich for eIF3 phosphopeptides and tandem mass spectrometry to identify phosphorylated residues. We found that three CK2 consensus sequences in Nip1 are phosphorylated: S98, S99, and S103. Using in vitro kinase assays, we showed that CK2 phophorylates Nip1 and that a synthetic Nip1 peptide containing S98, S99, and S103 competitively inhibits the reaction. Replacement of these three Nip1 serines with alanines causes a slow growth phenotype.     

Yeast 18 S rRNA Is Directly Involved in the Ribosomal Response to Stringent AUG Selection during Translation Initiation

In eukaryotes, the 40 S ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely on the AUG start codon. Structural arrangement of the 18 S rRNA determines the overall shape of the 40 S subunit. Here, we present genetic evaluation of yeast 18 S rRNA function using 10 point mutations altering the polysome profile. All the mutants reduce the abundance of the mutant 40 S, making it limiting for translation initiation. Two of the isolated mutations, G875A, altering the core of the platform domain that binds eIF1 and eIF2, and A1193U, changing the h31 loop located below the P-site tRNA_i^Met, show phenotypes indicating defective regulation of AUG selection. Evidence is provided that these mutations reduce the interaction with the components of the preinitiation complex, thereby inhibiting its function at different steps. These results indicate that the 18 S rRNA mutations impair the integrity of scanning-competent preinitiation complex, thereby altering the 40 S subunit response to stringent AUG selection. Interestingly, nine of the mutations alter the body/platform domains of 18 S rRNA, potentially affecting the bridges to the 60 S subunit, but they do not change the level of 18 S rRNA intermediates. Based on these results, we also discuss the mechanism of the selective degradation of the mutant 40 S subunits.     

Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA

We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690-720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5'-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.     

The Hsp40 chaperone Jjj1 is required for the nucleo-cytoplasmic recycling of preribosomal factors in Saccharomyces cerevisiae

Ribosome biogenesis is a major conserved cellular pathway that requires both ribosomal proteins and many preribosomal factors. Most of the pre-60S factors are recycled into the nucleus; some of them shuttle between the nucleus and the cytoplasm while a few others, like Rei1, are strictly cytoplasmic and are mostly involved in the dissociation/recycling of the pre-60S shuttling factors. Here, we investigated the role of the Jjj1 Hsp40 chaperone in ribosome biogenesis. The absence of Jjj1 leads to a cold sensitive phenotype, a defect in the relative amount of the large ribosomal subunit with the appearance of halfmers, and to cytoplasmic accumulation of shuttling factors such as Arx1 and Alb1, which stay bound to the pre-60S particles. Jjj1 is, thus, a novel pre-60S factor involved in the last cytoplasmic steps of the large ribosomal subunit biogenesis. We report the biochemical association of Jjj1 and Rei1 to similar pre-60S complexes, their two-hybrid interactions, and their functional links. Altogether, these results indicate that Rei1 and Jjj1 share many common features. However, while the functions of Jjj1 and Rei1 partially overlap, we could distinguish specific role of the two proteins in Arx1/Alb1 and Tif6 recycling. We propose that Jjj1 is preferentially required for the release of Arx1 and Alb1 shuttling factors from the cytoplasmic pre-60S particles while Rei1 is preferentially involved in their recycling.     

Identification of a Translation Initiation Factor 3 (eIF3) Core Complex, Conserved in Yeast and Mammals, That Interacts with eIF5

Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni²⁺ affinity and gel filtration chromatography and obtained a complex of ≈600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNA_i^Met binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.     

Locking and unlocking of ribosomal motions

During the ribosomal translocation, the binding of elongation factor G (EF-G) to the pretranslocational ribosome leads to a ratchet-like rotation of the 30S subunit relative to the 50S subunit in the direction of the mRNA movement. By means of cryo-electron microscopy we observe that this rotation is accompanied by a 20 A movement of the L1 stalk of the 50S subunit, implying that this region is involved in the translocation of deacylated tRNAs from the P to the E site. These ribosomal motions can occur only when the P-site tRNA is deacylated. Prior to peptidyl-transfer to the A-site tRNA or peptide removal, the presence of the charged P-site tRNA locks the ribosome and prohibits both of these motions.