Ribosomes are the cellular machines that translate mRNAs into proteins. In eukaryotes, they consist of a small 40S and large 60S subunit, which carry the decoding and peptidyl transferase activity, respectively, and together comprise four ribosomal (r)RNAs (18S, 5.8S, 25S and 5S rRNA) and 78 ribosomal proteins in yeast. The synthesis of eukaryotic ribosomal subunits requires the concerted activity of ~200 assembly factors that drive ribosome biogenesis through a series of pre-rRNA cleavage, folding and modification reactions, which are coupled to the incorporation of ribosomal proteins (Henras et al., 2015; Woolford and Baserga, 2013; Zemp and Kutay, 2007). Initial steps of 40S biogenesis occur in the nucleolus, which leads to the formation of the first stable assembly intermediate, called the 90S pre-ribosome (Dragon et al., 2002; Grandi et al., 2002; Kornprobst et al., 2016), within which many of the early assembly steps for the 40S take place. This process requires between 50–70 different ribosome biogenesis factors (RBFs) (Woolford and Baserga, 2013; Grandi et al., 2002), which were shown by recent cryo-electron microscopy (cryo-EM) analysis to engulf the nascent pre-18S rRNA (Kornprobst et al., 2016; Sun et al., 2017; Chaker-Margot et al., 2017). Following early maturation steps, the pre-40S moiety detaches and is subsequently exported to the cytoplasm, containing only a handful of biogenesis factors including Pno1, Tsr1, Enp1, Ltv1, Nob1, Dim1 and Rio2 (Schäfer et al., 2006; Schäfer et al., 2003). Once in the cytoplasm, it has been proposed that assembly factors physically block the association of the translation machinery by occupying functional sites on the 40S subunit (Strunk et al., 2011). Structural insights into the architecture of pre-40S particles have previously been obtained through cryo-EM analysis, using preparations from both yeast and human cells (Strunk et al., 2011; Johnson et al., 2017; Larburu et al., 2016). In combination with RNA-protein crosslinking data, these structures have allowed the approximate positioning of most of the biogenesis factors on the late pre-40S particles (Strunk et al., 2011; Granneman et al., 2010). However, in contrast to recent higher resolution structures obtained for the early 60S intermediates (Greber, 2016), no late pre-40S structures with atomic resolution are available. Accordingly, detailed insight into the molecular interactions of the RBFs and the conformation of the pre-rRNA in late 40S pre-ribosomes was lacking.

To gain a better understanding of the small ribosomal subunit biogenesis on a molecular level we purified late pre-40S particles via the well-defined biogenesis factor Ltv1 (Schäfer et al., 2006; Johnson et al., 2017), using Ltv1-Flag-TEV-ProteinA (FTpA) as bait (Figure 1—figure supplement 1A). With this strategy, we obtained a high yield of homogeneous pre-40S particles, which were used for single particle cryo-EM (Figure 1—figure supplement 1B–C). After classification we obtained a major class containing the stably bound RBFs Enp1, Tsr1, Rio2 and Pno1 (Figure 1—figure supplement 1D) but lacking a number of late binding ribosomal proteins (RACK1, uS10, uS14, eS10, eS26 and uS3) (Ferreira-Cerca et al., 2007). This main class could be refined to an average resolution of 3.6 Å, with the local resolution ranging from 3.5 Å in the core to approximately 8 Å for flexible regions (Figure 1—figure supplement 2). We built atomic models for Tsr1 and Pno1, and were able to model Enp1 and Rio2 on a secondary structure level (Figure 1A and Figure 1—figure supplements 2–3). In addition, the structure of the pre-18S rRNA revealed very distinct conformational differences, as compared to the mature state (Heuer et al., 2017), of functionally important regions including all three tRNA binding sites (A,P and E) and the entire mRNA path. We found that two major rRNA condensation steps still have to happen for these sites to mature: one in the head/beak region and the other in the central region of the 18S rRNA (Figure 1B).

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The characteristic tertiary structure of the head rRNA (h28 to h43) is mainly determined by three-way junctions (Mohan et al., 2014). We observe that in the pre-18S rRNA only one such junction is not yet formed, namely that which connects h34, h35 and h38 (Figure 1C and Figure 1—figure supplement 4A). It joins three blocks of rRNA, which all contain parts of functionally important regions: one block contains h33 of the beak and h34, a central element in the formation of the A-site decoding center, while the block comprising h29-h32 and h38-h42 contains key residues for mRNA binding and accommodation of anticodon-arms for all three tRNAs. The third block (h35-h37) contains h36, which forms important tertiary interactions between head and body and is part of the central region of the 18S rRNA (Wimberly et al., 2000) (see below). Due to the absence of this junction, these blocks are shifted relative to each other and relative to the body, preventing the formation of the actives sites. Notably, formation of this junction requires the incorporation and stabilisation of the late associating ribosomal proteins uS3, uS10 and uS14 (Lescoute and Westhof, 2006), which are absent from the pre-40S particle (Figure 2—figure supplement 1).

The central region of the 18S rRNA comprises the central pseudoknot (CPK), a universally conserved structure that connects the head with the body via h28, h1 and h2. It provides a core structure around which major parts of the active A- and P-sites form, with the most central being h44 and h28. The tip of h44 contains two universally conserved adenosine bases (A1755/A1755, A1492/A1493 in E. coli) critical for mRNA decoding (Ogle et al., 2003) and the ‘neck helix’ h28 provides a hinge for head rotation, which is crucial for tRNA movement during elongation (Mohan et al., 2014; Korostelev et al., 2008). Formation of the CPK is a major structural landmark and we observe that, unlike in the 90S, the CPK is fully folded and the contact with the head (h36) has been established (Figure 1—figure supplement 4B–C). In contrast, we observe that the top of h44 is not yet base-paired, and the linker of h44 with h28 and h45 remains highly flexible. Notably, this linker forms major parts of the A and P sites in the mature state (Figure 1D and Figure 1—figure supplement 4B). Moreover, h44 is repositioned outwards relative to its mature position and h28 is tilted by 12 degrees in the direction of the beak (Figure 1—figure supplement 4B–C). Collectively, we observe that the pre-18S rRNA is still in a non-functional immature state since all elements forming the active decoding and mRNA interaction sites are prevented from adopting their mature fold (Figure 2).

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We next investigated the role that the RBFs play regarding the immature pre-18S rRNA conformation. The first RBF, Tsr1, shares a similar domain architecture (I-IV) to several translational GTPases with an additional N-terminal extension, which was unresolved in previous studies (Johnson et al., 2017; McCaughan et al., 2016). Tsr1 mainly binds to the region which forms the universal translation factor binding site on the small subunit. Tsr1 contacts the junction of h5 - h15 and uS12 via its domains I and III (Figure 3A). Specific interactions are established with h17 and to the N-terminal tail of eS30, which is yet to adopt its mature position (Figure 4—figure supplement 1). Further, domain IV of Tsr1 links the body with the immature head contacting h30 to h32, as well as the shifted h34. Notably, these rRNA structures are contacted from the opposite side by Enp1/Ltv1 (see below), resulting in the coordinated stabilization of the ribosomal beak in its immature conformation.

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Due to high local resolution, we were able to build the N-terminal part of Tsr1. It forms a 35 Å long α-helix, which pierces through the ribosome between h5 and h44. By reaching further it touches h11-h12 (Figure 3A–B), thus serving as a distance enforcing wedge for h44. Thereby, via a long distance effect, Tsr1 keeps the linker connecting h44 with h28 and h45 unfolded and immaturely positioned. To assess the functional significance of the interaction of the N-terminal helix of Tsr1 and h44 we generated reverse charge mutants (R54D, K55D, K57D and K59D) where combinations of 3 or more substitutions indeed resulted in a slow growth phenotype (Figure 3C). All mutants showed the same nuclear localization, but a decrease in association with pre-ribosomes as compared to wild-type Tsr1 (wt) (Figure 4—figure supplement 1A–B). These data suggest that the N-terminus of Tsr1 is important for both, the stabilization of h44 in its immature conformation and the association of Tsr1 with pre-ribosomes. We also assessed the consequences of abolishing the interaction between domain IV of Tsr1 and the head of the pre-40S by removing domain IV. This mutant no longer supported yeast cell growth but continued to interact with pre-ribosomal particles (Figure 4—figure supplement 1C–D). We therefore propose that domain IV is not necessary for the association of Tsr1 with the pre-ribosome, but rather plays an important role to stabilize the pre-40S head in its immature conformation.

Enp1 is one of the few assembly factors that is already present in the 90S particle and remains associated until the integration of uS3 during late pre-40S biogenesis in the cytoplasm (Kornprobst et al., 2016; Schäfer et al., 2003). We observe Enp1 binding to the tip of the bent h16 near the mRNA entry site (Figure 3D, 4A). From there it reaches over to bind h32 - h34, thus keeping the head in its immature conformation together with Tsr1. Notably, we observe that Enp1 binds the same rRNA elements as in the 90S (Kornprobst et al., 2016; Sun et al., 2017; Chaker-Margot et al., 2017), further suggesting an early stabilization of the ribosomal beak. Ltv1, Enp1 and Rps3 are known to form a stable protein complex (Schäfer et al., 2006). Most likely due to the absence of Rps3 in our structure, only the extra density on top of Enp1 likely corresponds to its interaction partner Ltv1, which is in agreement with previous structural studies (Strunk et al., 2011; Larburu et al., 2016). The binding site of Enp1/Ltv1 occupies the position of the as yet unincorporated protein eS10 (Figure 4A) and explains Enp1/Ltv1’s described role in facilitating uS3 integration at an adjacent site (Schäfer et al., 2006). It further suggests a role for Enp1 in the maturation of h34 and the h34-h35-h38 three-way junction.

Rio2 a RBF conserved in all archaea and eukaryotes (Geerlings et al., 2003; Vanrobays et al., 2003; Schäfer et al., 2003), is an essential serine kinase required for 40S maturation. It binds the pre-40S at the A and P site region with all three domains (Figure 3E). The N-terminal winged-helix-turn-helix motif (wHTH) contacts the tip of h18, which forms the ‘latch’ for the mRNA in the mature 40S together with uS3, h34 and uS12 (Schluenzen et al., 2000). The two-lobed kinase domains of Rio2, K1 and K2, are positioned close to h28 whereby K1 contacts the region, which serves as a hinge during the 40S head rotation (Mohan et al., 2014). Rio2’s K1 also contacts the 40S head via h30 at a position close to domain IV of Tsr1. Finally, K2 contacts h29 and h42, which forms the P-site for binding the (initiation) tRNA in the mature ribosome.

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We further identified Pno1, a factor that together with the endonuclease Nob1 controls one of the final events of 40S biogenesis, the maturation of the 18S rRNA through cleavage of the 3’-end at cleavage site D (Lamanna and Karbstein, 2009; Lamanna and Karbstein, 2011). This cleavage event is believed to be regulated by Pno1 (Vanrobays et al., 2004), which belongs to the family of single-stranded RNA binding proteins with KH-domains. It is located on the platform of the pre-40S (Figure 5A) where it interacts with uS11/uS1, the tilted rRNA h28, h45 and the 3’-end of the pre-18S rRNA. Importantly, in this position, Pno1 sterically hinders h28 from adopting its mature conformation and the binding of eS26 (Figure 4B). We were able to follow the 3’ rRNA end, bound by Pno1, up to the pre-terminal base (U1799, D cleavage occurs after A1800) in molecular detail: A multitude of interactions is formed by three α-helices of the KH2 domain of Pno1, which recognize the first two single-stranded bases (G1793 and A1794) as well as the stem of h45 (Figure 5B) including the last base of the h44-h45 linker (U1769), which later will form a part of the active P- and mRNA binding sites. Thus Pno1, like Tsr1 and Rio2, prevents compaction of the central region of 18S rRNA.

Figure 5

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Like other members of the KH family (Nicastro et al., 2015), Pno1-KH2 uses its hallmark GXXG-RNA binding motif to position four nucleobases (residues 1795–1798) in a hydrophobic pocket (Figure 5C–D). Interestingly, KH1, which lacks the signature GXXG sequence in yeast (Woolls et al., 2011), also contributes to 3’-binding and contacts the terminal 18S rRNA bases 1797–1799 via its α-helix h1. Sequence alignments for the 3’-end revealed not only that the UCAU sequence - that contactes KH2 - is conserved from yeast to humans, but also the surrounding bases up to the D-cleavage site. This suggests that specific binding of Pno1 and positioning of the 3’-end in a distinct conformation may be a universal feature of eukaryotic ribosome maturation. Notably, Pno1 is in an ideal location to sense any further maturation of 18S rRNA, in particular conformational changes of the close-by h28, which may allow Pno1 to productively present the D-site for cleavage by the neighbouring endonuclease Nob1. In addition, Pno1 may protect the 3’ end against further cleavage until the small ribosomal subunit is fully matured.

In conclusion, we have discovered that the collective association of a few ribosome biogenesis factors on the late pre-40S ribosome regulates final rRNA folding steps at functionally important sites, in particular at the decoding centre. It appears that the role of these factors is to temporarily maintain the 40S subunit in a translationally incompetent state during ribosome biogenesis, preventing premature substrate interaction or entry into cycles of translation, which would be error-prone and potentially harmful to the cell. We envision that removal of biogenesis factors and the maturation of these regions are inter-dependent and coordinated processes. Conditional stepwise removal of these biogenesis factors may therefore serve as checkpoints that ensure the structural integrity of the ribosomal subunit, and thereby fitness for translation.

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Author details

1. André Heuer

   Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Emma Thomson

   For correspondence

   heuer@genzentrum.lmu.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6144-4316

2. Emma Thomson

   Heidelberg University Biochemistry Center, Heidelberg University, Heidelberg, Germany

   Present address

   University of Sheffield, Sheffield, United Kingdom

   Contribution

   Resources, Data curation, Validation, Writing—review and editing

   Contributed equally with

   André Heuer

   Competing interests

   No competing interests declared

3. Christian Schmidt

   Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany

   Contribution

   Validation, Writing—review and editing

   Competing interests

   No competing interests declared

4. Otto Berninghausen

   Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany

   Contribution

   Data curation

   Competing interests

   No competing interests declared

5. Thomas Becker

   Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany

   Contribution

   Conceptualization, Supervision, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8458-2738

6. Ed Hurt

   1. Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany
   2. Heidelberg University Biochemistry Center, Heidelberg University, Heidelberg, Germany

   Contribution

   Conceptualization, Funding acquisition, Writing—review and editing

   For correspondence

   ed.hurt@bzh.uni-heidelberg.de

   Competing interests

   No competing interests declared

7. Roland Beckmann

   Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   beckmann@genzentrum.lmu.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4291-3898

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