Here, we have presented a protein–RNA interaction map for the late pre-40S ribosomes, providing insights into their architecture and maturation. Importantly, we found a good correlation between the CRAC data and previous protein–protein interaction and biochemical data, underlying the reliability of the method. We can now significantly extend the interaction maps as shown in .
Figure 5 Overview of known and predicted protein–protein interactions in pre-40S complexes. The interaction map depicts interactions between the various assembly factors and ribosomal proteins in pre-40S complexes. Black lines, physical interactions among (more ...)
No crystal structures are available for eukaryotic ribosomes, and the best available structure model for the yeast ribosome was generated using cryo-EM reconstructions and homology modelling (Spahn et al, 2001
). To relate the binding sites identified in the primary sequence to the 3D structure, we first identified the corresponding sequence in the archaeal rRNA and its position in the crystal structure, which was used to locate the binding site in the yeast structure model. A striking finding was that cross-linking sites for five of the late-acting 40S synthesis factors Rio2, Tsr1, Dim1, Nob1 (this work) and Prp43 (Bohnsack et al, 2009
) are located in close proximity to functionally important sequence elements in the 3′ region of the 18S rRNA. Intriguingly, the rRNA-binding sites appear to be located in proximity to ribosomal proteins previously shown to be required for D-site cleavage and/or efficient nuclear export of the pre-40S complex (Rps2, Rps3, Rps15 and Rps20, Rps0 and the C-terminus of Rps14) (Tabb-Massey et al, 2003
; Jakovljevic et al, 2004
; Leger-Silvestre et al, 2004
; Ferreira-Cerca et al, 2005
). This could reflect a general mechanism by which assembly factors prevent stable binding of ribosomal proteins before rRNA maturation steps are completed, as has been proposed for the association of Enp1 and Ltv1 with Rps3.
Notably, the binding sites identified predominately lie in the mature 18S rRNA region, rather than in the transcribed spacers, and are far more common over evolutionarily conserved regions than over the eukaryotic-specific insertion elements.
In Escherichia coli
, the emphasis in research into ribosome synthesis has been on the analysis of in vitro
reconstitution rather than in vivo
assembly. However, binding sites have been characterized for some factors. The RNA-binding sites for the bacterial orthologue of Dim1 (KsgA) bound to the 30S ribosomal subunit were determined by in vitro
directed hydroxyl radical cleavage and footprinting experiments (Xu et al, 2008
). In E. coli
, KsgA contacts rRNA regions in the 30S subunit that surrounds the modification sites in H45 including H11, 24, 27, 28 and 44 (Supplementary Figure 5
) and these interactions are proposed to be important in preventing premature interactions of pre-30S particles with the translation machinery. Yeast Dim1 can complement an E. coli ksgA
Δ mutant (Lafontaine et al, 1994
) and Dim1 cross-linking sites on the 18S rRNA included analogous positions (Supplementary Figure 5
). We conclude that both the methyl transferase function of Dim1 and its interactions with the rRNA are conserved in evolution.
Ribosome biogenesis in bacteria involves several different GTPases, which are proposed to act as ‘molecular switches', regulating the stepwise assembly and maturation of RNA–protein subcomplexes (reviewed in Culver, 2001
; Karbstein, 2007
; Connolly and Culver, 2009
). Two bacterial GTPases required for 16S rRNA processing (Era and RsgA/YjeQ) are genetically linked to KgsA (Inoue et al, 2006
; Campbell and Brown, 2008
). Cryo-EM microscopy studies revealed that some sites of Era interaction with the 16S rRNA are at positions analogous to the Tsr1-binding sites in yeast (H26, H28, H44, H45) (Sharma et al, 2005
). Similarly, RsgA also contacts the 3′ minor domain and GTP-bound RsgA causes structural rearrangements in H44 (Kimura et al, 2008
). The overlap in RNA-binding sites observed for Dim1 and Tsr1 strongly suggest that they interact directly in pre-40S complexes. We predict that Tsr1 and Dim1 together fulfill functions in ribosome assembly that are equivalent to KsgA and Era/RsgA in bacteria.
The Rio2 protein kinase is required for 20S–18S processing, but its targets are unknown. In the 40S structure model, the Rio2-binding site is located close to Rps15, () and pre-40S ribosomes that lack Rps15 fail to efficiently incorporate Rio2 and are not efficiently exported to the cytoplasm (Leger-Silvestre et al, 2004
; Zemp et al, 2009
). This suggests that these proteins interact directly. Human Rio2 kinase activity is required for release of hNob1, hLtv1 and hDim2 from pre-40S ribosomes (Zemp et al, 2009
) and the binding sites for yeast Rio2, Ltv1 and Nob1 are closely located in the head domain. These sites are also close to Rps16 and Rps18 (). The bacterial Rps18 homologue (S13) is phosphorylated at serine and threonine residues (Soung et al, 2009
) and this may also be the case for bacterial Rps16 (S9) (Traugh and Traut, 1972
), suggesting Rps16 and Rps18 as potential Rio2 substrates.
Pre-40S particles lack the prominent beak structure present in the mature subunit, implying large-scale structural reorganization during 40S maturation (Schafer et al, 2006
). Two 40S synthesis factors, Enp1 and Ltv1, were implicated in this reorganization but their actual roles were unclear. We report that Enp1 directly binds sequences in H33 that will form the beak. Ltv1 binds sequences in H41, which are located close to the beak, but also binds H16, which is more distantly located in the shoulder region of the 40S particle. If Ltv1 binds both sequences simultaneously, it would need to span the head–shoulder gap—a distance of some 87 Å in the mature 40S subunit. Ltv1 is ~53 kDa and, assuming a monomer with cylindrical shape and an average density of 0.73 cm3
/g, an 87 Å long Ltv1 protein would have a diameter of ~30 Å. Comparison of the cryo-EM maps for pre-40S and mature 40S revealed extra density the side of the head domain in pre-40S particles, close to the location predicted for Ltv1 () (Schafer et al, 2006
). The volume of this region would be in good agreement with the presence of a protein of ~53 kDa (B Böttcher, personal communication).
To better facilitate the interpretation of the cryo-EM images, we manually docked the 40S structure model (1s1h) (Spahn et al, 2001
) onto the 40S cryo-EM map (mesh model in ) (Schafer et al, 2006
) and overlaid this with the pre-40S cryo-EM map (transparent blue). This provided a reasonable estimation of location of ribosomal proteins and RNA structures in pre-40S pre-ribosomes. In the pre-40S EM map, the shoulder formed by H16 is absent or poorly defined, however, the extra density appears to be located parallel to Rps3 and just above H16. We therefore predict that this density corresponds to Ltv1, although we cannot exclude the possibility that multiple copies of Ltv1 are present.
The CRAC data revealed that Dim1, Tsr1 and Rio2 bind 18S rRNA regions that are important for the association of translation factors, tRNAs and 60S subunit joining. In the case of Dim1 and Tsr1, these are conserved to E. coli
and in both bacterial and eukaryotic ribosomes are incompatible with binding to the mRNA, 60S subunit, initiator tRNA and translation factors. Dissociation of each of these proteins from the pre-40S particles would therefore be required for translation to commence. Pre-40S complexes were recently reported to associate with polysomes (Soudet et al, 2010
), particularly after depletion of Nob1 or the Rio1 kinase. These conditions prevent 18S maturation, resulting in a very substantial 20S pre-rRNA accumulation, which is readily visible by ethidium bromide staining of total RNA. We predict that not all of this large pool of accumulated pre-40S particles can be associated with the ribosome synthesis factors, and their absence may explain the ability of the defective pre-40S ribosomes to engage with the translation machinery (Soudet et al, 2010
Nob1 is the PIN-domain endonuclease that cleaves site D at the 3′ end of 18S rRNA (Fatica et al, 2003
; Pertschy et al, 2009
). Unexpectedly, the major binding site identified for Nob1 was located in H40, distinct from the cleavage site. Binding to and cleavage of site D requires the PIN domain (Fatica et al, 2004
; Lamanna and Karbstein, 2009
; Pertschy et al, 2009
). This indicates that H40 recognition involves a different region of Nob1, most likely the C-terminal, Zn-ribbon putative RNA-binding domain, potentially leaving the PIN domain free to recognize the cleavage site.
Nob1 associates with 90S pre-ribosome early in 40S subunit synthesis pathway, but cleaves site D only in very late pre-40S particles after nuclear export (Fatica et al, 2003
; Pertschy et al, 2009
). Structure probing revealed that the region containing the D-cleavage site is readily accessible to chemical modification within pre-ribosomes, both in vitro
and in vivo
. This indicates that it is largely unstructured and unbound by proteins, and should therefore be accessible to the nuclease. What then prevents Nob1 from cleaving site D in early pre-ribosomes? The pre-40S cross-linking data reported here and by Bohnsack et al (2009)
identified four putative enzymes interacting with the decoding centre and with H44 (Tsr1, Rio2, Dim1 and Prp43), strongly implying that this region undergoes restructuring; as does the homologous region in bacteria. Notably, Dim1 also binds early, nuclear pre-ribosomes, but methylates the 3′ end of 18S rRNA much later in the pathway, after export to the cytoplasm. Restructuring might not only be essential for the correct folding of the 3′ domain of the 18S rRNA, but also to allow cleavage at site D to occur. On the basis of 3D modelling, we speculate that only when the structure of 3′ domain of 18S is close to its final conformation, can Nob1 access and cleave site D. In 3D models, the Tsr1- and Dim1-binding sites are in close proximity to the 3′ end of the 18S rRNA in the pre-40S and it is conceivable that both proteins could interfere with D-site cleavage by sterically hindering Nob1 binding to the cleavage site. We therefore predict that both RNA restructuring and protein remodelling steps in the 3′ region of the 18S rRNA are necessary for Nob1-dependent cleavage at site D.
Cryo-EM studies showed that binding of yeast translation initiation factors eIF1-eIF1A to the small subunit induces a conformational change that leads to a connection between the head and the shoulder, mediated by Rps3 and H16 (Passmore et al, 2007
). This interaction stabilizes the head domain and prevents the ‘mRNA latch' from forming prematurely, making the mRNA-binding channel more accessible to the large cap-binding protein complex attached to the 5′ end of the mRNA (Passmore et al, 2007
). We propose that Ltv1 binding to both Rps3 and H16 locks the head into the pre-ribosome conformation, whereas Enp1 binding to Rps3 and H33 directly prevents formation of the beak structure (). The 40S subunit is characterized by structural changes during the translation cycle, and it appears that large-scale structural changes also feature it its maturation.