the pre-rRNAs. This information would be of key importance in establishing a detailed blueprint for ribosome assembly and lend focus to all future characterization of these proteins. With the aim of generating a map of protein–RNA interac- tions within pre-ribosomes, we performed a systematic UV cross-linking and cDNA analysis (CRAC) (Granneman et al, 2009) as outlined in Supplementary Figure 2. The best characterized, and simplest, pre-ribosomes are the late pre-40S particles. Here, we report the rRNA-binding sites for six factors that are retained in late pre-40S subunits. The ﬁndings provide insights into the 40S ribosome synthesis pathway and offer many avenues for future analyses. Results UV cross-linking of pre-40S-associated proteins We tested eight factors known to associate with late pre-40S complexes; Dim1, Dim2/Pno1, Enp1, Ltv1, Nob1, Rio1/ Rrp10, Rio2 and Tsr1 and identiﬁed binding sites for all except Dim2 and Rio1 (see Supplementary Table 1 for refer- ences). Rio1 was poorly cross-linked, whereas Dim2 was cross-linked but numerous attempts failed to reproducibly identify a clear RNA target sequence (data not shown). None of the C-terminal HTP fusions detectably impaired cell growth (Supplementary Figure 3A). Pre-rRNA processing was also unaffected in all strains except Nob1-HTP, which conferred a mild 20S-processing defect (Supplementary Figure 3B). HTP-tagging Nob1 at the N-terminus did not noticeably affect Nob1 function (Supplementary Figure 4C). CRAC experiments were performed 2–5 times, and we mon- itored the enrichment of tagged proteins in TEV and nickel eluates by western blot analysis (data not shown). To be considered a bona ﬁde RNA-binding site, a nucleotide sequence had to be signiﬁcantly enriched in every experiment. The locations of identiﬁed cross-linked RNA sequences are plotted in Figure 1. Sanger sequences of 50–80 cDNA clones obtained from independent experiments were aligned to a yeast non-coding RNA database using both Blast and Novoalign to align the fragments to the reference sequences and Novoalign was used to locate mutations and calculate percentage of mutations (see Materials and methods). The locations of the hits obtained for each protein using Novoalign are shown aligned against the 18S rRNA sequence, annotated with the predicted secondary structure, in Supplementary Tables 4–9. Cross-linking sites were precisely identiﬁed by the presence of multiple point deletions or substitutions at a speciﬁc position in sequence reads, or a minimal RNA-binding site was determined from overlapping sequences. Except for Tsr1 and Dim1 (see below), there was little overlap between major peaks in the histograms for each protein, showing that these peaks represent unique RNA-binding sites. Figure 1B shows the results of three independent CRAC experiments performed with an untagged strain, which served as a negative control. The most abun- dant contaminants (asterisks in Figure 1; Supplementary Figure 3B) were derived from regions near the 30 end of the 25S rRNA (position B5800 in rDNA). These were almost always observed in CRAC experiments (Granneman et al, 2009), but generally represented a larger fraction of the sequences recovered with proteins that cross-linked less efﬁciently to RNA. & 2010 European Molecular Biology Organization Towards a structural framework for 40S assembly S Granneman et al Enp1 and Ltv1 bind the rRNA near the beak structure A major structural rearrangement in pre-40S complexes is the formation of the characteristic ‘beak’ structure, which is shaped by protrusion of helix 33 (H33). Cryo-EM and bio- chemical studies revealed that beak formation requires a cascade of phosphorylation and dephosphorylation events in the cytoplasm, leading to the stable association of Rps3 and release of assembly factors Ltv1 and Enp1 (Schafer et al, 2006). Premature formation of the rigid beak structure is likely to hinder nuclear export of pre-40S complexes, as complexes lacking Ltv1 or Hrr25, the kinase responsible for Enp1, Rps3 and Ltv1 phosphorylation, are not efﬁciently exported to the cytoplasm (Schafer et al, 2006; Seiser et al, 2006). Regulation of the timing of beak structure formation is therefore important. Among all Enp1-associated sequence reads mapped to the rDNA, 64% included the sequence of H33 (Figure 1A; Supplementary Table 5). Deletions and point mutations were found in the internal loop of H33 (nt 1256–1259), pinpointing a cross-linking site (Figure 2B) and positioning Enp1 directly in the beak. Cross-linking to the adjacent H34 was observed less frequently (Figures 1A and 2B). Cryo-EM reconstruction images indicated that in pre-40S particles H33 was ﬂipped sideways (Schafer et al, 2006) and it seems probable that this correlates with the binding of Enp1 to H33. Most RNAs cross-linked to Ltv1 mapped to H16 and H41/ 41A (Figures 1A and 2B; Supplementary Table 6). Mutations identiﬁed in the terminal loop of H16 and in a bulge near H41A reveal the precise cross-linking sites (Figure 2B, nt 453– 454, 1490–1491). Cross-links to H21, H39 and H40 were found less frequently (Figure 1A). In the yeast 40S structure model (Figure 3A), H41A is located in close proximity to the beak and to Rps3, consistent with two-hybrid interactions reported between Rps3 and Ltv1 (Ito et al, 2001), whereas H16 is more distantly located in the shoulder region. Simultaneous binding of Ltv1 to both H16 and H41A would require that Ltv1 span the gap between the head and the shoulder (Figure 3C). Although we cannot exclude the possibility that these sites are not occupied simultaneously, it is notable that the cryo-EM structure does indeed show appropriate density to correspond to Ltv1 bound across this region (Figure 3B) (see Discussion). Collectively, these CRAC results are in good agreement with previously published biochemical and genetic data, and support the model that release of Enp1 and Ltv1 is directly linked to beak structure formation (see Figure 3C). Rio2 binds the 18S rRNA at the mRNA tunnel in pre-40S complexes Rio1 and Rio2 are serine kinases involved in processing of 20S pre-rRNA in the cytoplasm. Rio2, but not Rio1, is stably associated with pre-40S complexes (Vanrobays et al, 2003), probably explaining why only Rio2 was detectably cross- linked to pre-rRNA (Supplementary Table 1). Rio2 preferen- tially cross-linked to a terminal loop in H31 (nt 1194–1196) (Figures 1A and 2B; Supplementary Table 8). Loss of mod- iﬁcation of residue U1191 in the H31 loop was recently reported to inhibit site D cleavage (Liang et al, 2009), suggesting that this may inﬂuence the binding or activity of Rio2. H31 is located in the head of the mature 40S subunit, in a region also likely occupied by Rps15, Rps16, Rps18, Rps20 The EMBO Journal VOL 29 | NO 12 | 2010 2027