A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease
2011; Springer Nature; Volume: 30; Issue: 19 Linguagem: Inglês
10.1038/emboj.2011.256
ISSN1460-2075
AutoresSander Granneman, Elisabeth Petfalski, David Tollervey,
Tópico(s)RNA Research and Splicing
ResumoArticle2 August 2011Open Access Source Data A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease Sander Granneman Corresponding Author Sander Granneman Wellcome Trust Centre for Cell Biology and Centre for Systems Biology at Edinburgh, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Elisabeth Petfalski Elisabeth Petfalski Wellcome Trust Centre for Cell Biology and Centre for Systems Biology at Edinburgh, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author David Tollervey Corresponding Author David Tollervey Wellcome Trust Centre for Cell Biology and Centre for Systems Biology at Edinburgh, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Sander Granneman Corresponding Author Sander Granneman Wellcome Trust Centre for Cell Biology and Centre for Systems Biology at Edinburgh, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Elisabeth Petfalski Elisabeth Petfalski Wellcome Trust Centre for Cell Biology and Centre for Systems Biology at Edinburgh, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author David Tollervey Corresponding Author David Tollervey Wellcome Trust Centre for Cell Biology and Centre for Systems Biology at Edinburgh, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Author Information Sander Granneman 1, Elisabeth Petfalski1 and David Tollervey 1 1Wellcome Trust Centre for Cell Biology and Centre for Systems Biology at Edinburgh, University of Edinburgh, Edinburgh, Scotland *Corresponding authors: Centre for Systems Biology at Edinburgh (CSBE), University of Edinburgh, CH Waddington Building, Mayfield Road, Kings Buildings, Edinburgh EH9 3JD, Scotland. Tel.: +44 131 651 9082; Fax: + 44 131 651 9068; E-mail: [email protected] Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Mayfield Road, Kings Buildings, Edinburgh EH9 3JR, Scotland. Tel.: +44 131 650 7092; Fax: +44 131 650 7040; E-mail: [email protected] The EMBO Journal (2011)30:4006-4019https://doi.org/10.1038/emboj.2011.256 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The 5′-exonuclease Rat1 degrades pre-rRNA spacer fragments and processes the 5′-ends of the 5.8S and 25S rRNAs. UV crosslinking revealed multiple Rat1-binding sites across the pre-rRNA, consistent with its known functions. The major 5.8S 5′-end is generated by Rat1 digestion of the internal transcribed spacer 1 (ITS1) spacer from cleavage site A3. Processing from A3 requires the 'A3-cluster' proteins, including Cic1, Erb1, Nop7, Nop12 and Nop15, which show interdependent pre-rRNA binding. Surprisingly, A3-cluster factors were not crosslinked close to site A3, but bound sites around the 5.8S 3′- and 25S 5′-regions, which are base paired in mature ribosomes, and in the ITS2 spacer that separates these rRNAs. In contrast, Nop4, a protein required for endonucleolytic cleavage in ITS1, binds the pre-rRNA near the 5′-end of 5.8S. ITS2 was reported to undergo structural remodelling. In vivo chemical probing indicates that A3-cluster binding is required for this reorganization, potentially regulating the timing of processing. We predict that Nop4 and the A3 cluster establish long-range interactions between the 5.8S and 25S rRNAs, which are subsequently maintained by ribosomal protein binding. Introduction During ribosome subunit biogenesis in eukaryotes, the nascent pre-rRNA can either undergo cotranscriptional cleavage by the small subunit processome or be transcribed into the 35S pre-rRNA, which is cleaved within the 90S pre-ribosome complex. In either pathway, the early cleavages separate the pre-40S and pre-60S complexes, which are then matured independently (Figure 1A). During 60S maturation, internal transcribed spacers 1 and 2 (ITS1 and ITS2) are removed from the pre-rRNA by endonuclease cleavage followed by exonuclease digestion to yield the mature 5.8S and 25S rRNA. The 60S ribosome maturation pathway involves several intermediate particles and a many different proteins. Several late steps in 60S maturation have recently been characterized (see Lo et al, 2010 and Panse and Johnson, 2010), but much less is known about early pre-60S particles or the actual function of most of the protein components. Understanding of yeast ribosome assembly will be greatly facilitated by the recent determination of the crystal structure of the mature 80S ribosomes (Ben-Shem et al, 2010). Figure 1.Pre-rRNA processing and protein interactions. (A) Schematic representation of pre-rRNA processing in yeast. The locations of processing sites on the 35S pre-rRNA are indicated. The positions of oligonucleotides used for northern hybridization (020) and primer extension (007) are shown. In the nucleolus, the 35S pre-rRNA, part of a 90S-sized complex called the SSU processome or 90S pre-ribosome, is processed at sites A0, A1 and A2, leading to the formation of 43S and 66S pre-ribosomes. Proteins studied in the CRAC analysis and their association with pre-ribosomal complexes are indicated. The 43S pre-ribosome is exported to the cytoplasm where Nob1 cleaves at site D, yielding the mature 18S rRNA and 40S subunit. 66S pre-ribosomes containing 27SA2 pre-rRNA are either processed at A3 or at B1L, which requires the presence of Nop4. Pre-ribosomes containing 27SA3 pre-rRNA are exonucleolytically trimmed by the Rat1, Rrp17 and Xrn1 5′–3′ exonucleases, yielding the 27SBS pre-rRNA. This maturation step requires the presence of Nop12, Nop7, Nop15 and Erb1 (depicted as coloured circles). After this step, the 27SB is cleaved at C2 by an unknown endonuclease, followed by exonucleolytic and endonucleolytic trimming of 7S by Rrp6 and Rrp44, two components of the exosome complex. Pre-ribosomes containing the 6S pre-rRNA are exported to the cytoplasm and matured by Ngl2 after which Nop12 and Nop4 dissociate. (B) Overview of known and predicted protein–protein interactions in 66S pre-ribosomes. The interaction map depicts interactions between the various assembly factors and r-proteins in 66S pre-ribosomes. Black lines; physical interactions among proteins shown to be part of subcomplexes or interacting as recombinant proteins (Krogan et al, 2004; Miles et al, 2005). Dashed red lines; yeast two-hybrid interactions (Ito et al, 2001; Miles et al, 2005). Red lines; interactions from protein-fragment complementation assays (PCAs), which detect proteins located within ∼80 Å (Tarassov et al, 2008). Dashed black lines; protein–protein interactions predicted from our CRAC data. Note that these may be mediated by RNA. Download figure Download PowerPoint Early in the eukaryotic lineage, the 5.8S rRNA was derived from the 5′-end of an ancestral 23S rRNA-like molecule by insertion of the ITS2 region. Reflecting this, the 5.8S rRNA associates with the 25S rRNA via two regions of extended base pairing between the 5′-region of 5.8S (nts 4–13) and 25S rRNA (nts 404–413), and between the 3′-region of 5.8S (nts 139–155) and 25S rRNA (nts 4–19). 5.8S–25S base pairing is established before or at C2 cleavage, since the 7S pre-rRNA remains associated with 25S rRNA following deproteinization (DT, unpublished). However, secondary structure models of the ITS1 region of the 27SA pre-rRNAs strongly predict that the 5′-region of 5.8S is base paired to the 3′-end of ITS1 in a stem-loop that is incompatible with base pairing to 25S rRNA, implying the presence of a conformational switch (Yeh et al, 1990). It is currently unclear whether the transfer of the 5′-region of 5.8S from ITS1 base pairing to 25S base pairing is provoked by exonuclease digestion of ITS1 from A3 to B1S, or is required for this processing to take place. The 5′-end of the yeast 5.8S rRNA is heterogeneous due to the use of two alternative processing pathways (Henry et al, 1994). The major, short form of the rRNA (5.8SS) has a 5′-end at site B1S (Figure 1A). This is generated by exonuclease digestion from an upstream cleavage site (A3), which is the target for cleavage by RNase MRP. Processing from A3 to B1S involves two exonucleases: the Rat1–Rai1 heterodimer (Schmitt and Clayton, 1993; Henry et al, 1994; Lygerou et al, 1996; Xue et al, 2000) and Rrp17 (Oeffinger et al, 2009). In addition, the cytoplasmic exonuclease Xrn1 can degrade the A3 -B1S region when Rat1 is inactive (Henry et al, 1994), but probably does not contribute to normal processing (El Hage et al, 2008). Formation of the 5′-end of the less abundant 5.8SL rRNA does not require RNase MRP or the 5′-exonucleases and is believed to involve an unidentified endonuclease (Faber et al, 2006). Similarly, 5′ heterogeneity is observed for 5.8S rRNA in metazoans, plants and other eukaryotes (Henry et al, 1994), suggesting the use of conserved processing pathways. In addition to the exonucleases, A3 processing requires components of the pre-60S particles (Dunbar et al, 2000; Pestov et al, 2001; Gadal et al, 2002; Oeffinger et al, 2002; Fatica et al, 2003; Oeffinger and Tollervey, 2003; Miles et al, 2005). Loss of these factors (including Rlp7, Cic1/Nsa3, Erb1, Nop7 and Nop15) leads to accumulation of the 27SA3 pre-rRNA and reduced synthesis of 27SBS and mature 5.8SS without concomitant loss of 27SBL or 5.8SL. A further factor, Nop12, is non-essential but required for efficient 25S rRNA synthesis (Wu et al, 2001) and, together with Brx1 and Ebp2 is predicted to be physically close to Rlp7, Cic1/Nsa3, Erb1, Nop7 and Nop15 within the pre-60S particle (Tarassov et al, 2008; Figure 1B). The role of Nop12 in 5.8S rRNA maturation was not assessed (Wu et al, 2001). A further protein, Nop4 (Nop77) was predicted to bind in this region (Tarassov et al, 2008), but its depletion confers a different phenotype, with specific inhibition of cleavage at both A3 and B1L (Bergès et al, 1994; Sun and Woolford, 1994, 1997). To better understand the roles of the non-enzymatic ribosome synthesis factors implicated in 5.8S 5′-maturation, we identified their sites of interaction with the pre-rRNA using the crosslinking and analyses of cDNA (CRAC) crosslinking technique (Supplementary Figure S1; Granneman et al, 2009). Unexpectedly, binding sites for five factors required for exonucleolytic removal of the A3-B1S fragment clustered near the 3′-end of the 5.8S rRNA (E-site), not the 5′-end. In contrast, Nop4, required for endonucleolytic cleavages in ITS1, crosslinked near the 5′-end of 5.8S. This suggests that a ribonucleoprotein complex, hereafter referred to as 'A3 cluster', assembled near the 3′-end of 5.8S rRNA is required for the activity of the 5′-exonucleases acting at the 5′-end of 5.8S, whereas endonucleolytic cleavage in ITS1 requires Nop4 binding to the 5′-end of 5.8S. High-throughput CRAC on the Rat1 5′-exonuclease indicated significant crosslinking at the 3′-end of 5.8S and in ITS2 and we furthermore demonstrate that Rat1 can bind the 5′-end of 25S independently of Nop4 and A3-cluster protein Nop15. We propose that A3-cluster proteins act to coordinate events at the 3′- and 5′-ends of 5.8S rRNA, ensuring that 5′-end maturation of 5.8S precedes ITS2 cleavage. Results Rat1 is associated with multiple sites in the pre-rRNA Rat1 has several distinct targets in the pre-rRNA; it accurately processes the 5′-ends of the mature 5.8S (A3-B1S) and 25S rRNAs (C2-C1) and completely degrades the excised A0-A1 and A2-A3 pre-rRNA fragments (Figures 1 and 2). In addition, Rat1 functions in the 5′-maturation of intronic snoRNAs, surveillance of mRNAs with defects in splicing and 5′-capping, and transcription termination on both Pol I and Pol II genes. Given this large number of substrates, we anticipated that Rat1 binding would be very transient. To maximize the recovery of RNA binding sites, we therefore developed a novel UV crosslinking system that allows UV irradiation of large volumes of cells actively growing in culture medium. This crosslinking device (the 'Megatron') consists of a 1.2-m metal tube with a central 205 W, 254 nm UV lamp, and can irradiate 2.5 l of culture in ∼1 min (Supplementary Figure S1A). Cells expressing HTP-tagged Rat1 and a non-tagged negative control strain (BY4741) were UV irradiated in the Megatron. A3-B1S processing also requires the 5′-exonuclease Rrp17 and the Rat1 cofactor Rai1 (Fang et al, 2005; Oeffinger et al, 2009). Both were tested as HTP-tagged constructs but neither gave usable crosslinking efficiencies. Illumina-Solexa deep sequencing of two independent Rat1 CRAC cDNA libraries revealed that around a quarter of the reads mapped to rDNA repeats. A multiple sequence alignment of rDNA-mapped reads is shown in Supplementary Table S2. As expected, many other putative Rat1 substrates were identified, which will be discussed elsewhere (manuscript in preparation). Figure 2.Rat1 crosslinking sites over the pre-rRNAs. (A) Rat1 crosslinks primarily to spacer regions in the pre-rRNA. A histogram that displays the distribution of rDNA-mapped reads along the entire rDNA sequence is shown. The red line indicates 100 000 averaged hits from two independent Rat1 CRAC experiments. The blue line indicates the distribution of 10 000 rDNA hits from the negative control experiment. The asterisks indicate frequent contaminants. The rDNA is schematically represented below the x axis, with processing sites included. The y axis displays the total number of times a nucleotide within an RNA fragment was mapped to the rDNA sequence. (B) The dashed lines point to expanded views of hits over the 5′-ETS and ITS1-25S region with schematics showing Rat1 substrates. Positions of potential crosslinking sites in spacer regions are shown in Supplementary Figure S9. Download figure Download PowerPoint Prominent peaks of Rat1 crosslinking were observed over the 5′-region of the 5′-ETS (Figure 2B), which is degraded by pathways including endonuclease cleavage (Lebreton et al, 2008; Schaeffer et al, 2009; Schneider et al, 2009). These data indicate that, like its human counterpart Xrn2 (Wang and Pestov, 2010), Rat1 is involved in degradation of the cleaved 5′-ETS fragments. A very prominent peak was present at 3′-end of the 5′-ETS, directly upstream of the A1 cleavage site, in agreement with the reported role of Rat1 in degradation of the excised A0-A1 fragments (Petfalski et al, 1998). Inspection of the regions surrounding the known Rat1 pre-rRNA processing substrates, A3-B1 in ITS1 and C2-C1 in ITS2 (Figure 2) revealed apparent similarities. High levels of crosslinking were seen immediately 5′ to the A3 and C2 cleavage sites; over the A2-A3 region in ITS1 and over the 3′-region of 5.8S and between the 3′-end of 5.8S (site E) and cleavage site C2 in ITS2. Reads including the 3′-region of 5.8S predominately extended through site E, at least 2 nt into ITS2 (Supplementary Figure S2A), indicating that binding occurred on the pre-rRNA, rather than on mature 5.8S rRNA. Substantial Rat1 crosslinking was also observed 3′ to the Rat1 targets, over the 5′-regions of the mature 5.8S (helices H3 and H4) and 25S rRNAs (H11) (Figure 2). In contrast, crosslinking was much lower over the Rat1 processing substrates A3-B1 and C2-C1. The intermediates in A3-B1 and C2-C1 processing are almost undetectable in wild-type yeast indicating high processivity during Rat1 processing. Finally, hits located at H66, H79 and H99 were also frequently found in negative control experiments (marked with asterisks in Figures 2A, B and 3C) and were therefore considered background. Figure 3.Overview of CRAC results and locations of protein–RNA interaction sites in the 25S and 5.8S rRNA secondary structures. (A) Results from 2 to 5 independent CRAC experiments. (B) Results from untagged strain. (C) Illumina-Solexa results from Nop4 (red line) and negative control (untagged strain; blue line). Sequences were aligned to the rDNA reference sequence using blast and plotted using gnuplot. Locations of mature rRNA sequences, spacers and cleavage site are indicated below the x axis. The y axis displays the total number of times each nucleotide within an RNA fragment was mapped to the reference sequence. The location of the peaks in the secondary structure of the rRNA is indicated with helix (H) numbers (Klein et al, 2004). The asterisks indicate frequent contaminants. (D) Locations of minimal binding sites for the ribosome synthesis factors are displayed on the 5.8S/25S rRNA secondary structures (http://www.rna.ccbb.utexas.edu/) and the 'ring model' for yeast ITS2 structure (Joseph et al, 1999; Cote et al, 2002). Large 25S rRNA domains are indicated with dashed boxes. The 5.8S rRNA sequence is coloured red. Locations of r-protein binding sites are boxed, based on their locations in the yeast 60S crystal structure (Ben-Shem et al, 2010) and previous genetic studies (van Beekvelt et al, 2000). Two Rat1 binding sites in helices 3/5 and 11 are shown in light blue. Crosslinking sites in the spacer regions are shown in Supplementary Figure S9. Download figure Download PowerPoint Rat1 also participates in degradation of the excised A2-A3 spacer fragment (Petfalski et al, 1998). This may contribute to Rat1 crosslinking over this region, but mutational analyses (below) indicate that this is not the major source. The location of site C2 was originally inferred from fingerprinting of in vivo labelled RNA and predicted to lie within the G133–G136 region (Veldman et al, 1980). However, Rat1-associated sequences frequently extended ∼8 nt further 3′ to terminate at U140 and A141 in ITS2 (Supplementary Figure S2A and C). This end was previously detected by primer extension in a rat1 mutant (Geerlings et al, 2000) and the same end is observed in the wild-type (see below). 7S pre-rRNA was immunoprecipitated using HTP-tagged Nop7 and 3′-ends were mapped by RNA cloning (Supplementary Figure S2B). The results confirmed that the 3′-end of 7S predominately falls at U140 and A141. We conclude that the major endonuclease cleavage site lies between A141 and G142, as proposed previously (Geerlings et al, 2000), which we refer to herein as site C2. We conclude that Rat1 is associated with sites flanking its target regions for pre-rRNA processing. Binding sites for Nop7, Nop12, Erb1, Cic1 and Nop15 cluster around the 3′-end of the 5.8S rRNA Based on published data (Miles et al, 2005; Tarassov et al, 2008), a group of synthesis factors were predicted to physically interact in early pre-60S particles: Brx1, Cic1/Nsa3, Erb1, Ebp2, Nop4, Nop7, Nop12, Nop15, Pwp1 and Ytm1 (see Figure 1B for interaction map). Except for Rlp7 and Ebp2, all of these factors were tested in CRAC analyses, and six (Cic1, Erb1, Nop4, Nop7, Nop12 and Nop15) efficiently crosslinked to RNA. The locations of crosslinking sites along the rDNA are indicated in Figure 3A and C, and are displayed on the predicted secondary structures of 25S, 5.8S and ITS2 in Figure 3D. CRAC experiments were performed 2–5 times and to be considered a bona fide RNA binding site, a nucleotide sequence had to be significantly enriched in every experiment. As negative controls, four independent CRAC experiments were performed with the non-tagged parental strain (Figure 3B). Sanger sequencing of Nop4 CRAC cDNA libraries had indicated multiple pre-rRNA binding sites and therefore high-throughput Solexa sequencing was performed together with a negative control to obtain sufficient sequence depth for each binding site (Figure 3C). Recovered sequences that overlapped with control peaks in H66, H79 and H99 at the 3′-end of the 25S were considered background (asterisks in Figure 3). Crosslinking sites were precisely identified by the presence of multiple point deletions or substitutions at a specific position in sequence reads (see Granneman et al, 2009), or a minimal RNA binding site was determined from overlapping sequences. In CRAC analyses, Erb1 was crosslinked to the 5′-region of the 25S rRNA over H21 and H22 (Figure 3A and D). In the yeast 60S structure, this region forms a compact structure, probably associated with Rpl42, Rpl8, Rpl28 and Rpl15 (Figure 4A and B; Ben-Shem et al, 2010). The average length of RNAs crosslinked to Erb1 was relatively long (59 nt), possibly because this structure confers protection during the RNase digestion used to generate the cloned fragments. Figure 4.Location of Nop4, Nop7, Erb1 and Nop12 and RNA binding sites in 60S crystal structure. (A, B) Images, rotated by 90°, showing the protein neighbourhood around the 3′-end of the yeast 5.8S rRNA. The 5.8S rRNA is shown in wheat color, with binding sites for Nop12 (orange and blue), Nop7 (red) and Erb1 (purple) indicated as coloured nucleotide strands. Images were generated using pymol. Ribosomal proteins (Rpl15, Rpl17, Rpl25, Rpl26, Rpl35 and Rpl37) binding to this region are indicated as surface representations. Helix numbers are indicated with 'H'. The double arrows indicate the distance (in Å) between the 3′-end region of 5.8S and the Nop7 and Erb1 binding sites. (C, D) Images showing the location of the Nop4 (blue) binding sites that surround the 5′-end of 5.8S. Ribosomal proteins Rpl15, Rpl17 and Rpl37 that bind in this region are indicated as surface representations. Helix numbers are indicated with 'H'. (E, F) Images showing the location of the Nop7 (red) binding site in the structure of the 25S rRNA (wheat color) and 5.8S 3′-end (light blue). Superimposed are structures for r-proteins Rpl35 (orange), Rpl25 (dark purple) and Rpl15 (green), Rpl26 (yellow) and Rpl17 (light purple). The polypeptide exit tunnel is indicated. The double arrow indicates the distance (in Å) between the 3′-end of 5.8S and the base of the helix containing the Nop7 binding site. Download figure Download PowerPoint Nop7 predominately crosslinked to H54 in 25S rRNA (Figure 3A and D). This site is within Domain III, in close proximity to the binding sites for Rpl25 and Rpl35 (Figures 3D, 4A and B). Nop7 is genetically linked to Rpl25 (Oeffinger et al, 2002) and cells lacking Rpl35A accumulate 27SA3 pre-rRNA and are defective in ITS2 processing (Babiano and de la Cruz, 2010). In the yeast 60S structure, the N-terminal domain of Rpl25 makes extensive contacts with 5.8S, whereas the C-terminal region mainly contacts Domain III of 25S (Figure 3D). Both CRAC and genetic data, therefore, support direct interactions between Nop7, Rpl25 and Rpl35. Notably, Rpl35, together with Rpl17 and Rpl26, binds in the vicinity of the peptide exit tunnel in mature 60S subunits (Figure 4E and F). Cic1 and Nop15 crosslinked preferentially to the 5′-end of the ITS2 spacer region (Figure 3A and D). Although many of the RNAs crosslinked to Nop15 and Cic1 originated from the same region, analysis of shorter fragments and specific mutations in the RNAs allowed us to distinguish the respective RNA binding sites (Supplementary Figure S3). Cic1 also consistently crosslinked to H10 in 25S (Figure 3A), albeit less frequently. High-throughput data suggested that Cic1 and Nop15 interact (Tarassov et al, 2008), and the locations of their RNA binding sites strongly support their direct association in pre-60S ribosomes. The major Nop12 binding sites were located at H8 (Figure 3A), near the 3′-end of the 5.8S rRNA, and in H10, which is formed by base pairing between the 3′-end of 5.8S and the 5′-end of the 25S rRNA (Figure 3D, ITS2 proximal stem). Thus, Nop12 could have a role in formation or stabilization of the interaction between the 5.8S and 25S rRNAs. The proximity of Nop12 binding sites to A3-cluster protein crosslinking sites prompted us to re-analyze the role of Nop12 in 5.8S processing. Because cells lacking Nop12 are cold-sensitive (Wu et al, 2001) pre-rRNA processing was analysed in nop12Δ cells grown at 18 °C for 24 h. Similar to A3-cluster-depleted cells, nop12Δ cells accumulated 35S and 27SA3 pre-rRNAs (Supplementary Figure S4B and C, lanes 11 and 12), albeit modestly. We conclude that Nop12 is a novel A3-cluster protein. Nop4 crosslinking sites were dispersed in the primary rRNA sequence and mapped to 25S rRNA domains II and III (Figure 3C and D). In the yeast 60S crystal structure, these sites are ∼30–80 Å apart and cluster near the 5′-end of 5.8S (Figure 4C and D). Nop4 is an ∼80-kDa protein and could conceivably contact many of these sites simultaneously, bringing domains II and III in proximity to each other. The Nop4 binding sites in H26, H32, H33 and H47 are close to 5.8S rRNA and to binding sites for Rpl17, Rpl35 and Rpl37. We predict Nop4 directly contacts these ribosomal proteins. The binding site in H60 is adjacent to Rpl19 binding sites (Figures 3D, 4C and D). Consistent with previous data (Bergès et al, 1994), Nop4 depletion leads to delayed cleavage at site A2, with more severe inhibition at A3, B1L and concomitant decrease in 27SB levels (Supplementary Figure S4B and C, lanes 7 and 8). The 27SB1L/27SB1S ratio was not significantly altered in these cells (Supplementary Figure S4D, compare lane 2 with lane 1), suggesting Rat1 can process residual 27SA3 generated in the absence of Nop4. We conclude that cleavage at A3 and B1L requires Nop4 binding near the 5′-end of 5.8S, whereas Rat1-dependent processing from A3 requires A3-cluster proteins binding near the 3′-end of 5.8S. Validation of CRAC data To validate the CRAC data, we performed immunoprecipitation experiments with HTP-tagged proteins and the non-tagged parental strain and analysed co-precipitated RNAs by northern blot analysis, primer extension analysis and EtBr staining (Figure 5). The data were quantified by calculating fold enrichment over background levels observed in experiments with untagged strains (Supplementary Figure S5). Consistent with previous reports, tagged Nop7, Cic1 and Nop15 co-precipitated 27SA/B and 7S pre-rRNAs (Nissan et al, 2002; Fatica et al, 2003; Oeffinger & Tollervey, 2003; Figure 5B and C). HTP-tagged Nop7 also associated with 6S pre-rRNA, a 5.8S rRNA precursor that is 3′-extended by only 8–9 nt (Figure 5B, lane 12). These observations are consistent with the CRAC data and indicate that ITS2 may be a major determinant for pre-60S binding by Cic1 and Nop15, but not by Nop7. Figure 5.Nop4, Nop7, Nop12, Erb1 and Nop15 associate with 66S pre-rRNAs. Immunoprecipitations were performed with HTP-tagged proteins and the non-tagged parental strain (−lanes). (A) Schematic representation of pre-rRNA species that are detected by oligonucleotides 020 and 007. Red lines indicate the positions of these oligonucleotides on the rDNA. RNAs were resolved on 1.2% agarose and 8% polyacrylamide/7 M urea gels and detected by northern hybridization (B) or primer extension (C) using oligonucleotides 020 and 007, respectively (Supplementary Table S1). Input and supernatant indicate 0.1% of RNA extracted from cell lysates and supernatants after immunoprecipitation. Note that in the northern blots shown in (B) longer exposures are shown for inputs and supernatants. Rat1-1/xrn1Δ primer extension products were loaded (C, lane 15) as markers for 26S and 5′-extended (5′-ext) 25S. Download figure Download PowerPoint The association of Nop4, Nop12 and Erb1 with pre-rRNA species has not previously been analysed in detail. Both Nop4 and Nop12 co-precipitated 27SA/B and 7S pre-rRNAs, albeit in modest amounts compared with other proteins tested (Figure 5B and C, lanes 11 and 13). Nop12 and Nop4 also reproducibly showed modest enrichment of mature 5.8S and 25S (Figure 5A and B, lanes 11 and 13; Supplementary Figure S5A), suggesting that these proteins remain associated with pre-ribosomes after removal of ITS2. HTP-tagged Erb1 efficiently precipitated 27S pre-rRNA, but precipitated low amounts of 7S and 5′-extended 25S pre-rRNAs (25S' and 26S) compared with other proteins tested (Figure 5B and C, lane 10; Supplementary Figure S5B and C), implying that Erb1 dissociates from pre-ribosomes following cleavage of 27SB pre-rRNA at site C2. In the 25S rRNA secondary structure, the Erb1 and Nop7 binding sites appear distant from the 3′-end of 5.8S and ITS2 (Figure 3D). However, in the yeast 60S structure (Ben-Shem et al, 2010), these sites are in the vicinity of the Nop12 binding sites and ∼30–50 Å (equivalent to 10–15 nt) from the 3′-end of 5.8S rRNA (Figure 4A and B). Nop7 and Erb1 directly interact in pre-ribosomes (Tang et al, 2008), suggesting their participation in long-range interactions bringing domains III and I of 25S close to the 3′-end of 5.8S (Figure 4B). Collectively, UV crosslinking, protein–protein interaction and pre-rRNA processing data strongly indicate that A3-cluster proteins physically and functionally interact with the pre-60S region surrounding the 3′-end of 5.8S and with ITS2. Altered Rat1 binding in strains depleted of Nop4 or Nop15 Sites of Rat1 crosslinking may reflect targets undergoing active degradation, pre-rRNA docking sites directed by protein association or both. To distinguish between these possibilities, Rat1 crosslinking was analysed in strains depleted of Nop4 or the A3 cluster protein Nop15. Rat1-HTP strains carrying PGAL::3xHA-NOP4 or PGAL::3xHA-NOP15 were compared with the parental Rat1-HTP strain. Cells were harvested at an OD600 of ∼0.5 after 12 h of depletion in glucose-containing minimal medium. At this depletion time point, only modest defects in growth and pre-rRNA processing were observed (Supplementary Figure S6A and B). Crosslinked RNAs were cloned using barcoded 5′ linkers (Suppleme
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