Yeast Polypeptide Exit Tunnel Ribosomal Proteins L17, L35, and L37 are Necessary to Recruit Late-assembling Factors Required for 27SB Pre-rRNA Processing

Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing Michael Gamalinda 1 , Jelena Jakovljevic 1 , Reyes Babiano 2 , Jason Talkish 1 , Jesu ´s de la Cruz 2 and John L. Woolford Jr 1, * 1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA and 2 Departamento de Gene ´tica, Universidad de Sevilla, Sevilla, E-41012, Spain Received August 13, 2012; Revised October 14, 2012; Accepted November 5, 2012 ABSTRACT Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2. INTRODUCTION Ribosomes are complex macromolecular machines that catalyze the fundamental process of translation. Assembly of ribosomes is a complicated multi-step process that involves transcription, folding, modification and processing of pre-rRNAs, as well as the concomitant assembly of ribosomal proteins (r-proteins) (1–4). In eukaryotes, assembly of both 40S and 60S ribosomal subunits begins in the nucleolus with the transcription of pre-35S and pre-5S rRNA precursors by RNA polymerase I (Pol I) and RNA polymerase III (Pol III), respectively (Figure 1A). Assembly proceeds through a dynamic flux of protein–protein, protein–rRNA and rRNA–rRNA interactions toward final maturation in the cytoplasm (1,6–9). More than 200 trans-acting assembly factors facilitate these remodeling events that trigger the successive nucleolytic removal of transcribed spacer sequences and the hierarchical addition of r-proteins into nascent subunits (6,10) (Figure 1A and B). Pioneering in vitro reconstitution studies of bacterial 30S subunits established the hierarchical succession of small subunit r-protein assembly with 16S rRNA (11–13). rRNA structural probing showed that binding of an initial subset of r-proteins causes structural rearrangements in 16S rRNA. These rearrangements form binding sites that allow subsequent association of another set of r-proteins and stabilize rRNA configurations (14–19). What emerged from these studies is the principle that ribosome synthesis is highly cooperative and occurs in parallel intermediate pathways (20–22). However, ribosome assembly in vivo is much more complex, involving (i) coordination of transcription with assembly, (ii) removal of spacer sequences, (iii) export of pre-ribosomes from the nucleus to the cytoplasm and (iv) controlled action of assembly factors. The discovery of more than 200 ribosome biogenesis factors has led to extensive investigations on how they facilitate processing of pre-rRNAs and structural rearrangements to steer maturation of assembling pre-ribosomal complexes. Although the field has learned a great deal from studying them, trans-acting factors are *To whom correspondence should be addressed. Tel: +1 412 268 3193; Fax: +1 412 268 7129; Email: [email protected] Published online 24 December 2012 Nucleic Acids Research, 2013, Vol. 41, No. 3 1965–1983 doi:10.1093/nar/gks1272 ßThe Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]. only one part of the grand design that is ribosome biogenesis. Ribosomes are multimolecular complexes wherein r-proteins embellish a core of rRNA; hence, r-proteins likely serve as molecular scaffolds necessary not only for ribosome function but also assembly. Comprehensive studies of how r-proteins facilitate ribosomal subunit biogenesis are greatly underrepresented. Recent work has identified for which steps in pre-rRNA processing different r-proteins are required, but it is not clear exactly how they participate in these steps (23–35). It is presumed that they do not function directly in pre-rRNA processing; r-proteins are thought to have structural rather than enzymatic functions. The roles of r-proteins in establishing pre-ribosomal particles and recruitment of assembly factors and other r-proteins to enable subsequent maturation are even less well studied. Thus far, examination of how r-proteins affect the composition of pre-ribosomes to propel pre-rRNA maturation has only been reported for a handful of r-proteins from both r-subunits (36–39). Hence, more studies of this kind are needed to fully comprehend the assembly process. Initial systematic investigation of 60S subunit assembly categorized r-proteins based on their requirement for early, intermediate, and late pre-rRNA maturation steps [(30), unpublished data]. Following up on Po ¨ll et al. (30), we recently characterized in more detail a subset of r-proteins that function early in ribosome assembly, during the exonucleolytic trimming of ITS1 sequences in 27SA 3 pre-rRNAs (39). In this work, we have focused on r-proteins L17, L35 and L37, which had been implicated in removal of ITS2, and are adjacent to each other in the structure of mature 60S subunits (40–42) (Figure 2). L17 and L35 are eukaryotic homologs of bacterial L22 and L29, respectively, whereas L37 does not have a bacterial homolog. Together, these three r-proteins interact with all six secondary structure domains of 5.8S/25S rRNA, having multiple contact sites with each domain (40–42). They may therefore be important in promoting and stabilizing long-range inter-domain interactions that ultimately influence the architecture of assembling ribosomes. Thus, these r-proteins may help create local or global structural prerequisites for pre-rRNA processing and association of proteins. They are also of interest because, together with the nonessential r-protein L26 (43), L17, L35 and L37 are missing from pre-ribosomes in mutants unable to process early pre-rRNA intermediates (39,44). This is attributed to misfolding of rRNA domains to which they bind, as well as reflecting the coupling of prerRNA processing with r-protein assembly. In addition, Figure 1. Ribosome assembly pathway in Saccharomyces cerevisiae.(A) Pre-rRNA processing pathway indicating exonucleolytic trimming and endonucleolytic reactions. Note that cotranscriptional processing at the A 3 site also occurs (5) (not shown). (B) Maturation of pre-ribosomal particles from the nucleus to the cytoplasm. 1966 Nucleic Acids Research, 2013, Vol. 41, No. 3 since these r-proteins lie around the rim of the nascent polypeptide exit tunnel, one might be able to relate the construction of this rRNP neighborhood to intermediate pre-rRNA maturation steps. We wanted to take a comprehensive look at the function of these three r-proteins and how they affect ribosome assembly. We tested their role in recruiting assembly factors to begin to understand how their absence leads to a distinct pre-rRNA processing defect. We have compared the phenotypes upon depleting each of these r-proteins and found that they exhibit very similar defects in biogenesis of 60S subunits even though they are not interdependent for association with pre-ribosomal particles (pre-rRNPs). Our data indicate that these r-proteins affect recruitment of a specific set of assembly factors necessary for 27SB pre-rRNA processing, and in their absence pre-rRNPs are gradually turned over. MATERIALS AND METHODS Generation of yeast strains and in vivo depletion of r-proteins Yeast strains used in this study are derivatives of either JWY6147 (MATa ura3-52 trp1-1 lys2-801 his3-200 leu2-1) or BY4741 (MATa his31 leu20 ura30 met150) as listed in Supplementary Table S1. Gene disruptions, GAL1 promoter fusions, and 3xHA-, 13xMYC- and TAP-tagged genes were constructed using polymerase chain reaction (PCR)-based chromosomal modifications (45,46). Oligonucleotide primers for PCR and plasmids used in this study are available upon request. Like most yeast r-proteins, L17, L35 and L37 are encoded by duplicate (paralogous) genes (47). These r-protein pairs have highly similar, or identical, amino acid sequences (Supplementary Figure S1). Conditional null mutant strains of RPL17 (GAL-HA-RPL17;MATa ura3-52 trp1-1 lys2-801 his3-200 leu2-1 rpl17b:: KANMX6 rpl17a::GAL-HA-RPL17A TRP1) and RPL35 (GAL-HA-RPL35;MATa ura3-52 trp1-1 lys2-801 his3-200 leu2-1 rpl35b::KANMX6 rpl35a::GAL-HA- RPL35A TRP1) were constructed by first disrupting one r-protein gene copy. The promoter of the more highly expressed copy of each duplicate r-protein gene (48) that confers a stronger phenotype upon deletion (data not shown) was the GAL1 promoter. Thus, we replaced the complete open reading frame (ORF) of RPL17B/ YJL177W or RPL35B/YDL136W with the KANMX6 cassette that confers resistance to the drug geneticin (G418). PCR-generated KANMX6 amplicons from the Figure 2. Localization of L17, L35, and L37 around the polypeptide exit tunnet (PET). (Top left) Location of L17, L35, and L37 in the structure of mature 60S ribosomal subunits (PDB: 3U5D and 3U5E) (41). CP denotes the central protuberance. (Top right) The 60S ribosomal subunit structure is viewed from the rim of the polypeptide exit tunnel looking straight through the subunit. 5.8S rRNA is in black, 25S rRNA is in grey, r-proteins of interest are shown as colored spheres. Six r-proteins (L22, L24, L26, L31, L38 and L39) adjacent to L17, L35 and L37 are nonessential. (Bottom, left to right) rRNA domains with which r-proteins L17, L35 and L37 interact. 5.8S rRNA is shown in black, while domains I, II, III, IV, V and VI of 25S rRNA are shown in yellow, blue, orange, green, red, and purple, respectively. Nucleic Acids Research, 2013, Vol. 41, No. 3 1967 pFA6a-kanMX6 plasmid were transformed into JWY6147, transformants were screened for G418 resistance, and replacements were verified by genomic PCR. From these single gene disruption backgrounds, the endogenous promoter of the other r-protein gene copy in its genomic locus (RPL17A/YKL180W or RPL35A/ YDL191W) was replaced with a GAL1 promoter cassette together with the TRP1 selectable marker. This cassette also contains a 3xHA-epitope to place an HA-tag at the N-terminus of the respective r-proteins, to enable their detection via western blotting. PCR modules containing GAL1 promoter sequences were transformed into the single r-protein gene deletion strains; transformants were selected for growth in medium containing galactose but lacking tryptophan. Trp+ transformants were screened for correct integration of the GAL1 promoter and the N-terminal HA-epitope fusion by western blotting with anti-HA antisera. After numerous attempts, creating a chromosomal fusion of the GAL1 promoter to either gene copy of RPL37 proved to be unsuccessful. For this reason, an RPL37 conditional mutant strain (pGAL-RPL37;MATa his31 leu20 ura30 met150 rpl37a::HIS3MX6 rpl37b::KANMX4 pGAL-RPL37A LEU2) derived from BY4741 was used for characterization. The rpl37 mutant strain used has both chromosomal copies of RPL37 (RPL37A/YLR185W and RPL37B/YDR500C) deleted and contains a plasmid bearing a conditional allele of RPL37A, without an in-frame N-terminal HA epitope fusion [procedure described in (26) and (30)]. C-terminal 3xHA-, 13xMYC- and TAP-fusion cassettes for tagging genes by transformation were PCR-amplified from plasmids bearing the sequences of the tags and a selectable marker (45,46). His+ or Ura+ transformants were screened by immunoblotting with anti-HA, -MYC or anti-TAP antisera to validate correct cassette integration. Conditional null mutant strains were grown at 30Cin either rich or synthetic minimal medium containing 2% galactose to express GAL1 fusion genes, or shifted to medium containing 2% glucose to repress expression of the r-protein gene. Cloning of yeast r-protein genes into appropriate vectors Unique restriction sites were incorporated 800–1000 nucleotides upstream and 50–100 nucleotides downstream of the start and stop codons of r-protein genes, respectively, via a two-step PCR reaction. Plasmids from the Yeast Genomic Tiling Collection (Open Biosystems) that bear intact RPL17A,RPL35A and RPL37A ORFs as the template and oligonucleotides used were designed to contain palindromic sequences recognized by restriction enzymes that produce sticky ends (oligonucleotide sequences available upon request). Upstream and downstream PCR oligonucleotides to amplify each r-protein gene contained restriction sites as follows: BamHI and HinDIII for RPL17A; BamHI and SalI for RPL35A; and, BamHI and EcoRI for RPL37A. Resulting PCR products were digested with the corresponding restriction enzymes and then ligated into vectors digested with the same restriction enzymes. RPL17A- and RPL35A-con- taining PCR amplicons were cloned into pRS315 (LEU2) whereas the cassette bearing the RPL37A ORF was cloned into pRS316 (URA3). Correct plasmid constructs were verified by sequencing. Sucrose gradient centrifugation Pre-ribosomes, 40S and 60S r-subunits, 80S ribosomes, and polyribosomes were fractionated using a modified protocol derived from Deshmukh et al. (49). Briefly, 100 ml of logarithmically growing yeast cultures were treated with 5 mg cycloheximide 10 min prior to harvesting. Forty A 254 units of whole-cell lysates were loaded into 7–47% (w/v) sucrose gradients and subjected to centrifugation at 27 000 rpm using a Sorvall AH-629 swinging bucket rotor. Fractions were collected and A 254 was monitored using a Teledyne ISCO Foxy R1 density gradient fraction collector. Steady-state analyses of whole-cell mRNAs, pre-rRNA intermediates and mature rRNAs Steady-state levels of pre-rRNAs were assayed by primer extension and northern hybridization protocols adapted from Horsey et al. (50). An oligonucleotide (51) complementary to a region immediately upstream of the C 2 cleavage site in ITS2 was used to assay for 50-ends of 27S pre-rRNA intermediates by primer extension. The same oligonucleotide was used to assay for 7S pre-rRNA by northern blotting. Transcription of GAL1- driven RPL17A,RPL35A and RPL37A genes in expressive and repressive conditions was also monitored by northern hybridization using oligonucleotides complementary to respective mRNAs (oligonucleotide sequences available upon request). U2 snRNA was used as loading control in all northern hybridization assays. To detect amounts of mature 25S and 18S rRNAs, total RNA was extracted from cells, separated on a 1% agarose gel, and visualized by ethidium bromide staining. Signal intensities of mature rRNAs were quantified using Image Gauge software (Fujifilm). Kinetic pulse-chase labeling analysis Metabolic pulse-chase experiments to assay the kinetics of pre-rRNA processing in vivo were carried out as described in Horsey et al. (50) with the following modifications: yeast cultures at OD 610 = 0.4–0.5 were pulse-labeled with [ 3 H-methyl]-methionine for 5 min then chased with an excess of non-radioactive methionine. RNA was extracted from samples collected at 0, 2, 5, 10, 20, 40 and 60 min after chase. Equal counts were loaded onto agarose-formaldehyde gels for each RNA sample from GAL-HA-RPL17 and pGAL-RPL37 strains grown in galactose-containing medium or shifted for 16–17 h to glucose-containing medium, respectively. Affinity purification of assembly intermediates TAP-tagged assembly factors were used to affinity-purify pre-ribosomes from whole-cell extracts using the 1968 Nucleic Acids Research, 2013, Vol. 41, No. 3 single-step affinity purification protocol described by Sahasranaman et al. (44). Silver staining and western blotting Proteins from whole-cell extracts or purified pre-ribo- somes were separated by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) in 4% to 20% Tris–Glycine precast gels (Invitrogen) after which a standard silver-staining protocol was followed. Proteins on SDS-PAGE profiles were labeled based on comigration with silver-stained proteins previously identified by mass spectrometry, decrease in protein band intensity when expression of the corresponding gene is turned off, or shift in mobility of epitope-tagged versions of the corresponding protein. Detection of proteins by western blotting was carried out using the protocol adapted from Sahasranaman et al. (44). Nog2, which comigrates with IgG on 4% to 20% precast Novex gels, was assayed using 4% to 12% Bis–Tris precast gels (Invitrogen). Co-immunoprecipitation of pre-rRNAs with GFP-tagged r-proteins Pre-rRNAs copurifying with GFP-tagged L17 and L37 were assayed using the one-step GFP-TrapÕ_A protocol (Chromotek) as previously described (43). Briefly, RNA enriched from purified pre-ribosomes containing the GFP-tagged protein was extracted, and pre-rRNAs were assayed by northern hybridization following procedures described by Venema et al. (52). Autoradiographs were analysed using an FLA-5100 imaging system (Fujifilm) at the Biology Service (CITIUS) from the University of Seville. Fluorescence microscopy Export of pre-ribosomal particles from the nucleolus through the nucleoplasm to the cytoplasm was assayed by tracking the localization of L25-eGFP or S3-eGFP as previously described (36,53–55). For this purpose, each conditional mutant strain was transformed with a plasmid carrying either L25- or S3-eGFP, and Nop1-mRFP (55,56). Cells were imaged using a Carl Zeiss LSM-510_META_UV_DuoScan inverted spectral confocal microscope and analysed using ImageJ (57). Timing of r-protein assembly was addressed by transforming YCplac111-RPL17B-eGFP and YCplac111- RPL37B-eGFP into rpl17 and rpl37 null strains, respectively. Each strain was then cotransformed with either pRS316-GAL-NMD3100 or the pRS316-GAL-NMD3FL plasmid, expressing a GAL promoter fusion of a dominant negative truncation or a wild-type allele of the NMD3 gene, respectively (58). Images were acquired using a Leica DMR microscope equipped with a DC camera and processed using Adobe Photoshop 7.0. In vivo ITS2 structure probing ITS2 secondary structure was assayed by in vivo dimethyl sulfate (DMS) probing using a protocol adapted from Dutca et al. (59). Conditional null strains were grown in expressive and repressive media as indicated above, to OD 610 = 0.4–0.5. Ten milliliter aliquots of yeast cultures were treated with 200 ml of a fresh dilution of DMS (Sigma Aldrich) in 95% ethanol (1:4 v/v) to a final concentration of 50 mM. Treated cells were incubated with shaking at 30C for 2 min. Reactions were quenched by placing the tubes on ice and adding 5 ml of 0.6 M 2-mercaptoethanol and 5 ml of water-saturated isoamyl alcohol. As a control for the effectiveness of the stop reaction, a control sample was included where DMS treatment was done after the addition of 2-mercaptoethanol and isoamyl alcohol. Cells were pelleted by centrifugation at 5000 gfor 5 min, and the liquid phase was carefully removed. Cell pellets were suspended in 5 ml 0.6 M 2-mercaptoethanol, and centrifuged again. Total RNA was immediately extracted from the cells. Nucleotide modifications were assayed by primer extension and gel electrophoresis as described earlier, using oligonucleotides complementary to ITS2 (available upon request). RESULTS Previous evidence indicates that both L17 and L35 are required for processing of the ITS2 spacer separating mature 5.8S and 25S rRNA sequences (30,31,44). The role of L37 in ribosome assembly has not been previously reported; however, our initial characterization led us to believe that L37 is also involved in the same assembly step. In cells where transcription of RPL35 is repressed, hence depleting the protein, mature 60S subunits are not produced as consequence of a block in 27SB pre-rRNA processing (31) (Supplementary Figures S2 and S3). Nucleocytoplasmic export is impaired when L35 is depleted (Supplementary Figure S3), and pre-60S ribosomal particles are gradually turned over (31). Furthermore, L35 assembles into the pre-60S ribosomal particles containing 27SA 2 pre-rRNA (66SA 2 assembly intermediates; Figure 1B). However, despite its role in 27SB pre-rRNA processing, no information is available on how L35 affects the structure of ITS2 or recruitment of specific assembly factors or r-proteins into pre-rRNPs. Here, we start by filling the gap in our knowledge of the roles of L17 and L37 in production of mature r-subunits, pre-rRNA processing, and when they are stably loaded into preribosomes. We then proceed to show the effects of depleting L17, L35 or L37 on the structure of ITS2, and the association of trans-acting factors and other r-proteins with pre-rRNPs. Depletion of r-proteins L17 and L37 is detrimental to cellular growth and synthesis of 60S subunits To assess the function of the essential L17 and L37 r-proteins (48,60) in ribosome biogenesis, we utilized conditional null mutant systems (GAL-HA-RPL17 and pGAL-RPL37) where the corresponding r-protein gene is expressed under the control of the GAL1 promoter (see ‘Materials and Methods’ section). These strains failed to grow on glucose-containing solid medium (Figure 3A) where transcription of GAL1-driven r-protein genes is repressed (Supplementary Figure S2), and stopped dividing Nucleic Acids Research, 2013, Vol. 41, No. 3 1969 6–9 h after shifting from galactose to glucose-containing liquid medium (Figure 3B). Consistently, significant reduction of the total cellular amounts of HA-L17 within this timeframe is evident (Supplementary Figure S2). Plasmids bearing wild-type RPL17A and RPL37A alleles complemented the growth defect of GAL-HA-RPL17 and pGAL-RPL37 strains on glucose-containing solid medium, respectively (Figure 3A). To test the effect of depleting each protein on production of ribosomes, we assayed levels of mature rRNAs and ribosomal subunits. Upon depletion of either L17 or L37, steady-state levels of 25S rRNA relative to 18S rRNA decreased. In each case, the effect was rapid; the ratio dropped within 1.5 h after shifting from galactose to glucose (Figures 4A and Supplementary Figure S4). Consistent with these observations, amounts of free 60S r-subunits were dramatically reduced relative to free 40S subunits in each of the two strains where L17 or L37 were depleted (Figure 4B). In addition, halfmer polyribosomes were present upon depletion of r-proteins, indicative of a deficit in 60S r-subunits. These observations indicate that L17 and L37 are specifically required for the proper formation of mature 60S r-subunits. L17 and L37 are required for processing of 27SB pre-rRNA and formation of stable 66S assembly intermediates To test how these r-proteins affect formation of mature rRNAs, we assayed for the synthesis and turnover of pre-rRNAs by pulse-chase experiments using [ 3 H-methyl]-methionine in GAL-HA-RPL17 and pGAL-RPL37 strains. Under permissive conditions where L17 and L37 are expressed, 35S pre-rRNA was rapidly processed into 27S and 20S precursors, which Figure 3. Depletion of either L17 or L37 leads to cellular growth arrest. (A) Growth on solid medium of each conditional null mutant strain relative to the wild-type control strains JWY6147 or BY4741. GAL-HA-RPL17,pGAL-RPL37, wild-type strains and conditional strains episomally expressing wild-type alleles of r-protein genes were grown in galactose-containing (Gal) liquid medium and diluted to an OD 610 of 0.5. Ten microliters of 10- to 100 000-fold serial dilutions were spotted on galactose-containing (Gal) and glucose-containing (Glu) solid medium, and then incubated at 30C. (B) Logarithmically growing GAL-HA-RPL17 and pGAL-RPL37 cells were retained in galactose-containing media or transferred to glucose-containing liquid media at 30C. Strains were diluted as necessary to maintain logarithmic growth, and cell densities (OD 610 ) were continuously measured. 1970 Nucleic Acids Research, 2013, Vol. 41, No. 3 were subsequently converted to mature 25S and 18S rRNAs, respectively (Figure 5A). However, in repressive conditions where L17 and L37 are depleted, synthesis of 25S rRNA was inhibited relative to 18S rRNA. Upon depletion of either r-protein, 27SB pre-rRNA persisted even after 10 min of chase, indicating a delay in processing of this precursor species. Interestingly, we observed a gradual decrease in 27SB pre-rRNA to undetectable levels 20 min after the chase began, suggesting efficient turnover of this precursor (Figure 5A). 23S pre-rRNA was also detected in each mutant, albeit less so in the absence of L37, suggesting a delay in processing at sites A 0 ,A 1 and A 2 . The abundance of this intermediate, previously found in many 60S r-subunit assembly mutants (28,53,61), is most likely a secondary consequence of inefficient recycling of assembly factors required for the early cleavage steps, which fail to be released from aberrant 66S pre-ribosomes [discussed in (62)]. We also assayed steady-state levels of pre-rRNA intermediates in r-protein depleted strains by primer extension and northern hybridization. Upon depletion of either L17 or L37, we found no differences in amounts of the primer extension stops at the A 2 and A 3 sites. However, we observed a significant accumulation of the primer extension stop at the B 1S site and a more modest accumulation at the B 1L site (Figure 5B). This might be a manifestation of the faster relative rate of turnover of abortive 66S intermediates containing 27SB 1L pre-rRNA in mutants lacking L17 or L37 (28). Northern blot analysis showed that levels of 7S pre-rRNAs decreased in each mutant (Figure 5C). Therefore, this indicates that the observed increase in B 1L and B 1S ends represent 27SB pre-rRNAs. Finally, consistent with previous data, L17-depleted cells also accumulated 50truncated intermediates caused by Rat1 exonucleolytic digestion past the B 1S site during 27SA 3 pre-rRNA processing (44) (Figure 5B). We conclude that L17 and L37 are required for 27SB pre-rRNA processing at the C 2 site in ITS2; in their absence, abortive intermediates are formed but subsequently targeted for turnover. Interestingly, these observations are very similar to the pre-rRNA processing Figure 4. Cells depleted of L17 or L37 exhibit a deficit of 60S ribosomal subunits. (A) Total RNA was extracted from GAL-HA-RPL17 and pGAL-RPL37 strains shifted to glucose-containing media at specified time points. Samples were separated on agarose gels, and stained with ethidium bromide. Ratios of 25S relative to 18S mature rRNA levels for at least three biological replicates are shown in the graph. (B) Polysomes from GAL-HA-RPL17 and pGAL-RPL37 strains grown in galactose-containing media and shifted to glucose-containing media for 17 h were sedimented through 7 to 47% sucrose gradients. 40S and 60S r-subunits, 80S ribosomes and polyribosomes were monitored at A 254 using a Teledyne ISCO Foxy R1 density gradient fraction collector. Arrows indicate halfmers. Nucleic Acids Research, 2013, Vol. 41, No. 3 1971 phenotype we previously observed upon depletion of L35 r-protein (31). Aberrant pre-ribosomes lacking either L17 or L37 are retained in the nucle(ol)us Ribosome maturation is initiated in the nucleolus, continues in the nucleoplasm, and is completed following export of the pre-40S and pre-60S ribosomal particles to the cytoplasm. Although efficient nucleocytoplasmic export of pre-40S particles requires minimal structural reorganization, pre-60S ribosomal particles undergo stringent structural and compositional rearrangements prior to export (6,7,63). To investigate the subcellular location of assembly intermediates in the absence of L17 and L37, we tracked the localization of GFP-tagged L25, a well-established 60S subunit reporter (64). Importantly, L25 is successfully assembled into pre-ribosomes in the absence of L17 or L37 (Figure 9C), and thus is a valid reporter in these mutants. As expected for an r-protein, we observed cytoplasmic localization of L25-eGFP in wild-type cells grown in galactose-containing medium (Figure 6A). However, GAL-HA-RPL17 and pGAL- RPL37 cells shifted from galactose- to glucose-containing medium exhibited increased intensity of L25-eGFP fluorescence, but not the 40S reporter S3-eGFP (Figure 6A). L25-eGFP signal in either L17- or L37-depleted cells largely colocalizes with the nucleolar Nop1-mRFP (53) (Figure 6B). This indicates the failure to form pre-60S ribosomal particles competent for transit to the nucleoplasm and subsequent nuclear export and maturation in the cytoplasm. Consistent with this, it has been reported that assembling pre-60S complexes are released to the nucleoplasm upon completion of 27SB pre-rRNA processing (53). We conclude that upon depletion of either L17 or L37, the intranuclear transport of pre-60S complexes from the nucleolus to the nucleoplasm is blocked. Moreover, particles that are released from the nucleolus are not exported to the cytoplasm. Two possible scenarios might account for the latter observation. First, in the absence of L17 or L37, 66S pre-rRNPs might traffic to the nucleoplasm but are possibly not in a conformation competent for export through the nuclear pore. Another more specific scenario Figure 5. Depletion of L17 or L37 leads to a defect in processing of 27SB pre-rRNA and its eventual turnover. (A) Synthesis and turnover of pre-rRNAs were assayed by pulse-chase experiments. GAL-HA-RPL17 and pGAL-RPL37 cells were grown in galactose- or glucose-containing medium lacking methionine for 16–17 h. Cells were pulse-labeled with [ 3 H-methyl]-methionine for 5 min and chased with an excess of nonradioactive methionine. Total RNA was extracted, and equal numbers of radioactive counts were loaded onto denaturing agarose gels, transferred to nitrocellulose membranes, and exposed to X-ray films. (B) Total RNA was extracted from GAL-HA-RPL17 and pGAL-RPL37 strains grown in galactose or shifted to glucose for 16–17 h at 30C. Samples were subjected to primer extension analysis of 50-ends of 27S and 7S pre-rRNAs. The asterisk indicates 50-truncated pre-rRNAs formed specifically in the absence of L17. (C) Total RNA was extracted as described earlier, and 7S pre-rRNAs were assayed by northern hybridization. U2 was probed to serve as a loading control. 1972 Nucleic Acids Research, 2013, Vol. 41, No. 3 is that L17 and L37 serve as platforms for the export machinery; hence in their absence, export is impaired. Indeed, some pre-60S export factors localize in close proximity to the polypeptide exit tunnel (65–67). Recently, cryo-EM analyses revealed that the export factor Arx1 interacts directly with r-proteins in this region, including L19, L25, L26 and L35 (68). L17 and L37 stably assemble into 27SB-containing pre-60S complexes in the nucleolus The timing of assembly of r-proteins with nascent ribosomes has been broadly categorized into early and late maturation steps by pulse-labeling experiments (69). L17 and L37 were among those r-proteins that associate early with assembling ribosomes. We took orthogonal approaches to investigate in further detail the timing of incorporation of L17 and L37 into pre-60S particles: (i) determining the localization of the GFP-tagged r-proteins in an NMD3100 dominant negative mutant defective for nucleocytoplasmic export of pre-60S ribosomal particles (58,70), (ii) assaying pre-rRNA intermediates that copurify with GFP-tagged L17 and L37 r-proteins by northern hybridization. Figure 6. Depletion of L17 and L37 results in retention of pre-ribosomes in the nucleus. (A)GAL-HA-RPL17 and pGAL-RPL37 cells expressing either the 60S subunit reporter L25-eGFP or the 40S subunit reporter S3-eGFP were grown in galactose- or shifted to glucose-containing selective medium to an OD 610 of 0.4–0.6. Localization of GFP signal in wild type and mutant cells was detected using a Carl Zeiss LSM- 510_META_UV_DuoScan inverted spectral confocal microscope. (B) Colocalization of L25-eGFP with the nucleolar marker Nop1-mRFP in GAL-HA-RPL17 and pGAL-RPL37 cells grown in galactose-containing media and shifted to glucose-containing media for 16 h. Nucleic Acids Research, 2013, Vol. 41, No. 3 1973 L35 and L37 conditional mutants that prevent 27SB pre-rRNA processing. Moreover, identifying where B factors interact with pre-rRNAs or pre-rRNPs would enable us to further understand how r-proteins together with cis- and trans-acting factors work together to enable processing of 27SB pre-rRNA at ITS2. To fully comprehend this processing step, it is also a big challenge to identify the elusive endonuclease for C 2 cleavage or to determine whether this cleavage could be a result of autocatalysis. The rate of turnover of pre-ribosomes in the absence of L17, L35 or L37 is slower than in r-protein mutants affecting earlier steps in pre-rRNA processing Previously, our analysis of L7 and L8 mutants, which affect an early step of pre-rRNA processing, showed substantially rapid turnover of pre-rRNAs (39). In contrast, L17, L35 or L37 mutants exhibit more delayed turnover of unprocessed 27SB pre-rRNA (31) (Figure 5A). These defective pre-rRNA intermediates are presumably targeted for degradation by a surveillance machinery (106–108). At this point, it is not clear how pre-ribosomal particles are detected as being aberrant and what events trigger turnover in assembly mutants. Nevertheless, the general picture that emerges is that gross misfolding of rRNA may lead to a more rapid turnover of assembly intermediates, as seen in the L7 and L8 mutants. Pre-ribosome composition is affected to a far greater extent in the L7 or L8 mutants than in the L17, L35 or L37 mutants. When L7 or L8 are depleted from pre-ribosomes, the association of several proteins is affected: early- and late-assembling B factors, including Nsa2 and Nog2; A 3 factors including Nop15 and Cic1 that bind ITS2, and Erb1 and Nop7 that bind 25S rRNA domains I and III, respectively; r-proteins L17, L26, L35 and L37 that primarily bind 5.8S/25S rRNA domain I, in addition to other rRNA domains; and a number of r-proteins adjacent to L7 and L8 that that bind to 25S domain II and I, respectively (39). This suggests that L7 and L8 are necessary to establish the structure of several rRNA neighborhoods, nearby or farther away. The unstructured assembly intermediate in the absence of L7 or L8 may signal instantaneous turnover of pre-rRNAs, via the exosome or the exonuclease Rat1, which is present in preribosomes of L7 or L8 mutants (39). In stark contrast, upon depletion of L17, L35 or L37, thus far only Nsa2 and Nog2 are observed to be decreased in pre-ribosomes, whereas all the other r-proteins and assembly factors that fail to associate with pre-ribosomes in the absence of either L7 or L8 are still present. Strikingly, even adjacent r-proteins in the neighborhood surrounding the polypeptide exit tunnel are unaffected in L17, L35 and L37 mutants. This suggests that fewer pre-rRNA regions may be misfolded, accounting for the slower turnover of aberrant assembly intermediates in L17, L35 or L37 mutants. In this case, the unprocessed ITS2 might be the target for turnover by the exosome. Additionally, the A 3 factor Cic1, a known proteasome adaptor (109), which remains associated with ITS2 of unprocessed 27SB intermediates in the absence of L17, L35 or L37, might also signal turnover. Hence, correct timing of the removal of ITS2 might yet be another quality control checkpoint to ensure that aberrant 27SB- containing pre-rRNPs are eliminated. SUPPLEMENTARY DATA Supplementary Data are available at NAR online: Supplementary Table 1, Supplementary Figures 1–7, Supplementary Method and Supplementary References [111–113]. ACKNOWLEDGEMENTS The authors are grateful to the members of the Woolford and de la Cruz laboratories for stimulating discussions and critical reading of the manuscript. They are also thankful for the generous gifts of antibodies from Drs. M. Fromont-Racine, F. Lacroute, P. Linder, D. Goldfarb, J. Maddock, M. McAlear, E. Tosta, J. Brodsky, J. Warner, L. Lindahl, A. Johnson, S. Rospert and K. Siegers. They thank the following people for generously providing strains and plasmids: P. Milkereit, E. Hurt, M. N. Hall and A. Jacobson. They also thank H. Teng for confocal microscopy training and the Molecular Biosensor and Imaging Center at Carnegie Mellon for use of their confocal microscope. FUNDING National Science Foundation [MCB 0818534 to J.L.W.]; the Spanish Ministry of Science and Innovation and European Regional Development Fund [BFU2010- 15690]; the Andalusian Government [CVI-271, and P08-CVI-03508]. Funding for open access charge: NSF [MCB 0818534]. J.T. is a recipient of the Richard King Mellon Foundation Presidential Graduate Fellowship in the Life Sciences and the Semon H. Stupakoff Scholarship. Conflict of interest statement. None declared. REFERENCES 1. Henras,A.K., Soudet,J., Gerus,M., Lebaron,S., Caizergues- Ferrer,M., Mougin,A. and Henry,Y. (2008) The post-transcriptional steps of eukaryotic ribosome biogenesis. Cell. Mol. Life Sci.,65, 2334–2359. 2. Woodson,S.A. (2008) RNA folding and ribosome assembly. Curr. Opin. Chem. Biol.,12, 667–673. 3. Staley,J.P. and Woolford,J.L. 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