A yeast homolog of chromatin assembly factor 1 is involved in early ribosome assembly

Brief Communication 1885

A yeast homolog of chromatin assembly factor 1 is involved in early ribosome assembly Sigrid Schaper*¶, Micheline Fromont-Racine†, Patrick Linder‡, Jesu´s de la Cruz‡, Abdelkader Namane§ and Moshe Yaniv* Cells have a recurrent need for the correct assembly of protein-nucleic acid complexes. We have studied a yeast homolog of the smallest subunit of chromatin assembly factor 1 (CAF1), encoded by YMR131c and termed “RRB1” [1]. Unlike other yeast homologs, Msi1p, and Hat2p, Rrb1p is essential for cell viability. Impairment of Rrb1p function results in decreased levels of free 60S ribosomal subunits and the appearance of half-mer polysomes, suggesting its involvement in ribosome biogenesis. Using tandem affinity purification (TAP [2]) combined with mass spectrometry, we show that Rrb1p is associated with ribosomal protein L3. A fraction of Rrb1p is also found in a protein-precursor rRNA complex containing at least ten other earlyassembling ribosomal proteins. We propose that Rrb1p is required for proper assembly of preribosomal particles during early ribosome biogenesis, presumably by targeting L3 onto the 35S precursor rRNA. This action may resemble the mechanism by which CAF1 assembles histones H3/H4 onto newly replicated DNA. Addresses: *Unite´ Virus Oncoge`nes, URA 1644 du CNRS, De´partement des Biotechnologies, Institut Pasteur, 25, Rue du Docteur Roux, 75724 Paris cedex 15, France. † Unite´ Ge´ne´tique des Interactions Macromole´culaires, De´partement des Biotechnologies, Institut Pasteur, 25, Rue du Docteur Roux, 75724 Paris cedex 15, France. ‡ De´partement de Biochimie Me´dicale, Centre Me´dical Universitaire, Universite´ de Gene`ve, 1211 Gene`ve 4, Switzerland. § Laboratoire de Chimie Structurale des Macromole ´ cules, URA 2185 du CNRS, De´partement Biochimie et Ge´ne´tique Mole´culaire, Institut Pasteur, 28, Rue du Docteur Roux, 75724 Paris cedex 15, France. Present address: ¶ Otto-Warburg-Laboratories, Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, D-14195 Berlin, Germany. Correspondence: Sigrid Schaper E-mail: [email protected] Received: 30 August 2001 Revised: 12 October 2001 Accepted: 15 October 2001

Results and discussion Early-associating ribosomal proteins copurify with Rrb1p

RRB1 (YMR131c) encodes a protein from the WD40 protein family, containing at least three Trp-Asp repeats in its C-terminal region. WD40 repeats are short structural motifs which represent a protein-protein interaction interface in this functionally diverse family of proteins [3]. In order to identify proteins interacting with Rrb1p, we introduced the TAP tag in-frame at the 3⬘ end of the RRB1 gene and purified the Rrb1p complex according to the TAP protocol [2]. This approach constitutes a convenient method to purify native protein complexes. The resulting RRB1-TAP strain displayed a thermosensitive and reversible phenotype; growth was slightly reduced at 25⬚C and severely reduced at 37⬚C compared to wild-type (data not shown). Separation of proteins by SDS-polyacrylamide gel electrophoresis and mass spectrometry analysis of individual bands revealed that the purified fraction contained two major polypeptides, one of them corresponding to the tagged Rrb1p, the other to the largest ribosomal protein L3 (Figure 1). This is in good agreement with recently published coimmunoprecipitation experiments that showed an association between these two proteins [1]. In addition, our Rrb1-TAP purification revealed reproducibly several substoichiometrically copurifying bands, corresponding to ribosomal proteins L4, L7, L8, L13, and S1, S3, S4, S11, S17, S24 (Figure 1a). Several r proteins associate with the nucleolar preribosomes at early steps during ribosome maturation, whereas others are assembled in the nucleus at later steps or are even added only in the cytoplasm [4, 5]. The 35S pre-rRNA associates with r proteins to form the nucleolar 90S preribosomal particle, from which the 66S and 43S preribosomes are formed [6, 7]. Interestingly, several of the ribosomal proteins detected in the Rrb1-TAP complex are suggested to associate early with the 35S precursor rRNA: L3, L4, L8, S3, S4, and S11 [5–7]. With exception of L7, which is attributed to the 66S preribosomal particle in the nucleus, the purified Rrb1-complex does not contain late-assembling r proteins, neither nuclear nor cytoplasmic. Consistent with that, we failed to detect the tagged Rrb1 in mature ribosomes or polysomes (see below).

Published: 27 November 2001 Current Biology 2001, 11:1885–1890 0960-9822/01/$ – see front matter  2001 Elsevier Science Ltd. All rights reserved.

Two additional nonribosomal proteins were identified: Hsh49p and an uncharacterized protein encoded by YFL046w. Hsh49p is a component of the splicing factor 3b (SF3b) complex associated with the U2 snRNP [8]. Since we did not detect any other component of the SF3b complex, Hsh49p may be a contaminant of the Rrb1-TAP

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Figure 1

complex due to its general RNA binding capacity [9]. Finally, some of the bands were degradation products of Rrb1p and L3 (*, Figure 1a). The substoichiometric appearance of ribosomal proteins in the Rrb1-TAP complex prompted us to test whether their copurification was dependent on the presence of RNA. Extracts from the RRB1-TAP strain were treated in parallel, either with a ribonuclease inhibitor or with 100 ␮g/ml ribonuclease A and were separately affinity purified. All of the substoichiometrically copurifying protein bands (except Rrb1p and L3 degradation products,*) were eliminated upon RNase A treatment (see arrowheads, Figure 1b), whereas L3 remained tightly associated with Rrb1p. Hence, it seems probable that Rrb1p and L3 form a binary complex that interacts with RNA which is bound by other early-assembling ribosomal proteins. An early precursor rRNA copurifies with TAP-tagged Rrb1p

L3 and other early-associating ribosomal proteins copurify with Rrb1-TAP. (a) The purified Rrb1-TAP complex was separated on a 16% SDS-polyacrylamide gel and subjected to a mass spectrometry analysis after Coomassie Blue staining. Identified proteins are indicated on the left. Unlabeled bands were nonidentifiable by mass spectrometry. Note that the designations A or B corresponding to the two gene copies for the r proteins L4, L7, L8, and S1 were omitted here because mass spectrometry analysis did not distinguish between the two gene products. Bands corresponding to degradation products of Rrb1p (*) or ribosomal protein L3 (**) are labeled with asterisks. (b) Two protein extracts containing the Rrb1-TAP complex were treated with ribonuclease inhibitor or ribonuclease A, respectively, and Rrb1-TAP complexes were purified as before. Complexes were separated on 16% SDS-polyacrylamide gels and silver stained. Arrowheads indicate the bands of the substoichiometrically copurifying ribosomal proteins which “disappear” upon the ribonuclease A treatment. A single asterisk

Evidence for the presence of RNA in the purified Rrb1TAP complex suggested that the copurifying RNA species could be a precursor rRNA. To test this possibility, we performed reverse transcriptase (RT)-PCR on the TAPpurified Rrb1p complex. Three sets of primers were used to amplify different regions of the 35S pre-rRNA transcript (see scheme, Figure 2a). First, we investigated whether a putative precursor rRNA was detectable in the final eluate of the purified Rrb1p complex. For this purpose, primer number 9 was used for the first-strand synthesis, followed by PCR amplification of the region covering the internal spacers ITS1 and ITS2 and the 5.8S rRNA of the precursor rRNA (using primers number 7 and number 10*). This primer combination would concomitantly allow for the distinction between early and later forms of precursor rRNA. A PCR product of the expected size (681 bp) was visible in the reaction on purified Rrb1-TAP complex but not in control reactions on the Sas5-TAP complex (a complex involved in silencing; S.S., unpublished data) or reactions without RT (Figure 2c). To discriminate between the three early precursor forms of the pre-rRNA (35S, 33S, or 32S; see Figure 2b), primer number 12 was used for the first-strand synthesis, followed by PCR amplifications of the region covering the A0 cleavage site of the 5⬘ETS and part of the 18S rRNA (primers number 1* and number 11) or the region including the A1 cleavage site and part of the 18S rRNA (primers number 13* and number 11), respectively. PCR products of the expected size were generated in the Rrb1TAP reaction, using primers 13* and 11 (527 bp) and primers 1* and 11 (658 bp) (Figure 2c), but not in the control reactions. These results indicate that the Rrb1p-

indicates degradation products of Rrb1p. The band labeled with two asterisks is a degradation product of L3, possibly due to a protease contamination of ribonuclease A.

Brief Communication 1887

Figure 2 An early precursor rRNA copurifies with the Rrb1-TAP complex. (a) Structure of the 35S pre-rRNA and processing sites (adapted from [5]). This precursor contains the sequences for the mature 18S, 5.8S, and 25S rRNAs that are separated by the two internal transcribed spacers ITS1 and ITS2 and flanked by two external transcribed spacers 5⬘ETS and 3⬘ETS. Bars represent mature rRNA species; lines represent the transcribed spacers. The primers used are labeled with their corresponding numbers. Numbered primers with an asterisk indicate that the primers have the reverse orientation. (b) The first two processing steps of the 35S pre-rRNA at the A0 and the A1 cleavage sites (see [5] for a complete scheme). (c) RT reaction and PCR. Purified Rrb1-TAP complex (2 ␮l) was used to amplify different regions within the precursor rRNA. Purified Sas5-TAP complex (2 ␮l) or 2.5 ␮l (2.5 ␮g) total extracted yeast RNA (Rt) was used in the control reactions. The multiple slower migrating bands in the reactions on total RNA are probably due to nonspecific annealing of the primers. PCR products were resolved on a 1% agarose gel and visualized by ethidium bromide staining. The bands corresponding to products from PCR on genomic DNA (D), using the same sets of primers, are labeled at either side of the gel. The diverse sets of primers used are indicated on top of the panel and are described in the text.

L3 complex contains a precursor rRNA which corresponds to the 35S form.

Impaired Rrb1p function results in a 60S ribosomal subunit deficiency

To further confirm the presence of the 35S precursor rRNA, we used a primer hybridization assay with primers annealing specifically in the 35S, 33S, and matured forms of precursor rRNA. RNA was extracted from the final Rrb1-TAP and Sas5-TAP eluates or from an Rrb1-TAP preparation that was subjected to RNase A treatment during the course of TAP-purification (see above) and spotted onto a nylon membrane. Serially diluted total yeast RNA was applied for comparison. The membrane was hybridized successively with primers specific for the 35S pre-rRNA (number 15), 33S pre-rRNA (number 14), and the 25S rRNA (number 9) (Figure 3). All three hybridizations gave a positive signal of comparable intensity, indicating that the 35S precursor form was copurifying with the Rrb1-TAP complex.

To investigate further Rrb1p involvement in ribosome biogenesis, we performed polysome profile analyses with the thermosensitive rrb1-TAP strain or the normal growing RRB1-HA strain (Figure 4). rrb1-TAP, RRB1-HA, or the isogenic wild-type strains were grown at 30⬚C, and derived extracts were resolved in sucrose gradients. The rrb1-TAP strain showed a deficit of 60S versus 40S ribosomal subunits, an overall decrease in 80S ribosomes (free couples and monosomes) and polysomes, and an accumulation of half-mer polysomes (Figure 4b), whereas the RRB1-HA strain showed a wild-type profile (Figure 4c). Half-mer polysomes generally correspond to a 43S complex, consisting of a 40S ribosomal subunit with attached initiation factors, being stalled at the first start codon and waiting for the addition of a 60S ribosomal subunit [10]. The appearance of half-mer polysomes in combination with a net deficit of free 60S versus 40S ribosomal subunits has

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Figure 3

Primer hybridization assay. RNA was extracted from the final Rrb1TAP eluate or from a parallel preparation that was subjected to RNase A treatment during the course of TAP-purification or from a Sas5-TAP purification, heated briefly, and spotted (2 ␮l) onto a nylon membrane. For comparison, 2 ␮l total extracted RNA corresponding to the amounts indicated were spotted on the same membrane. Hybridizations were performed for 1 hr at 42⬚C, using successively 32 P-labeled oligonucleotides number 15, number 14, or number 9 (see scheme, Figure 2a).

been described for mutants defective in the 60S r proteins L3 (D. Kressler and P.L., unpublished data), L5 [11], and L16 [12], and for mutant genes defective in pre-rRNA processing and 60S ribosomal subunit assembly (see [5, 13] and references therein). Western blot analysis of the gradient fractions revealed that Rrb1-HA was very abundant in the top (low molecular weight) fractions and with lower concentration in the 40S to 80S fractions but was not detectable in the polysome fractions; L3 was detected in fractions corresponding to 60S subunits, mature 80S ribosomes, and polysomes (Figure 4c). Rrb1p was recently shown to localize throughout the nucleus with enrichment in the nucleolus [1], supporting that Rrp1p is not associated with the mature ribosome. In the Western blot of the Rrb1-TAP polysome profile, Rrb1-TAP was found in the top five to six fractions (Figure 4b). Unexpectedly, L3 was detectable only at the top of the gradient (fractions 1 through 4, Figure 4b). Only little amounts of L3 protein were detectable in this polysome profile, probably due to an inefficient trichloroacetic acid precipitation and degrada-

tion of L3 when nonincorporated into ribosomes. We believe that the partially defective Rrb1-TAP strain provides an experimental advantage to enrich the Rrb1-L3 assembly intermediate and to “freeze” the ribosomal assembly process at a very early stage, presumably before completion of the assembly of the 90S precursor particle. Both, Rrb1p and L3 sedimented here as low molecular weight material, further supporting the existence of a free Rrb1-L3 complex. We propose that Rrb1p forms a direct complex with L3 and assists its interaction with the rRNA precursor. The ten substoichiometrically copurifying r proteins may represent an early Rrb1p-associated assembly intermediate (on the way toward the 90S preribosome) that is less abundant in the cell. We regard these copurifying r proteins as specific for the Rrb1/L3-associated pre-rRNA, since no cytoplasmic-assembling r protein was detectable in the purified Rrb1-TAP complex(es). In one case, the ribosomal protein L4 is reported to interact specifically only with 35S pre-rRNA and not with mature 25S rRNA in vitro [14]. We postulate that the isolated subset of r proteins represent very early-assembling r proteins, characteristic of an early form of the preribosomal particle. This subset includes L13, S1, S17, and S24, four r proteins which were previously not assigned to early or later forms of ribosome assembly. In conclusion, our results indicate that, in addition to the previously reported involvement of Rrb1p in regulation of L3 expression and stability [1], Rrb1p plays a direct role in early 60S ribosomal subunit assembly. We propose that yeast Rrb1p functions as a chaperone for assembly of ribosomal protein L3 onto the precursor rRNA transcript. The specificity of Rrb1p for ribosomal protein L3 raises the possibility that all ribosomal proteins are targeted onto precursor rRNA in an ordered and highly specific manner by different trans-acting assembly factors. Although the pre-rRNA processing pathway and its intermediates have been fairly well elucidated, the order and process by which the rRNAs and the r proteins are assembled into mature ribosomal subunits is still poorly understood. A low-resolution picture of yeast ribosome assembly was achieved by studying in vivo the incorporation of individual r proteins [4] and by assessing the order of dissociation in vitro through increasingly stringent washes [15, 16]. Currently available data are, however, far from being sufficient to create a definitive assembly pathway for the different ribosomal proteins. Understanding the mechanism of ribosome assembly on the molecular level and identifying the functional specificity of ribosomal transacting assembly factors are important goals for the future.

Materials and methods Purification of the Rrb1-TAP complex A 6 liter yeast culture was grown in SC-Trp medium at 25⬚C to an optical density at 600 nm (OD600) of 2.0. Cells were harvested by centrifugation and broken using a French-press (P ⱕ 1100 Pa). All subsequent steps were performed exactly as described ([2]; http://www.embl-heidelberg. de/ExternalInfo/seraphin/TAP.html). Before loading the eluted fractions

Brief Communication 1889

Figure 4 The temperature-sensitive rrb1-TAP strain exhibits a deficit in free 60S ribosomal subunits and accumulation of half-mer polysomes. (a) The wild-type strain MGD353-13D and (b) the rrb1-TAP strain were grown at 30⬚C. Cells were harvested at an OD600 of 0.8, and cell extracts were resolved in 7%–50% (w/v) sucrose gradients. Gradients were analyzed by continuous monitoring at A254. Sedimentation is from left to right. The peaks of free 40S and 60S ribosomal subunits, 80S free couples/ monosomes, and polysomes are indicated. Half-mers are labeled with asterisks. (c) Analysis of the sedimentation of Rrb1-TAP in the sucrose gradient shown in (b). Collected fractions were precipitated as described [21], resolved on a 15% SDS polyacrylamide gel, and immunoblotted using PAP-soluble antibody complex. The Rrb1-TAP signal and the positions of the 40S, 60S, and 80S ribosomal particles are indicated.

of the second affinity chromatography step onto a 16% SDS-polyacrylamide gel, samples were precipitated with five volumes of ice-cold acetone and resuspended in loading buffer. Proteins bands were visualized by Coomassie staining, cut out, and processed for mass spectrometry analysis (see Supplementary material).

at 42⬚C, using successively 32P-labeled oligonucleotides number 15 (5⬘TCGGGTCTCTCTGCTGC-3⬘), number 14 (5⬘-GTTTGGAAACAGCTG AAATTCC-3⬘), or number 9 (see above). The membrane was washed for 15 min at 95⬚C in 0.2% SDS in-between individual hybridizations to remove efficiently the previous probe.

For the purification of the complex in presence or absence of RNA, a protein extract was prepared from a 6 liters culture and split in two halves. One of the extracts was incubated with 100 ␮g/ml Ribonuclease A (Boehringer) for 1 hr at 4⬚C, followed by the dialysis step for 3 hr as described [2]. The other half was treated with 0.67 U/␮l RNasin Ribonuclease inhibitor (Promega). Following affinity purification, steps were carried out as described above. Complexes were separated on 16% SDSpolyacrylamide gels and protein bands were revealed by silver staining.

Polysome analysis

RT-PCR and primer hybridization The presence of a precursor rRNA in the purified Rrb1-TAP complex was detected by RT-PCR using the following primers: number 9 (5⬘CCTCCGCTTATTGATATGC-3⬘) or number 12 (5⬘-CCCAAAGTTCAA CTACGAGC-3⬘), respectively, for the first-strand synthesis, and number 1* (5⬘-CCGGCAGCAGAGAGACC-3⬘), number 7 (5⬘-GGCCAGCAAT TTCAAGTTAA-3⬘), number 10* (5⬘-CTCCATCTCAGAGCGGAG-3⬘), number 11 (5⬘-GACCCGAATGGGCCCTG-3⬘), and number 13* (5⬘GCTTGTTGCTTCTTCTTTTAAG-3⬘) for the diverse amplification reactions. The final eluate (2 ␮l) of purified Rrb1-TAP complex or, as controls, 2 ␮l of purified Sas5-TAP complex or 2.5 ␮l (2.5 ␮g) of total RNA were used as templates for the first-strand synthesis. For the RT reaction and PCR amplification with primers numbers 1* and 11, it was necessary to deproteinize the eluted Rrb1-TAP RNA by phenol/chloroform/isoamyl alcohol (25:24:1) extraction and to heat the RNA to 90⬚C for 1 min prior to the RT reaction. Reverse Transcriptase Superscript II was purchased from GibcoBRL. For primer hybridizations, RNA was extracted from the final Rrb1-TAP eluate, resuspended in 10 ␮l, heated to 90⬚C for 1 min, and quick chilled on ice. This preparation (2 ␮l) or a parallel preparation that was subjected to RNase A treatment during the course of TAPpurification or extracted RNA from a Sas5-TAP purification were spotted onto a Hybond-N⫹ membrane (Amersham) and UV crosslinked. For comparison, 2 ␮l total extracted RNA corresponding to the amounts indicated were spotted on the same membrane. Prehybridization and hybridizations were performed in Rapid-hyb buffer (Amersham) for 1 hr

Polyribosome preparation and polysome analysis were done as described previously [17, 18]. Gradient analysis was performed with an ISCO UV-6 gradient collector with continous monitoring at A254. For Western analysis, proteins in each fraction were precipitated with 10% trichloroacetic acid exactly as described [18]. Presence of Rrb1-TAP protein in the fractions was detected by immunoblotting using PAPsoluble complex (Sigma) and the ECL detection system (Amersham). Rrb1-HA was detected with a rat monoclonal antibody (clone 3F10, Boehringer) and L3 with a mouse monoclonal antibody (a gift from J.R. Warner).

Supplementary material A discussion of the Rrb1p protein homology with known chromatin assembly factors and with uncharactrized ORFs from different species can be found with this article online at http://images.cellpress.com/ supmat/supmatin.htm.

Acknowledgements We thank B. Se´raphin for ingredients and comprehensive information on the TAP procedure. We are grateful to B. Dujon and his group for gifts of strains; W. Zachariae for the plasmid pWZV89; J.R. Warner for the anti-L3 antibody; and J.-C. Rousselle for help with mass spectrometry. We thank our colleagues A. Klochendler-Yeivin, B. Bourachot, and C. Saveanu for many helpful discussions regarding diverse aspects of this study; and J. Weitzman for critically reading the manuscript. S.S. acknowledges a long-term fellowship from the Human Frontier Science Program. This work was also supported by the Swiss National Science Foundation and the Ministe`re de l’Education Nationale, de la Recherche et de la Technologie. Initial parts of this study were also supported by the Deutsche Forschungsgemeinschaft.

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