Nucleolin functions in the first step of ribosomal RNA processing
1998; Springer Nature; Volume: 17; Issue: 5 Linguagem: Inglês
10.1093/emboj/17.5.1476
ISSN1460-2075
Autores Tópico(s)RNA Research and Splicing
ResumoArticle2 March 1998free access Nucleolin functions in the first step of ribosomal RNA processing Hervé Ginisty Hervé Ginisty Laboratoire de Biologie Moléculaire Eucaryote, Institut de Biologie Cellulaire et de Génétique du CNRS, UPR 9006, 118 route de Narbonne, 31062 Toulouse, Cedex, France Search for more papers by this author François Amalric François Amalric Laboratoire de Biologie Moléculaire Eucaryote, Institut de Biologie Cellulaire et de Génétique du CNRS, UPR 9006, 118 route de Narbonne, 31062 Toulouse, Cedex, France Search for more papers by this author Philippe Bouvet Corresponding Author Philippe Bouvet Laboratoire de Biologie Moléculaire Eucaryote, Institut de Biologie Cellulaire et de Génétique du CNRS, UPR 9006, 118 route de Narbonne, 31062 Toulouse, Cedex, France Search for more papers by this author Hervé Ginisty Hervé Ginisty Laboratoire de Biologie Moléculaire Eucaryote, Institut de Biologie Cellulaire et de Génétique du CNRS, UPR 9006, 118 route de Narbonne, 31062 Toulouse, Cedex, France Search for more papers by this author François Amalric François Amalric Laboratoire de Biologie Moléculaire Eucaryote, Institut de Biologie Cellulaire et de Génétique du CNRS, UPR 9006, 118 route de Narbonne, 31062 Toulouse, Cedex, France Search for more papers by this author Philippe Bouvet Corresponding Author Philippe Bouvet Laboratoire de Biologie Moléculaire Eucaryote, Institut de Biologie Cellulaire et de Génétique du CNRS, UPR 9006, 118 route de Narbonne, 31062 Toulouse, Cedex, France Search for more papers by this author Author Information Hervé Ginisty1, François Amalric1 and Philippe Bouvet 1 1Laboratoire de Biologie Moléculaire Eucaryote, Institut de Biologie Cellulaire et de Génétique du CNRS, UPR 9006, 118 route de Narbonne, 31062 Toulouse, Cedex, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1476-1486https://doi.org/10.1093/emboj/17.5.1476 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The first processing step of precursor ribosomal RNA (pre-rRNA) involves a cleavage within the 5′ external transcribed spacer. This processing requires sequences downstream of the cleavage site which are perfectly conserved among human, mouse and Xenopus and also several small nucleolar RNAs (snoRNAs): U3, U14, U17 and E3. In this study, we show that nucleolin, one of the major RNA-binding proteins of the nucleolus, is involved in the early cleavage of pre-rRNA. Nucleolin interacts with the pre-rRNA substrate, and we demonstrate that this interaction is required for the processing reaction in vitro. Furthermore, we show that nucleolin interacts with the U3 snoRNP. Increased levels of nucleolin, in the presence of the U3 snoRNA, activate the processing activity of a S100 cell extract. Our results suggest that the interaction of nucleolin with the pre-rRNA substrate might be a limiting step in the primary processing reaction. Nucleolin is the first identified metazoan proteinaceous factor that interacts directly with the rRNA substrate and that is required for the processing reaction. Potential roles for nucleolin in the primary processing reaction and in ribosome biogenesis are discussed. Introduction Ribosome biogenesis is a complex process which involves the transcription of a large rRNA precursor, its maturation and assembly with ribosomal proteins (Hadjiolov, 1985; Eichler and Craig, 1994). Biogenesis of large amounts of ribosomes that are needed throughout the life of a cell requires an efficient coordination of different steps which take place in the nucleolus. In mammalian cells, rRNA is transcribed as a large precursor of 47S which undergoes multiple post-transcriptional nucleotide modifications (Maden, 1990) and nucleolytic processing steps to yield the mature 18S, 5.8S and 28S rRNA species (Eichler and Craig, 1994). Two classes of processing events can be distinguished. In the first, the nucleolytic cleavages lead to the formation of the 5′ and 3′ extremities of the mature rRNA species. The second type of cleavages take place within the 5′ and 3′ external transcribed spacers (ETSs). After completion of rRNA transcription, a rapid processing occurs within the 5′ ETS of rRNA and it is followed by cleavages within the 3′ ETS (Miller and Sollner-Webb, 1981; Gurney, 1985). The first processing step within the 5′ ETS, called the early or primary cleavage of pre-rRNA, has been the most studied. It occurs so fast that, for some time, it was believed to represent the transcription initiation site (Urano et al., 1980; Bach et al., 1981; Miller and Sollner-Webb, 1981). Although this processing does not lead directly to the formation of a mature rRNA species, it is well conserved in several species and can occur at various positions within the 5′ ETS: +650/+657 in mouse (Miller and Sollner-Webb 1981), +414/+419 in human (Kass et al., 1987), +105 in Xenopus laevis (Mougey et al., 1993a), +1700 in Physarum polycephalum (Blum et al., 1986) and +609 in Saccharomyces cerevisiae (Hugues and Ares, 1991). Despite the conservation of this early processing event, its role in ribosome biogenesis is still unknown. One interesting aspect of this cleavage reaction is that it can be reproduced accurately with an in vitro transcribed RNA and a cell extract. Deletional analyses on the mouse pre-rRNA have shown that a minimal RNA which contains five nucleotides upstream and 200 nucleotides downstream of the cleavage site can be processed efficiently (Craig et al., 1987, 1991). Further deletions from the 3′ end of this RNA result in a progressive decrease of the cleavage efficiency (Craig et al., 1987, 1991). Although the ETS nucleotide sequences have greatly diverged, this 200 nucleotide segment downstream of the first cleavage site is 80% conserved between mouse and human (Miller and Sollner-Webb, 1981; Miesfeld and Arnheim, 1982; Kass et al., 1987) and can fold in a large stem–loop structure (Michot and Bachellerie, 1991). Even more strikingly, an 11 nucleotide sequence located at nt +658/+668 in the mouse pre-rRNA is perfectly conserved in mouse, human, Xenopus borealis and X.laevis (with one mismatch) (Kass et al., 1987). Deletion of these conserved residues abolished the processing reaction, suggesting that they play an as yet undetermined but important role (Kass et al., 1987). While a X.laevis pre-rRNA substrate is cleaved efficiently in a mouse cell extract, the converse is not true (Mougey et al., 1993a). This suggests that some factors have been conserved between these two species that might be involved in the recognition of this conserved nucleotide sequence. Unlike in yeast rRNA processing, for which numerous trans-acting factors have been identified by genetic analysis (Maxwell and Fournier, 1995; Tollervey, 1996), much less is known about the processing of higher eukaryotic pre-rRNAs. Processing-competent RNAs assemble in a large complex characterized by a sedimentation coefficient of ∼20S (Kass and Sollner-Webb, 1990; Mougey et al., 1993a). UV cross-linking experiments identified a number of proteins whose identity and implication in the cleavage reaction remain to be determined. Elegant experiments in X.laevis oocytes demonstrated that formation of a structure observed by electron microscopy at the terminal ends of the ribosomal transcripts (terminal balls) observed on Miller's Christmas trees (Miller and Beatty, 1969) was correlated directly to the presence of sequences required for the primary processing activity (Mougey et al., 1993b). This suggests that these terminal balls might represent the primary processing complexes. Since these structures have been observed in all tissues and organisms examined, this further suggests that the primary processing, or the formation of this complex at the 5′ ETS, serves an important function. Micrococcal nuclease treatment of a processing-competent extract abolishes the cleavage reaction, suggesting that, in addition to proteins, the reaction requires a nucleic acid component (Kass et al., 1990). Several small nucleolar ribonucleoparticles (snoRNPs) are known to be involved in this reaction (Kass et al., 1990; Enright et al., 1996). In particular, the U3 small nucleolar RNA (snoRNA), one of the most abundant snoRNAs, has been found associated with both the pre-rRNA substrate and the cleavage product (Kass et al., 1990). A U3-depleted extract processes an rRNA substrate inefficiently (Kass et al., 1990; Mougey et al., 1993a; Enright et al., 1996), demonstrating that this snoRNA is important for the primary processing. Depletion of other snoRNAs (U14, U17 and E3) also significantly reduces the in vitro 5′ ETS processing (Enright et al., 1996). The role of these snoRNAs in the processing reaction is still not understood. The observation that an almost complete depletion of U3 (98%) followed by the depletion of several other snoRNAs does not lower the primary processing activity below a basal level (Kass et al., 1990; Enright et al., 1996) suggests that these snoRNAs could have a stimulatory but not essential role in the processing. To understand this function better, it would be particularly interesting to know how U3 and other snoRNAs implicated in the processing reaction interact with the pre-rRNA, since no evolutionarily conserved complementarity exists between these snoRNAs and the rRNA. Here we have identified nucleolin as one of the cross-linked proteins which interacts with the RNA substrate. Nucleolin is one of the major phosphoproteins of the nucleolus. We show that the interaction of nucleolin with the RNA substrate is required for the processing reaction in vitro. Moreover, increasing the level of nucleolin stimulates the processing activity of the S100 cell extract. This nucleolin-dependent activation requires the U3 snoRNA. Interestingly, we show that the N-terminal domain of nucleolin is required for an interaction with the U3 snoRNP. Nucleolin has been implicated in the regulation of different steps of ribosome biogenesis such as transcription and ribosome assembly. The requirement for an interaction between nucleolin and the pre-rRNA for the primary processing reaction opens up new insights into possible functions for this early cleavage in ribosome biogenesis. Results Nucleolin interacts with a processing-competent rRNA substrate Pre-rRNA undergoes a series of cleavages resulting in the production of mature 18S, 5.8S and 28S species. The first cleavage, called the early or primary processing cleavage, takes place within the 5′ ETS and can be reproduced efficiently in a cell extract system with an in vitro transcribed RNA. A radiolabeled RNA corresponding to nucleotides +541 to +1250 of the mouse rRNA (RNA541/1250, Figure 1A) was in vitro transcribed using T7 RNA polymerase and incubated in a hamster S100 extract. This RNA541/1250 is processed relatively efficiently to a 600 nucleotide product (Figure 1B, lane 3) corresponding to a cleavage at position +651/657 of the pre-rRNA. In vivo and in vitro, the primary processing occurs either at +651 or +657. Because of the low resolution of the analysis system used, only one band could be observed, but primer extension analysis confirmed that both sites were used (data not shown). Cleavage efficiency (20–50% of the input rRNA substrate) varies slightly from one extract to another (see, for example, Figures 1B, 2B, 3A, 4A, 5C and 6A). This might indicate that a limiting component required for the cleavage reaction is present in these different extract preparations. In order to identify proteins involved in the primary processing, RNA541/1250 was incubated in the cell extract, then subjected to UV cross-linking. After RNase digestion, cross-linked proteins were revealed by SDS–PAGE. This processing-competent rRNA associates with a defined number of proteins (100, 85, 75 and 52 kDa) (Figure 1C, lane 1) and is consistent with previous cross-linking experiments which used 4-thiouridine-substituted RNA (Kass and Sollner-Webb, 1990). This pattern of cross-linked proteins strikingly resembles the detection of nucleolin and its in vivo maturation products by Western blot analysis (Bugler et al., 1982; Bourbon et al., 1983b), and led Kass and Sollner-Webb (1990) to suggest that nucleolin might be the 100 kDa protein which interacts with this RNA. An immunoprecipitation of UV cross-linked proteins with an anti-nucleolin antibody was performed to test this hypothesis (Figure 1C, lane 3). This experiment shows that the anti-nucleolin antibody could precipitate the 100 kDa cross-linked protein, identifying it as nucleolin. The 52 kDa protein was also significantly retained, and may represent either a degradation product of nucleolin or an associated protein which is co-immunoprecipitated. These data establish that nucleolin interacts with this processing-competent rRNA substrate. Figure 1.Nucleolin interacts with an rRNA precursor in processing extracts. (A) Schematic representation of the mouse rDNA transcription unit and surrounding spacer regions. The region between −170 and +3000 is enlarged. The arrowhead indicates processing sites at +651 and +657. The T7 RNA polymerase-transcribed substrate RNA541/1250 used in our experiments is indicated. (B) A standard processing assay is shown. Ten fmol of the radiolabeled RNA541/1250 was added to the mouse cell extract (lane 3) and, following a 45 min reaction, RNA was extracted and electrophoretically resolved. In lane 2, the RNA precursor was incubated for the same period without extract. (C) Immunoprecipitation of UV cross-linked proteins. Cell extract was incubated for 30 min before the addition of 500 fmol of radiolabeled RNA541/1250. After 45 min, samples were UV cross-linked as described in Materials and methods and analyzed by SDS–PAGE (lane 1) or after immunoprecipitation without (lane 2) or with (IP, lane 3) an anti-nucleolin antibody. In lane 3, the 52 kDa labeled protein appears to migrate slightly faster than in lane 1 because of the IgG present in the immunoprecipitation sample which migrates just above this protein. (D) Interaction of labeled NRE (lane 1) and NS (lane 2) RNAs with cellular proteins under processing reaction conditions. The NRE is a 68 nucleotide RNA that binds nucleolin with high affinity (Ghisolfi et al., 1996). A single mutation within the consensus selected sequence drastically reduces nucleolin interaction and gives rise to the NS RNA (Serin et al., 1997). RNAs were incubated in the extract for 45 min, then subjected to UV cross-linking performed as described in Materials and methods. Download figure Download PowerPoint Figure 2.Interaction of nucleolin with the rRNA substrate is required for the processing activity. Increasing amounts of RNA competitors were added to the cell extract 30 min before the addition of the RNA substrate (RNA541/1250). The reaction was then allowed to proceed for 45 min. For each amount of competitor, a cross-linking assay (A) and a processing assay (B) were performed. In lanes 2–4 and 6–8, the NRE and NS RNAs were used as competitor, respectively. Lanes 1 and 5, no RNA competitor was added; lanes 2 and 6, 2 pmol of RNA competitor; lanes 3 and 7, 6 pmol; lanes 4 and 8, 20 pmol. The amount of nucleolin which is present in the extract for each reaction is ∼2.5 pmol (data not shown). Download figure Download PowerPoint Figure 3.Nucleolin stimulates the processing activity. (A) In vitro processing assay. Labeled RNA541/1250 was processed in a mouse cell extract that had been pre-incubated without (lane 2) or with 5 (lane 3) or 10 pmol (lane 4) of purified nucleolin. Lane 1 shows the RNA541/1250 substrate. In lanes 5–11, 10 pmol of purified nucleolin and increasing amounts of RNA competitors were added to the mouse cell extract. After 30 min of incubation, labeled RNA541/1250 was added and incubated for 45 min. The amounts of specific (NRE) or non-specific (NS) RNA competitor added to the reaction are 20 (lanes 6 and 9), 40 (lanes 7 and 10) and 80 pmol (lanes 8 and 11). In lane 5, no competitor was added. (B) UV cross-linking experiment. Aliquots of the in vitro processing reaction shown in (A) were subjected to UV cross-linking and treated with RNase, and the labeled proteins were analyzed by SDS–PAGE. Download figure Download PowerPoint Figure 4.Exogenous nucleolin can restore the processing activity of an NRE-treated extract. (A) In vitro processing assay. Ten (lane 3), 20 (lane 4) and 40 pmol (lanes 5–9) of NRE RNA competitor, and 2.5 (lane 6), 5 (lane 7), 10 (lane 8) and 20 pmol (lane 9) of nucleolin were incubated for 15 min in the extract before the addition of labeled precursor RNA541/1250. After a 60 min incubation, the reaction was stopped and RNA analyzed on a 6% polyacrylamide gel. (B) UV cross-linking experiment. Aliquots of the in vitro processing reaction shown in (A) were subjected to UV cross-linking, treated with RNase and the labeled proteins were analyzed by SDS–PAGE. Download figure Download PowerPoint Figure 5.The N-terminal domain of nucleolin is required for the processing activity. (A) Schematic representation of the structure of nucleolin and p50. p50 is a recombinant protein where the N-terminal domain of nucleolin has been deleted. The four RNA-binding domains (RBD 1–4) and the glycine/arginine-rich (RGG) domain of nucleolin are represented. (B) UV cross-linking assay. p50 (lane 2, 20 pmol; lane 5, 10 pmol; lane 6, 20 pmol; lane 7, 40 pmol) and 5 pmol of purified nucleolin (lanes 4–7) were incubated for 30 min in the mouse cell extract before the labeled RNA541/1250 was added. After the reaction, the samples were UV cross-linked as described in Materials and methods and resolved by SDS–PAGE. (C) In vitro processing. The same experimental protocol and the same amount of purified proteins were used in this processing assay as in the UV cross-linking assay shown in (B). After the reaction, RNA was extracted and loaded on a 6% denaturing polyacrylamide gel and then autoradiographed. Download figure Download PowerPoint Figure 6.Nucleolin and U3 are both required for processing activity. (A) Processing assay in mouse U3-depleted cell extract. Cell extract was incubated for 30 min in the absence (lanes 1–4) or presence (lanes 5–8) of an oligonucleotide U364–79 complementary to the U3 snoRNA. Half of the reaction was then removed and used for a Northern blot analysis (B). To the remaining reactions, increasing amounts of nucleolin were added and incubated for an additional 30 min. Then, labeled RNA541/1250 was added and the reaction was allowed to proceed for 45 min. The amounts of nucleolin added were 5 (lanes 2 and 6), 10 (lanes 3 and 7) and 20 pmol (lanes 4 and 8). (B) Northern blot analysis. To verify that U3 snoRNA was depleted in the cell extract, a Northern blot was performed using the labeled U364–79 oligonucleotide as a probe. (C) UV cross-linking on the non-depleted (lanes 1–4) or depleted (lanes 5–8) extract with the labeled RNA541/1250 was performed in the presence of increasing amounts of nucleolin. Lanes 1–8 correspond to the same lanes as in (A) and (B). Download figure Download PowerPoint Nucleolin–rRNA interaction is required for the primary rRNA processing The RNA-binding properties of nucleolin have been studied extensively (Olson et al., 1983; Bugler et al., 1987; Ghisolfi et al., 1996; Serin et al., 1996, 1997). Up to now, only one nucleolin RNA target constituted by a small stem–loop structure (nucleolin recognition element: NRE, Ghisolfi et al., 1996) has been identified. The first two RNA-binding domains of nucleolin are required for this specific interaction (Bouvet et al., 1997; Serin et al., 1997). To determine if the NRE RNA interacts with nucleolin of the S100 extract, labeled NRE was incubated in the extract then subjected to a UV cross-linking experiment (Figure 1D). As a control, we used an NRE mutant (NS) with a single point mutation within the RNA loop (Serin et al., 1997). This mutant shows a reduced affinity for nucleolin (Bouvet et al., 1997; Serin et al., 1997). The NRE RNA is strongly cross-linked with a 100 kDa protein (lane 1) compared with the NRE mutant (lane 2). The p52 protein previously found cross-linked to RNA541/1250 interacts with the same efficiency with the NRE and NS RNAs, suggesting that this is probably an abundant RNA-binding protein which interacts non-specifically with RNA. The simple cross-linking pattern obtained with NRE (compare lanes 1 of Figure 1C and D) and the fact that a single point mutation in this RNA abolishes cross-linking with the p100 suggest that the interaction between this protein and the NRE RNA is highly specific. To test if the interaction of the 100 kDa protein with the processing-competent substrate RNA541/1250 observed in Figure 1C was related to the nucleolin RNA-binding specificity, an increasing amount of competitor RNA (NRE) was added to the processing reaction and an aliquot of this reaction was subjected to UV cross-linking (Figure 2A). Addition of nucleolin RNA target, NRE, results in a loss of the 100 kDa protein cross-link (lanes 2–4), whereas the 52 kDa signal remains mostly unchanged. In the same competitor range, NS RNA is unable to prevent the interaction of the 100 kDa protein with the rRNA substrate. Altogether, these results show that this protein possesses nucleolin RNA-binding specificity, which is in agreement with the immunoprecipitation experiment (Figure 1C) identifying the 100 kDa protein as nucleolin. These results also indicate that it is unlikely that the 52 kDa protein is a nucleolin degradation product which contains nucleolin RNA-binding domains since it is cross-linked with both the NRE and NS RNA (Figure 1D), and its interaction with the RNA541/1250 is not competed efficiently by the NRE RNA (Figure 2A). We next asked whether the interaction of nucleolin with the RNA541/1250 substrate was required for the processing reaction. RNA was extracted from the second half of the processing reaction used in Figure 2A and analyzed on a denaturing gel (Figure 2B). In the presence of an excess of nucleolin RNA target (lanes 2–4), the processing reaction is reduced drastically, whereas in the presence of the non-specific RNA (lanes 6–8), the efficiency of the cleavage reaction is unchanged. These results demonstrate that the interaction of nucleolin with the rRNA substrate is correlated with the ability of the extract to support the cleavage reaction. Exogenous nucleolin stimulates the primary processing If the interaction of nucleolin with the pre-rRNA substrate is a limiting step in the primary processing reaction, then addition of an excess of nucleolin to the extract theoretically would stimulate processing. To test this hypothesis, an increasing amount of purified nucleolin protein was added to the extract before the addition of radiolabeled precursor RNA541/1250. After the incubation period, analysis of the cleavage efficiency showed that the addition of exogenous nucleolin stimulated the processing reaction (Figure 3A, lanes 2–4). The level of stimulation depends on the basal level of activity present in the extract (see, for example, reactions with other extract preparations in Figures 5C and 6A), but in each case addition of exogenous nucleolin increases the processing activity of the extract (2- to 5-fold). Remarkably, this nucleolin-induced processing activity is correlated with an increased cross-linking of RNA541/1250 substrate with nucleolin (Figure 3B, lanes 2–4). The specificity of this nucleolin-dependent activation was again tested with the nucleolin NRE RNA target. In the presence of an increasing amount of NRE, both the nucleolin–RNA541/1250 substrate interaction and the processing activity are progressively lost (Figure 3A and B, lanes 6–8) whereas even in the presence of high levels of non-specific RNA competitor (NS), cleavage activity and nucleolin rRNA interaction are still observed (Figure 3A and B, lanes 9–11). In agreement with the results presented above, these data indicate that nucleolin interaction with the precursor rRNA is required for the processing reaction. Quantification of the amount of nucleolin present in the extract (∼2.5 pmol, data not shown) indicates that it is in excess relative to the added rRNA substrate. However, most of this nucleolin is engaged in large complexes (>20S, data not shown). Therefore, the 'free' nucleolin available for the interaction with the RNA541/1250 is likely to be limiting. The addition of purified nucleolin increases the pool of nucleolin available for the interaction with the rRNA substrate, and thus increases the primary processing reaction of this added RNA. Exogenous nucleolin can restore the processing activity of an NRE-treated extract The interaction between the NRE RNA sequence and nucleolin is highly specific in vitro (Ghisolfi et al., 1996; Bouvet et al., 1997; Serin et al., 1997), and within the S100 processing extract (Figures 1D, 2 and 3). However, up to now, we could not exclude that the addition of the NRE RNA to the extract titrates a protein other than nucleolin. To demonstrate conclusively that the interaction of nucleolin with the pre-rRNA substrate is required for the processing reaction, we added increasing amounts of purified nucleolin to an NRE-treated extract (Figure 4). As shown previously (Figure 2), addition of increasing amounts of NRE RNA to the extract prevents the cleavage reaction (Figure 4A, lanes 3–5) and the interaction of endogenous nucleolin with the RNA substrate (Figure 4B, lanes 3–5). When increasing amounts of purified nucleolin are added to this processing-deficient extract, the full processing activity of the extract is progressively restored (Figure 4A, lanes 6–9). Addition of an excess of nucleolin is still able to stimulate the processing activity above the efficiency of the untreated extract (Figure 4A, compare lanes 2 and 9). This cleavage activity is again accompanied by an increase of nucleolin cross-linking with the RNA substrate (Figure 4B, lanes 6–9). This addback experiment is in agreement with data shown in Figures 2 and 3 where, in the presence of an excess of nucleolin, more NRE competitor is required for the inhibition of the processing reaction. Altogether, these experiments show that the inhibitory effect of the NRE RNA is related directly to nucleolin and strengthen our previous data (Figures 2 and 3) that the interaction of nucleolin with the pre-rRNA substrate is required for the processing reaction. Several nucleolin domains are required for its activity A recombinant protein, p50 (Figure 5A), encoding the four RNA-binding domains and the RGG motif of nucleolin is sufficient to account for the RNA-binding affinity and specificity of the full-length protein (Serin et al., 1997). We therefore tested if this domain was sufficient to stimulate the processing reaction. The p50 protein was added to the cleavage reaction before addition of the RNA541/1250 precursor, then half of the sample was subjected to UV cross-linking (Figure 5B, lanes 1–2), and the remaining reaction sample analyzed for the processing activity (Figure 5C, lanes 1–2). As the p50 protein interacts with the rRNA substrate, a decrease of nucleolin cross-linking is observed. An 8-fold excess of p50 over the nucleolin present in the extract is sufficient to prevent nucleolin interaction with the RNA substrate (Figure 5B, lane 2). Remarkably, this decrease of nucleolin binding is perfectly correlated with a decrease of rRNA processing (Figure 5C, lane 2). When exogenous nucleolin is added to the extract at the same time as the p50 protein, the two proteins compete for the interaction with RNA541/1250, showing again that the interaction of nucleolin with the rRNA substrate is specific and saturable. As p50 interaction with RNA541/1250 increases, a simultaneous decrease of nucleolin–rRNA interaction and of the processing reaction is observed (Figure 5B and C, lanes 3–7). Therefore, these results not only confirm that nucleolin interaction with the rRNA substrate is required for the processing reaction, but also demonstrate that the C-terminal RNA-binding domains of nucleolin are not sufficient to support the processing reaction. The interaction of the p50 protein with the pre-rRNA, by impeding the binding of full-length nucleolin to the substrate, could prevent the correct assembly of the processing complex. The N-terminal end of nucleolin that is characterized by stretches of acidic residues might be required for the interaction of nucleolin with other components of the processing complex. U3 snoRNA is required for nucleolin activity The primary processing reaction requires not only the formation of a large protein complex (Kass and Sollner-Webb, 1990) but also several snoRNAs (Enright et al., 1996). The U3 snoRNA has been shown to be associated with the rRNA processing complex and also to be required for the processing reaction (Kass et al., 1990). U3 snoRNA depletion can be achieved easily by oligonucleotide-directed RNase H degradation (Figure 6B, lanes 5–8). This U3-depleted extract shows a reduced rRNA processing activity (Figure 6A, compare lanes 1 and 5; Kass et al., 1990). It recently has been suggested that snoRNPs might be stimulatory but not essential for the processing reaction (Enright et al., 1996). To determine if nucleolin was still able to stimulate the processing activity in the absence of U3 snoRNA, increasing amounts of nucleolin were added to a U3-depleted extract. Although nucleolin interaction with the rRNA substrate is not affected by the absence of the U3 snoRNA (Figure 6
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