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. 2002 May 15;30(10):2251–2260. doi: 10.1093/nar/30.10.2251

Identification of nucleolin and nucleophosmin as genotoxic stress-responsive RNA-binding proteins

Chonglin Yang 1, Dony A Maiguel 1, France Carrier 1,a
PMCID: PMC115285  PMID: 12000845

Abstract

Genotoxic stress (DNA damage) can elicit multiple responses in mammalian cells, including the activation of numerous cascades of signal transduction that result in the activation of cellular genes involved in growth control, DNA repair and apoptosis. In an earlier report, we have shown that DNA-damaging agents can also induce the RNA-binding activity of several specific proteins that favor a double stem–loop RNA structure. Here we report the purification and identification of nucleophosmin (NPM) and nucleolin as two genotoxic stress-responsive RNA-binding proteins. UV radiation induces the protein expression levels and RNA-binding activity of NPM while nucleolin RNA-binding activity increases after UV or ionizing radiation exposure. Moreover, we have identified 40 mRNA ligands that are potentially regulated by nucleolin, several of which are stress-responsive transcripts. In addition, our data indicate that activation of nucleolin RNA-binding activity by genotoxic stress is mediated by stress-activated protein kinase p38. Our findings suggest that activation of the RNA-binding properties of nucleolin and NPM is part of the cellular response to genotoxic stress.

INTRODUCTION

The cellular response to genotoxic stress includes the activation of several regulatory pathways. The activation of cell cycle checkpoints by DNA damage is probably the best understood response to genotoxic stress. It involves the activation of tumor suppressor genes such as p53 and pRb and their downstream effector genes (1). Activation of effector genes may also be mediated by post-transcriptional regulation that affects mRNA stability through the interaction of a mRNAs cis-element and a regulatory protein trans-element. Generally, these regulatory proteins belong to a family of RNA-binding proteins (RBPs) having one or more RNA recognition motifs and auxiliary domains of specific functions. Previous studies have shown that RBPs are involved in RNA processing, mRNA localization and mRNA translation as well as mRNA turnover (reviewed in 2). Moreover, RBPs also play active roles in modulating important cellular processes such as transcription, DNA replication and apoptosis (3).

In contrast to the cell cycle checkpoints, relatively little is known about the roles of RBPs in the cellular response to genotoxic stress. Recently, HuR, an ELAV-like RBP, was shown to stabilize p21 transcripts in response to UV radiation (4). MCG10, another RBP with a KH domain (hnRNP K homology domain), is up-regulated by p53 and suppresses cell proliferation by inducing either cell cycle arrest or apoptosis (5). Our recent studies have shown that a novel RBP, A18 hnRNP, plays a protective role in the genotoxic stress response by stabilizing the expression of transcripts involved in cell survival (6). In an earlier report (7), we demonstrated that DNA-damaging agents could activate the RNA-binding activity of specific proteins that favored a double stem–loop RNA structure. In that study (7), the HIV TAR RNA double stem–loop structure was used to detect stress-activated RBPs. Even though this structure is associated with viral proteins, it is reminiscent of the structure found in nascent transcripts (8) and was postulated to be a target of mammalian RBPs based on studies showing activation of the HIV LTR in a system defective for the viral Tat protein (9). Here, we report the purification and identification of two stress-responsive RBPs. The proteins were isolated on a stem–loop TAR RNA affinity column and identified as nucleolin and nucleophosmin (NPM). Our data indicate that these two RBPs are indeed stress responsive. The RNA-binding activity and the protein levels of NPM are induced by UV radiation while the RNA-binding activity of nucleolin increases after UV or ionizing radiation. Moreover, using our new in vitro binding assay (6), we have identified 40 potential mRNA ligands for nucleolin and we present evidence that the stress-activated protein kinase (SAPK) p38 is involved in the activation of nucleolin RNA-binding activity by genotoxic stress. Taken together, these data indicate that activation of NPM and nucleolin RNA-binding activity contribute to the cellular response to genotoxic stress.

MATERIALS AND METHODS

Cell culture and treatments

The hamster cell lines lung fibroblast cell line V-79 and ovarian CHO cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were grown and maintained in Ham’s F-12 medium containing 12% fetal calf serum. The human lymphoblast cell line GM0536 was obtained from the Cell Repositories of the Coriell Institute for Medical Research (Camden, NJ) and the cells were grown in RPMI 1640 containing 15% fetal calf serum. The human colorectal carcinoma cell line RKO was provided by A. J. Fornace Jr (NCI, NIH, Bethesda, MD) and the cells were grown in RPMI 1640 containing 10% fetal calf serum. Treatments of the cells were as described previously (10) except that the UV source was a Philips 30 W germicidal lamp and ionizing irradiation was performed with a 137Cs source at 5.5 Gy/min. Intensities of the UV lamp were determined with a UVX Radiometer (UVP Inc., Upland, CA) and the irradiation rate was 2.2 J m–2 s–1. The SAPK p38 inhibitor SB203580 was purchased from Upstate Biotechnology (Lake Placid, NY), dissolved in DMSO and applied to the cells 30 min prior to UV exposure. The inhibitor remained in culture for 4 h following UV irradiation. The cells were then harvested and processed as described in the text.

In vitro transcription

Two plasmids used in a previous study (7) were used to generate the 32P-labeled RNA transcripts. pT7TAR and pGA20 contain, respectively, the sequence +1–80 and +20–80 of HIV TAR driven by a T7 promoter (11). Both plasmids were linearized with HindIII and gel purified for in vitro transcription. In vitro transcription was performed with MAXI-scripts (Ambion, Austin, TX) according to the manufacturer’s recommendations.

Northwestern blotting

Northwestern blots were performed as previously described (7). Briefly, the proteins were separated by SDS–PAGE and blotted onto nitrocellulose membranes. The membranes were washed twice with RNA-binding buffer (RBB) (20 mM Tris–HCl, pH 7.5, 60 mM KCl, 1 mM MgCl2, 0.2 mM EDTA, 10% glycerol) and blocked with 5% milk in RBB containing 2 µg/ml yeast tRNA for 1 h at room temperature. The membranes were rinsed with RBB and transferred to the same buffer containing 0.25% milk, 2 µg/ml yeast tRNA, 10 U/ml RNase inhibitor ANTI-RNase (Ambion). 32P-labeled RNA (2 × 106 c.p.m./ml) was incubated with the membranes overnight and the membranes were washed four times with RBB containing 0.25% milk. Membranes were air dried and exposed to X-ray film at –70°C. Where indicated the levels of RNA-binding activity were evaluated with a phosphorimager (Molecular Dynamics Storm 820) with ImageQuant software.

Purification of RBPs

The RBPs were isolated from human colorectal carcinoma RKO cells treated with the alkylating agent methylmethane sulfonate (MMS) (Sigma, St Louis, MO). The RKO cells (100 p150 plates) were treated for 4 h with 100 µg/ml MMS. The cells were harvested and the nuclear proteins were extracted as previously described (12).

The nuclear proteins (76 mg) were diluted in RBB up to 50 ml and mixed overnight at 4°C with a biotinylated TAR RNA probe. The biotinylated TAR RNA probe was made with biotin-21-UTP (Clontech, Palo Alto, CA) and a T7-MEGA Shortscript in vitro transcription kit (Ambion) using the T7-TAR plasmid (7) as template. A trace amount (40 µCi) of [α-32P]UTP was added to the reaction to facilitate assessment of RNA synthesis. After digesting the DNA template, the unincorporated biotinylated UTP was separated from the RNA probe on a Nick column (Pharmacia, Piscataway, NJ) and the probe was precipitated. After mixing the RNA probe with the nuclear extract in RBB containing RNase inhibitor and tRNA, the mixture was passed three times over a Neutravidin agarose (Pierce, Rockford, IL) column equilibrated with RBB at 4°C. The column was washed extensively and the bound proteins were eluted with 0.2 M glycine. The eluted fractions were then neutralized to pH 7.0 with 1 M Tris–HCl, pH 9.0, and concentrated. The protein samples were loaded on a 1 mm thickness 10% polyacrylamide gel and briefly stained with Coomassie blue. Two proteins, of 40 and 102 kDa, eluting in the same fraction were cut from the gel, extensively destained and rinsed with 50% acetonitrile. The protein bands were digested separately in situ with trypsin and the resulting peptides resolved by microcapillary reverse phase HPLC (Harvard Microchemistry Facility, Cambridge, MA). The peptides eluting from the reverse phase HPLC column were directly channeled into a nano-electrospray ionization source of an ion trap mass spectrometer (Finnigan LCQ quadrupole). Sequences of 14 tryptic peptides were obtained from the 102 kDa protein. The 40 kDa protein was also trypsinized and 20 peptides were sequenced. The sequences of the resulting peptides were compared with the SWISS-PROT protein database through the BLAST search engine.

Recombinant proteins

The truncated nucleolin fragment (residues 284–707), which contains all four RNA-binding domains (RBDs) and the C-terminal RGG boxes, was amplified by PCR and cloned into the NdeI and XhoI sites of the bacterial expression vector pET21a (Novagen, Madison, WI). The expression vector was transformed into Escherichia coli strain BL21(DE3). Nucleolin expression was achieved by inducing 1 l of bacteria (OD600 0.5) with 0.4 mM IPTG at 30°C for 2 h. The cells were collected and suspended in 50 mM phosphate buffer (pH 7.4, 50 mM NaCl) and sonicated. The supernatant was loaded on a DEAE–Sephacel column and eluted with a 0.1–1 M linear NaCl gradient. Fractions containing nucleolin were collected and dialyzed against 10 mM Tris–HCl, pH 7.4, 200 mM NaCl. The samples were then batch purified on poly(G)–agarose (Sigma) and washed consecutively with 0.4, 0.6, 0.8 and 1 M NaCl. Finally, the proteins were eluted stepwise with 1.2, 1.5 and 2 M NaCl. The eluted fractions containing purified nucleolin were dialyzed against 50 mM phosphate buffer (pH 7.4, 50 mM NaCl).

The human NPM open reading frame (ORF) was obtained from P. K. Chan (Houston, TX). The ORF was amplified by PCR and cloned into the NdeI and XhoI sites of pET21a. This vector added six histidine residues at the C-terminus of the recombinant protein. Protein expression was performed as described above. For purification, the soluble proteins were loaded on a Ni–NTA column (Novagen, Madison, WI) and eluted with a stepwise gradient of 100–500 mM imidazole. The imidazole in the purified fractions was removed by gel filtration on a D-Salt Excellulose Desalting Column (Pierce, Rockford, IL) and exchanged into RBB.

RNA band shift

The RNA band shift assay was performed in RBB containing 32P-labeled RNA transcript (1 × 106 c.p.m.) and various amounts of recombinant protein in a final reaction volume of 30 µl. The reaction mixtures were incubated at 30°C for 30 min and run on a 7% native acrylamide gel in 0.5× TBE. The gel was dried and exposed to X-ray film at –70°C overnight.

Antibodies and western blots

The polyclonal antibody against nucleolin was generated in rabbits immunized with the truncated recombinant protein (residues 284–707). The polyclonal antibody against NPM was also generated in rabbits, but a synthetic peptide (residues 221–238) was used as antigen. Cellular extracts were separated by SDS–PAGE and blotted onto nitrocellulose membranes. Western blots were performed according to standard protocols with a 1/1000 dilution of the nucleolin antibody and a 1/500 dilution of the NPM antibody. Hybridization of the antibodies was detected by chemiluminescence (ECL; Amersham Pharmacia, Arlington Heights, IL) according to the manufacturer’s recommendations.

Selection of mRNAs by nucleolin

Human colon carcinoma (RKO) cells were cultured to 90% confluency in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were irradiated with UV at a dose of 20 J m–2 and then put back into culture for 2 h. The mRNAs were extracted by the acid phenol procedure (13). Selection of nucleolin mRNA ligands was performed as described (6). Briefly, recombinant nucleolin (40 µg/ml in carbonate buffer, pH 9.6) was coated overnight at 4°C in an immunotube (Nalgen Nunc International, Rochester, NY). The coated tube was washed and non-specific sites were blocked with 2% milk in PBS containing 2 µg/ml yeast tRNA for 1 h at room temperature. After washing the tube, the mRNAs (10 µg) were incubated in the nucleolin-coated immunotube for 2 h. Following extensive washing, the bound mRNAs were eluted stepwise with increasing concentration (0.5–2 M) of NaCl. The eluted mRNA was precipitated and used for cDNA synthesis.

The first strand cDNA synthesis was performed in a 20 µl reaction mixture at 42°C for 1 h with the SmartII cDNA synthesis oligo (Clontech) and Superscript reverse transcriptase (Gibco BRL, Rockville, MD). Five microliters of the reaction mixture was used to amplify the double-stranded cDNA by long distance PCR (95°C for 1 min, 65°C for 1 min and 68°C for 6 min) for 30 cycles. The amplified cDNA was purified and ligated with pGEM-T vector (Promega, Madison, WI) then transformed into E.coli DH5α. White colonies on LB plates containing IPTG/X-Gal were picked up for further analysis by PCR fingerprinting and sequencing was performed on non-redundant clones.

In vitro phosphorylation

The recombinant nucleolin (residues 284–707, 0.25–1.25 µg) was phosphorylated in vitro with 0.25 µg GST–SAPK p38 kinase (Upstate Biotechnology) and 30 µCi [γ-32P]ATP in 40 µl of reaction buffer containing 30 mM Tris–HCl, pH 7.4, 1 mM MgCl2, 1 mM EGTA. After the reaction was allowed to proceed for 30 min at 30°C, the samples were boiled and loaded on a 10% SDS–PAGE gel. The gel was dried and exposed to X-ray-sensitive film.

A similar experiment was repeated with cold phosphate (0.25 mM ATP). Increasing amounts of non-phosphorylated or phosphorylated nucleolin were run on a 10% SDS–PAGE gel, transferred to nitrocellulose and analyzed by northwestern blotting.

RESULTS

Activation of RNA-binding activity by genotoxic stress in different cell lines

We have previously shown (7) that activation of RNA-binding activities by genotoxic stress is protein and cell type specific. Moreover, our data (7) indicate that a double stem–loop RNA structure was favored by these stress-activated proteins. The double stem–loop structure of the HIV TAR RNA (Fig. 1) is reminiscent of the structure found in nascent transcripts that is recognized by several RBPs (8). We thus used this RNA probe to examine further the activation of RBPs in three different cell lines after exposure to DNA-damaging agents. As shown in Figure 2A, in the Chinese hamster V-79 lung fibroblast cells, five bands ranging from 35 to 102 kDa demonstrated constitutive RNA-binding activity (lane 1). The RNA-binding activity of the 48 and 50 kDa bands did not change significantly following exposure to stress (lanes 2–4). However, the RNA-binding activity of the 102 kDa protein was markedly increased following UV and X-ray irradiation of the cells, while the alkylating agent MMS did not affect it. Notably, the constitutive RNA-binding activity of a 35 kDa protein was not affected by UV radiation but was decreased after exposure to MMS or X-ray irradiation. In human lymphoblast cells, the 35 kDa protein was not detected under any of the conditions used (lanes 5–8). In contrast to V-79 cells, the RNA-binding activity of the 48 and 50 kDa bands was increased under all stress conditions (lanes 6–8). The RNA-binding activity of a 102 kDa protein was also activated following exposure to MMS and ionizing radiation (lanes 7 and 8) but not UV radiation (lane 6). In human colon carcinoma RKO cells, RBPs of similar molecular weight were also detected (lanes 9 and 10). As observed with the V-79 cells (lanes 1–4), the binding activity of the 35, 48 and 50 kDa proteins was not activated following stress (lanes 9 and 10). On the other hand, even though the constitutive binding activity of the 102 kDa protein was high in RKO cells (lane 9), this activity was increased after ionizing radiation (lane 10). The RNA-binding activity of an additional band of ∼80 kDa was also increased by this treatment. This band was not observed in any other cell line studied and could be a breakdown product of the 102 kDa protein. In addition, the RNA-binding activity of a 40 kDa protein was also clearly stimulated in RKO cells after exposure to UV and MMS (Fig. 2B). These data indicate that different DNA-damaging agents can activate RBPs. As reported before (7), the number of activated proteins and the level of activation is cell type dependent.

Figure 1.

Figure 1

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Predicted secondary structures of riboprobes used in the northwestern and gel shift assays. The wild-type TAR RNA (+1–82) double stem–loop structure is generated by pT7 TAR. Deletion of the first 19 nt of the TAR sequence results in an undefined structure without the typical double stem–loop conformation (pGA20).

Figure 2.

Figure 2

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(A) Northwestern blots of mammalian nuclear protein extracts (10 µg). The cells were either left untreated (C), exposed to 14 J m–2 UV light (UV), treated with 100 µg/ml MMS for 4 h (MMS) or exposed to 20 Gy ionizing radiation (XR). The protein extracts were run on 7.5% SDS–PAGE gels and transferred to nitrocellulose. The blot was hybridized to the TAR RNA probe as described in Materials and Methods. (B) Northwestern blots of human RKO cell nuclear extract (10 µg). The blot was processed as in (A) except that the proteins were run on 10% SDS–PAGE gels. RNA-binding activation of a 40 kDa protein is shown.

Purification and identification of two RBPs activated by genotoxic stress

In order to further investigate the potential role of these RBPs in the cellular response to genotoxic stress, we focused our efforts on the purification and identification of these proteins. We elected to purify the proteins from the RKO cells since these cells possessed the highest basal RNA-binding activity and that activity can still be stimulated by genotoxic stress (Fig. 2). To maximize our chances of purifying stress-activated proteins, we treated the cells with the alkylating agent MMS. We took advantage of the fact that these proteins bind avidly to the HIV TAR RNA (Fig. 2) (7) and designed an affinity column with this probe. Of the initial 76 mg of nuclear protein used, 12 µg of protein were purified with this column. The purified fraction was run on a 12% SDS–PAGE gel and stained with Coomassie blue. The major component of this fraction was a 40 kDa protein. An additional protein of ∼102 kDa representing approximately one-tenth of the amount of the 40 kDa protein was also detected. This represents >6000-fold purification for the 40 kDa protein and >60 000-fold purification for the 102 kDa protein. Both proteins were readily cut out of the gel, digested with trypsin and microsequenced. From the 102 kDa protein, 14 peptide sequences were obtained, while 20 peptides derived from the 40 kDa protein were sequenced. Six representative peptide sequences for each protein are shown in Table 1. A BLAST algorithm search revealed that all the peptide sequences derived from the 102 kDa protein corresponded to nucleolin, a nucleolar RBP with multiple functions (14). Our database search also revealed that the peptide sequences derived from the 40 kDa protein corresponded to NPM, a nucleolar protein involved in ribosome assembly (15).

Table 1. Sequence analyses of RBP tryptic digests.

Peptides from the 102 kDa protein Peptides from the 40 kDa protein
GYAFIEFASFEDAK DELHIVEAEAMNYEGSPIK
GFGFVDFNSEEDAK MTDQEAIQDLWQWR
GLSEDTTEETLK VDNDENEHQLSLR
NDLAVVDVR GPSSVEDIK
VEGTEPTTAFNLFVGNLNFNK ADKDYHFK
SATEETLQEVFEK MQASIEK
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Tryptic digest peptides from the 102 and 40 kDa proteins.

NPM is induced by UV radiation and binds hairpin RNA structures

Activation of specific RBPs by DNA-damaging agents could be the result of at least two possible events: either protein expression is induced and/or the binding activity of these proteins is increased as a result of protein modification. We first investigated whether NPM protein levels can be induced by UV irradiation. As shown by western blot (Fig. 3A), NPM expression is increased after UV treatment in a dose-dependent manner in two human cell lines, RKO and H1299. Interestingly, basal levels of NPM in RKO cells (lane 1) can be detected with NPM antibody while no RNA-binding activity was observed at that molecular weight range under these conditions (Fig. 2B, lane 1). These data suggest that activation of the RNA-binding activity of NPM is not only due to increased protein levels but also probably to other mechanisms triggered by genotoxic stress. Even loading of the protein samples was verified by hybridization to an actin antibody. It thus appears that NPM is indeed a genotoxic stress-induced RBP.

Figure 3.

Figure 3

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(A) Western blots performed on RKO and H1299 protein extracts. The cells were exposed to increasing doses of UV radiation. The position of NPM is indicated. The blots were stripped and hybridized with an actin antibody to evaluate loading consistency. (B) RNA band shift of recombinant NPM. Increasing amounts (0, 0.75, 1.25, 2.5 and 5.0 µg) of NPM were incubated with the indicated RNA probe. The position of the retarded complex is indicated (Shift).

It has been reported that, in vivo, NPM is associated with nucleolin (16). Nucleolin is known to bind specifically to RNAs with a hairpin structure (8) but no information regarding NPM binding to such a structure has been reported. To make sure that NPM was purified as a result of its RNA-binding property and not its association with nucleolin, an RNA mobility shift assay was performed with purified recombinant NPM. The data presented in Figure 3B show that a protein–RNA complex is formed between the recombinant protein and the TAR RNA probe in a dose-dependent manner (lanes 1–5). This result confirms the specificity of the TAR RNA column used in the purification protocol and indicates that NPM does bind to RNAs with a hairpin structure. Binding of NPM to RNAs could be structure specific since binding to pGA20, a probe lacking the first 20 nt of TAR RNA resulting in an undefined structure (Fig. 1), was reduced by 50% (lanes 6–10).

The RNA-binding activity of nucleolin is induced by genotoxic stress

The origin of nucleolin RNA-binding activation following exposure to DNA-damaging agents was also investigated. We treated CHO and RKO cells with either increasing doses of UV or ionizing (X-ray) radiation (Fig. 4). Nuclear protein extracts obtained from these cells were run on a SDS–PAGE gel and transferred to nitrocellulose. The membranes were first reacted with nucleolin antibody (western assay). Our data (Fig. 4A) indicate that after exposing the CHO cells to UV radiation, the protein levels of full-length nucleolin did not increase. Fragmentation (maturation) of nucleolin has been reported in different cell lines (17). Nucleolin fragmentation is cell type specific and sensitive to protease inhibitors (17). Our data (Fig. 4A) indicate that treatment of the cells with UV radiation did not significantly affect nucleolin fragmentation. When the blot was stripped and rehybridized with a TAR RNA probe (northwestern assay), the RNA-binding activity of a band of the size of full-length nucleolin (p102) increased up to 2-fold. No RNA-binding activity was detected for the nucleolin fragments in that cell line.

Figure 4.

Figure 4

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Western and northwestern blots of CHO and RKO cells. (A) The CHO cells were exposed to increasing doses of UV radiation and nuclear protein extracts (10 µg) were analyzed by western blot with nucleolin antibody. The position of nucleolin is indicated. The blot was stripped and analyzed by northwestern assay with the TAR RNA probe. The position of a 102 kDa protein is indicated. The fold increased RNA-binding activity is indicated and was calculated by phosphorimager and normalized to control (0 J m–2 UV). (B) As (A) except that the assays were performed with RKO cells exposed to increasing doses of ionizing radiation (XR).

Similar results were obtained with RKO cells exposed to ionizing radiation (Fig. 4B). The protein levels of full-length nucleolin did not increase after exposure to ionizing radiation (Fig. 4B, western assay) and no significant effect on nucleolin fragmentation was observed in that cell line. When the blot was stripped and rehybridized with a TAR RNA probe (northwestern assay), the RNA-binding activity of a band of the size of full-length nucleolin (p102) increased up to 1.5-fold. The RNA-binding activity of most fragments was only slightly noticeable in RKO cells after 10 Gy ionizing radiation and not detected after 25 Gy. A minor band of ∼80 kDa running just below the p102 band was detected in the western and northwestern blots after 25 Gy radiation (Fig. 4B). It does not appear that this protein fragment is the result of exposure to X-rays or other genotoxic treatment since the band was detected in the untreated sample in Figure 2 (lane 9) and was not present in other cell lines (Figs 2 and 4A). The low abundance of this protein and the variability in its detection may be the result of proteolysis. When present, this fragment seems to retain the capacity of the full-length protein to increase its RNA-binding activity following ionizing radiation (Figs 2 and 4B). These data indicate that ionizing radiation increases the RNA-binding activity of full-length nucleolin protein but does not significantly affect the binding activity of most nucleolin fragments. The RNA-binding activity of an unidentified protein of smaller molecular mass (30 kDa, p30) was also increased following X-irradiation. This protein may have run out of the lower percentage acrylamide (7.5%) gel presented in Figure 2 and was thus not observed in that figure.

Selection of mRNA ligands that are potentially regulated by nucleolin

In an effort to determine the possible cellular targets of nucleolin, we selected potential cellular mRNA ligands by our recently described immunotube technique (6). For this assay, we expressed and purified a truncated nucleolin (residues 284–707) that contains all the RBDs and the RGG box at its C-terminus (18) in E.coli. The recombinant nucleolin was coated on the inner surface of an immunotube and mRNAs prepared from UV-irradiated RKO cells were incubated and processed as described before (6). The specifically bound mRNAs were amplified, reverse transcribed, cloned and sequenced. A BLAST algorithm search was used to determine the identity of the non-redundant ligands.

Three groups of representative mRNA sequences are shown in Table 2. A substantial proportion of these sequences are stress responsive or stress related. Among them, human peroxiredoxin and glutathione peroxidase are oxidative stress responsive genes (19). They play a role in anti-apoptosis induced by radiation. Another protein of interest is the 90 kDa heat shock protein; its expression is not only increased after heat shock, but is also induced by oxidative stress (20). Human elongation factor 1 (eEF1) is up-regulated by homocysteine (21). Homocysteine can cause DNA strand breaks and apoptosis (22). The second group of potential ligands is constituted of ribosomal protein mRNAs, some of which are also stress responsive. For example, the mRNA expression of ribosomal protein 13 A (RPL13a) is induced by UVB or γ-radiation in rat keratinocytes (23) and RPS6 is induced by cold shock (24). The third group of mRNAs contains a variety of other transcripts (Table 2, Others) of diverse cellular function. Moreover, five sequences of unknown function and two novel sequences have been identified (data not shown). These data indicate that nucleolin may play a significant part in the post-transcriptional regulation of several stress-responsive transcripts as well as other transcripts involved in important cellular processes such as splicing, translation and tumor progression (Table 2).

Table 2. mRNAs selected by nucleolin.

Ribosomal proteins Stress-responsive proteins Others
RPS3a Peroxiredoxin 1 (PRDX1) Translational activator GCN1
RPS5 Glutathione peroxidase Mitochondrial COXII
RPS6 Ferritin L-chain ARP2/3 protein complex
RPS16 90 kDa heat shock protein Ketohexokinase
RPS17 Human elongation factor 1 Hypothetical protein FLJ 21884
RPS20 DNA-binding protein TAXREB107 Human HGTD-P
RPL7 ATP synthase γ-subunit Carbonyl reductase
RPL14 Laminin receptor Mitochondrial serine hydroxymethyltransferase
RPL23 Vacuolar H+-ATPase subunit Overexpressed breast tumor protein
RPL27 RPL13a Tumor transforming protein
RPL29   Acidic ribosomal phosphoprotein P0
RPL31   Translocon-associated protein
RPL35   Tubulin α
    Myosin light chain
    Human spliceosome associated protein TADA1
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To confirm the binding specificity of nucleolin to the selected transcripts, we performed northwestern analyses with recombinant nucleolin on three stress-responsive transcripts (Fig. 5) that were selected by nucleolin (Table 2). Our data indicate that nucleolin binds to eEF1, peroxideroxin and RPL13a transcripts in a dose-dependent manner (lanes 1–4). The binding specificity was verified by the inability of BSA to bind any of the transcripts (lanes 5).

Figure 5.

Figure 5

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Northwestern blots of recombinant nucleolin. Increasing amounts of the recombinant (residues 284–707, containing the four RBDs and the C-terminal RGG boxes) nucleolin was hybridized with eEF1 (600 nt), full-length peroxideroxin or full-length RPL13a transcript as described in Materials and Methods. BSA (1 µg) was used as a negative control. The blots were exposed for 48 h.

SAPK p38 activates nucleolin RNA-binding activity in response to stress

Since the protein levels of nucleolin did not change significantly after DNA damage (Fig. 4), post-translational modification of nucleolin in response to genotoxic stress may account for the increased RNA-binding activity. SAPK p38 has been shown to phosphorylate the RBP hnRNPA1 in response to UV radiation (25). We thus aimed at determining whether SAPK p38 could also be involved in nucleolin activation in response to genotoxic stress. We first investigated the capacity of SAPK p38 to directly phosphorylate nucleolin in vitro (Fig. 6A). Our data indicate that SAPK p38 can autophosphorylate (Fig. 6A, lane 1) and phosphorylate recombinant nucleolin (lane 2). No incorporation of 32P radiolabel was observed in the absence of SAPK p38 (lane 3). We then phosphorylated recombinant nucleolin with SAPK p38 and cold phosphate and compared the RNA-binding activity of the non-phosphorylated and phosphorylated nucleolin (Fig. 6B). The proteins were run on four different gels and the blots were processed and exposed simultaneously. Our data indicate that phosphorylated (Fig. 6B, lanes 5–8) nucleolin binds more efficiently to the eEF1 and TAR RNA probes than the non-phosphorylated (lanes 1–4) protein. Taken together these data indicate that phosphorylation of nucleolin by SAPK p38 is sufficient to increase nucleolin RNA-binding activity.

Figure 6.

Figure 6

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In vitro phosphorylation of nucleolin by SAPK p38. (A) In vitro phosphorylation of recombinant nucleolin (residues 284–707) by recombinant SAPK p38. The assay was performed as described in the text. (B) Northwestern analyses. Increasing amounts of recombinant (residues 284–707) unphosphorylated (lanes 1–4) or phosphorylated (lanes 5–8) nucleolin were hybridized to the indicated RNA probes. The blots were processed and exposed (12 h) simultaneously.

To determine whether SAPK p38 is involved in the UV activation of nucleolin RNA-binding activity in vivo, we treated CHO cells with SB203580, a specific inhibitor of SAPK p38, prior to exposing the cells to UV radiation (Fig. 7). Our data indicate that, as observed before (Fig. 4A), the protein levels of full-length nucleolin (Fig. 7A, lanes 1 and 2) did not change in response to UV irradiation. No significant nucleolin fragmentation was observed here, while fragmentation was observed in Figure 4A. This is probably due to the fact that nuclear proteins were used in Figure 4A while total cell extracts were used here. Our data (Fig. 7A) indicate that pretreatment of CHO cells with the p38 inhibitor had no effect on nucleolin protein levels (lanes 3) but reduced nucleolin UV-induced RNA-binding activity by 50% (Fig. 7B, 46 versus 23%). The induction of RNA-binding activity is similar to that observed in Figure 4 (1.5- to 2-fold) and the 1.7-fold activation observed for another RBP, MnSOD-BP, in the presence of herbimycin A (26). Taken together these data (Figs 6 and 7) indicate that SAPK p38 participates in the activation of nucleolin RNA-binding activity by genotoxic stress.

Figure 7.

Figure 7

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Western blots of and RNA-binding activity in CHO cells. The cells were pretreated for 30 min with SAPK p38 inhibitor (SB203580) and exposed to 20 J m–2 UV radiation. (A) Four hours later total proteins (30 µg) were extracted and analyzed by western blotting with either nucleolin or actin antibody. (B) Northwestern analyses were performed on the same samples and quantitated by phosphorimager. The averages of at least two experiments are shown and the values are expressed as relative percentage increase compared with the untreated (Control) sample.

DISCUSSION

We have previously reported that specific RBPs are activated under genotoxic stress (7); however, the role of these proteins, if any, in the cellular response to genotoxic stress is largely unknown. Here, we have identified and characterized nucleolin and NPM as two genotoxic stress-activated RBPs. Nucleolin was initially described as a nucleolar protein participating in ribosome biogenesis (reviewed in 14). Subsequent studies have shown that nucleolin is a multifunctional protein involved in several cellular processes such as transcription (27), attachment of genomic DNA to nuclear matrix and decondensation of chromatin (28). The predicted molecular mass of nucleolin according to its coding cDNA is 77 kDa (29); however, when run on a SDS–PAGE gel, it demonstrates an apparent mass of 100–110 kDa (14). This discrepancy has been attributed to the highly acidic N-terminal domain of this protein, which can contain as many as 38 aspartate and glutamate residues consecutively (30). Nucleolin is composed of three functionally different domains: the N-terminal acidic region that controls rDNA transcription, the central RBD consisting of four RNA-binding motifs that determine the RNA-binding specificity, and the C-terminal arginine/glycine-rich domain (RGG boxes) that regulates RNA or protein interactions (18). The first two RBDs have been shown to be sufficient to mediate RNA-binding activity (31). Whether these two RBDs are present in the different nucleolin fragments has not been determined, but this could account for the variability observed in the RNA-binding activity of these fragments. Nucleolin has also been identified as the human helicase IV that destabilizes DNA–DNA helices, DNA–RNA helices and RNA–RNA helices (32). Helicase IV is thus an important enzyme in homologous recombination and recombinational DNA repair. Due to its helicase activity and capacity to bind to hairpin RNA structures (8), nucleolin could also be involved in other DNA repair mechanisms, such as nucleotide excision repair or transcription-coupled repair. Activation of nucleolin in response to genotoxic stress could affect one or more of these mechanisms.

Nucleolin is subjected to post-translational modifications such as phosphorylation and methylation. Nucleolin phosphorylation is cell cycle dependent and can also be induced by hormones (33) and growth factors (34). Our results indicate that the increase in nucleolin RNA-binding activity following exposure to genotoxic stress is probably mediated by SAPK p38 (Figs 6 and 7). SAPK p38 has already been shown to mediate the translocation of hnRNPA1 in response to stress (25) and is involved in the activation of p53-mediated apoptosis in response to UV radiation (35). Activation of nucleolin RNA-binding activity thus appears to be a part of the cellular response to genotoxic stress mediated by the SAPK p38 pathway. It has been shown that in vitro phosphorylation of nucleolin by CKII and cdc2 can increase its helicase activity (32). It thus seems possible to speculate that the increased RNA-binding activity mediated by SAPK p38 phosphorylation could result in modification of nucleolin RNA helicase activity.

The increased RNA-binding activity of nucleolin after genotoxic stress implies that the RNA-binding property of nucleolin may be involved in the cellular responses to DNA damage. A recent report described a link between an RBP, DNA repair and tumor suppression (36) in response to genotoxic stress. The formation of a protein complex composed of tumor suppressors and CstF, an RBP required for the endonucleolytic cleavage of poly(A) tails, is increased by genotoxic stress (37). As a result, mRNA polyadenylation is repressed. Similarly, repression of rRNA synthesis has been observed in response to oxidative damage (38). Whether activation of nucleolin RNA-binding activity by genotoxic stress could facilitate the recruitment of other proteins involved in rRNA processing (37) or other cellular processes remains to be determined.

Several lines of evidence indicate that nucleolin is involved in the post-transcriptional regulation of mRNAs. For example, it has been shown that nucleolin is required for JNK-mediated interleukin-2 (IL-2) mRNA stabilization during T cell activation by interacting with the 5′-untranslated region of IL-2 mRNA (39). Nucleolin also specifically interacts with the 3′-untranslated region of the precursor mRNA of amyloid protein, a multifunctional protein related to Alzheimer’s disease (40). Therefore, another important function for nucleolin in the cellular response to genotoxic stress could be its participation in the stabilization of some mRNAs that are important for cell survival. Using an in vitro assay, we have identified 40 mRNAs (Table 2) that are potentially regulated by nucleolin, 11 of which are stress-responsive transcripts. The binding of nucleolin to the mRNAs of peroxiredoxin 1 and glutathione peroxidase is of particular interest since both proteins are antioxidants and prevent cells from undergoing apoptosis induced by radiation (19). Nucleolin could either regulate the stability of these mRNAs or be involved in the export of these transcripts from the nucleus to the cytoplasm (14). Our library was selective for stress-activated transcripts in RKO cells and thus the targets identified in Table 2 represent only a fraction of potential nucleolin targets.

Like nucleolin, NPM is a nucleolar protein involved in ribosome biogenesis (15). Our results indicate that NPM can also bind to RNA hairpin structures (Fig. 3B) and is induced by genotoxic stress (Fig. 3A). The induction of NPM by UV radiation is in good agreement with other studies showing a direct correlation between the levels of NPM and cell sensitivity to UV radiation (41). The increased RNA-binding activity of NPM (the 40 kDa protein shown in Fig. 2B) thus appears to be largely the result of increased protein expression. However, protein levels may not be sufficient to explain all the RNA-binding activity since under basal conditions (unstimulated) where low levels of NPM can be observed (Fig. 3A), no RNA-binding activity was detected (Fig. 2B). Post-translational modification of NPM following genotoxic stress may also account for the increased RNA-binding activity. In fact, it has been shown that NPM is phosphorylated after exposure to ionizing radiation (42).

NPM posseses an intrinsic RNase activity that preferentially cleaves poly(A), poly(U) and poly(C), but not poly(G) (43). Since NPM has been found to be associated with nucleolin (16), it is possible that both proteins work in concert in pre-rRNA processing. Our results have shown that both nucleolin and NPM are stress responsive (Figs 3 and 4). Their activation by genotoxic stress may thus link together the cellular response to DNA damage and rRNA processing in a manner analogous to CstF and inhibition of mRNA 3′ processing in response to DNA damage (36). There may be additional roles for NPM in the response to DNA damage. For example, it has been reported that NPM inhibits the DNA-binding and transcriptional activity of IRF-1, a tumor suppressor involved in apoptosis after DNA damage (44). We have also found that NPM physically and functionally interacts with the tumor suppressor p53 (D. A. Maiguel, unpublished observations). Further experiments are currently underway to advance our understanding of the roles that these two stress-activated RBPs are likely to play in the cellular response to genotoxic stress.

Acknowledgments

ACKNOWLEDGEMENTS

The authors would like to thank Dr Steven Hirschfeld for a careful reading of this manuscript and important discussions. This work was supported by the Institute of General Medicine, RO1GM57827-01 (F.C.) and an Initiative for Minority Student Development, GM55036 (D.A.M.).

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