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To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, P. O. Box 250509, Charleston, SC 29425. Tel.: 843-792-7475; Fax: 843-792-8565
* This work was supported by National Cancer Institute Grants CA 87553 (to E. K. S.) and CA 109254 (to D. J. F.) from the National Institutes of Health and an unrestricted grant from Antisoma Research Limited, UK (to D. J. F.). The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1. 1 Present address: Dept. of Chemistry and Biosciences, SASTRA University, SRC, Kumbakonam, India 612001. 2 Present address: School of Biology, Indian Institute of Science, Education, and Research, Kolkata, India 700106. 3 Present address: Dept. of Biophysics, Molecular Biology, and Bioinformatics, University of Kolkata, India 700009.
The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3′-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its half-life. Here, we examined the site on the bcl-2 3′-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5′ end of the 136-nucleotide bcl-2 AU-rich element (AREbcl-2). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to AREbcl-2. In RNA decay assays, AREbcl-2 transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of AREbcl-2 transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability.
Bcl-2, the prototype for its family, is an antiapoptotic protein. Its overexpression has been implicated in multiple cancers and associated with resistance to chemotherapy, making it an important prognostic factor, particularly in hematological malignancies. The Bcl-2 protein is often highly expressed in chronic lymphocytic leukemia (CLL),
) reported that Bcl-2 overexpression in CLL is related to bcl-2 mRNA stabilization.
It is becoming increasingly clear that mRNA stability is an important control point in the regulation of gene expression. In mammalian cells, regulation of mRNA turnover can dramatically alter the abundance of a particular mRNA without changes in transcription. One of the best characterized regulatory elements present in the 3′-untranslated region (3′-UTR) of mRNAs is the AU-rich element (ARE) (for a review, see Ref.
). These elements are usually composed of AUUUA sequences embedded in a U-rich stretch, and they act as potent mRNA-destabilizing sequences, targeting mRNAs for rapid decay. The bcl-2 mRNA contains an ARE in the 3′-UTR that plays a role in regulating its stability (
). The AREbcl-2 is a sequence of 136 nucleotides (nucleotides 921–1057) just downstream from the stop codon, containing two AUUUA pentamers and a UUAUUUAUU nonamer, which has also been shown to destabilize some mRNAs (
) subsequently found that stabilization of bcl-2 mRNA is, in part, the result of overexpression of nucleolin in the cytoplasm of CLL cells compared with normal CD19+ B cells. Also, Soundararajan et al. (
) have found that nucleolin and Bcl-2 proteins are highly expressed in the cytoplasm of MCF7 breast cancer cells but are not in nonmalignant MCF10A cells.
Nucleolin, also known as C23, is highly conserved among eukaryotes and is ubiquitously expressed. Despite being the most abundant protein in the nucleolus, it shuttles to the cytoplasm, where it participates in mRNA regulation, and it is also found in the plasma membrane (
). As a result, our current view of nucleolin is of a multifunctional protein involved in numerous cellular processes, such as proliferation and growth, transcription, cytokinesis, nucleogenesis, signal transduction, mRNA regulation, apoptosis, induction of chromatin condensation, and replication, to name a few (for a review, see Ref.
The unusual diversity in the biological functions of nucleolin can be understood, at least partially, from its complex protein structure. Nucleolin contains three structural domains: the N-terminal which contains highly acidic residues and a nuclear localization signal; the central domain which contains four ribonucleoprotein-type RNA binding domains (RBDs) that are determinants of RNA-binding specificity; and the C-terminal domain which contains arginine-glycine-glycine repeats (RGGs), participates in interactions with ribosomal proteins (
The presence of four RBDs in nucleolin may account for the diversity of target RNAs that are bound by this protein. Nucleolin binds to two different RNA motifs in preribosomal RNA (pre-rRNA), the nucleolin recognition element (NRE) (
). None of the four RBDs of nucleolin binds individually to fragments of pre-rRNA; however, a polypeptide containing the first two RBDs binds specifically to a short RNA containing the 18-nucleotide NRE (
). Although the contributions of the RBDs to binding to NRE and ECM RNAs is known, the contributions of the RBDs to binding of specific mRNAs are not known. In addition, the binding site of nucleolin on bcl-2 mRNA has not been mapped. To address these questions, the role of the four RBDs in binding of nucleolin to bcl-2 mRNA was assessed, and the binding site of nucleolin on the AREbcl-2 was mapped.
In mammalian cells, ARE-mediated mRNA decay starts with deadenylation of the 3′ poly(A) tail, followed by 3′-5′ exonucleolytic degradation by a complex of enzymes termed the exosome (for review, see Ref.
) have suggested that AUF1 plays a role in the turnover of bcl-2 mRNA. In this report we examined the ability of nucleolin to protect bcl-2 mRNA from exosome degradation and tested the hypothesis that nucleolin may compete with AUF1 for binding to bcl-2 mRNA. Based on our findings, we propose a model for how bcl-2 mRNA levels are regulated in normal cells and how, in some cancer cells, aberrant stabilization by nucleolin leads to up-regulation of the antiapoptotic Bcl-2 protein.
The RNase footprinting and gel shift RNA binding assays demonstrated that nucleolin binds to an ∼40-nucleotide binding site spanning nucleotides 36–77 at the 5′ end of the AREbcl-2. Gel shift binding assays (Fig. 2) showed that at increasing nucleolin protein concentrations, more than one nucleolin can bind to the AREbcl-2. Thus, the protected region of ∼40 nucleotides may result from binding of two or more nucleolin molecules. RNase protection of G114, which is outside of the primary binding site, may arise from G114 being spatially close to the 40-nucleotide sequence in the three-dimensional folded structure of the RNA. Alternatively, nucleolin binding could be inducing a conformational change in the transcript that results in G114 becoming susceptible to T1 cleavage and thus, G114 may not be in the nucleolin binding site.
Mutation analysis further showed that the AUUUA pentamers and the nonamer (UUAUUUAUU) sequence likely do not contribute significantly to the nucleolin binding energy. Thus, nucleolin does not appear to recognize typical ARE motifs in bcl-2 mRNA. Comparison of the sequence of the defined bcl-2 binding region with the NRE (
) mRNAs have been mapped, and although these mRNAs do not contain ARE motifs, there is a CU-rich sequence (CUCUCUUUC/AC) that is common to the three mRNAs. This sequence is not present in the bcl-2 ARE. Thus, it appears that nucleolin recognizes a different sequence within bcl-2 mRNA from other mRNAs. Also, it is likely that secondary and tertiary structures are important for nucleolin recognition because the binding site is a relatively large region containing potential stem and loop secondary structures. The finding that nucleolin binding to the ARE likely involves RBDs 1 and 2 suggests that the mode of binding may be similar to the interaction of nucleolin with the NRE, in which nucleolin binds to a stem and loop structure (
). This finding is consistent with the results reported by Ishimaru et al. (2009) which showed that the two proteins can bind concurrently to AREbcl-2 RNA in vitro. This supports the idea that nucleolin and HuR are present in common bcl-2 messenger ribonucleoprotein complexes involved in regulating the stability and translation of bcl-2 mRNA (
). Selective control of ARE mRNAs comes from the fact that ARE sequences are unique and are bound by specific ARE-binding factors, including stabilizing as well as destabilizing factors. Thus, the stability of an mRNA depends on the repertoire of RNA-binding proteins present in a particular cell under specific conditions. A number of studies have shown that abnormal control of mRNA stability can contribute to the development and/or maintenance of malignancies (for review, see Ref.
) demonstrated that AUF1 is involved in bcl-2 mRNA decay during apoptosis, specifically in response of Jurkat cells to UVC irradiation. Interestingly, when AUF1 was depleted from MV-4-11 S100 extracts, the rate of decay of bcl-2 mRNA was reduced (Fig. 5). Moreover, when nucleolin function was impaired by treatment of MV-4-11 cells with the aptamer AS1411, increased binding of AUF1 to bcl-2 mRNA was observed (Fig. 6) in the same time frame when lower bcl-2 mRNA levels have been detected (
), suggesting that nucleolin and AUF1 have opposing roles in the regulation of the bcl-2 mRNA stability. Even though our results suggest that AUF1 has a prominent role in stimulating bcl-2 mRNA degradation when nucleolin levels are low (Fig. 6), at this time one cannot rule out the possibility that both proteins could be present on the same bcl-2 mRNA molecule simultaneously, as has been shown with HuR and p37/AUF1 (
). Also, it is likely that other proteins, in addition to AUF1, play a role in the turnover of bcl-2 mRNA in some cells. For example, it was recently reported that Bcl-2 protein itself plays a role in regulating the decay of its cognate mRNA (
) demonstrated that CA repeats upstream of the ARE confer bcl-2 mRNA instability in the absence of an apoptotic stimulus in COS7 cells.
The AUF1 family consists of four splicing isoforms: the 45 kDa, which contains all exons; 42 kDa, which lacks exon 2; 40 kDa, which lacks exon 7; and 37 kDa, with both exons 2 and 7 deleted. The four isoforms have been described to differ in their ARE-binding affinities in vitro, with p37 having the highest affinity, followed by p42, p45, and finally by p40 (
) observed that UVC-induced apoptosis was associated with elevation of the levels of the p45 isoform of AUF1 and an increase in p45·bcl-2 ARE complex formation in Jurkat cells. Here, we have observed that only the 42-kDa isoform is UV-cross-linked to radio-labeled AREbcl-2 transcripts, even though all four isoforms are present in cytoplasmic extracts of MV-4-11 leukemia cells. Thus, bcl-2 regulation may involve an AUF1 isoform-specific mechanism in a cell type fashion.
Based on our findings, we propose a model (Fig. 8) of how bcl-2 mRNA decay may occur in normal cells as opposed to some cancer cells where nucleolin is overexpressed in the cytoplasm. According to the model, in normal cells bcl-2 mRNA is rendered intrinsically unstable through the action of AUF1 and possibly other factors, which bind to the ARE and recruit the exosome to the mRNA. Once the mRNA is deadenylated, it is rapidly degraded by the exosome (
). The bcl-2 mRNA decay pathway described here is likely a protective mechanism used by normal cells to avoid malignant transformation. A different scenario can be envisioned in cancer cells such as CLL cells. The presence of abnormal high levels of nucleolin in the cytoplasm may cause a shift in the balance of mRNA regulation toward stabilization rather than degradation of certain messages, such as bcl-2 mRNA.
In summary, our findings provide new insights into the mechanism of nucleolin-mediated overexpression of Bcl-2 in cancer cells, more specifically on the stabilization of bcl-2 mRNA. This information should aid efforts to exploit nucleolin as a target for developing new therapies active in a variety of cancers including acute myeloid leukemia and CLL.
We thank Dr. France Carrier for the purified GST-nucleolin and GST proteins and Dr. Nancy Maizels for the pMal-Nuc plasmids. We also thank David Burmeister for assistance with the ARE fragment binding assays, Dzmitry Fedarovich of the MUSC Protein Production Laboratory for the preparation of recombinant Nuc-His, and Dr. Visu Palanisamy, Dr. Jeff Wilusz, and John Anderson for helpful advice.