Mechanism of Regulation of bcl-2 mRNA by Nucleolin and A+U-rich Element-binding Factor 1 (AUF1)

Abstract

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 halflife. 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 (ARE bcl-2 ). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to ARE bcl-2 . In RNA decay assays, ARE bcl-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 ARE bcl-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), 5 even though there is no evidence of gene rearrangements that are known to up-regulate bcl-2 transcription. Recently, Otake et al.(1) 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 3Ј-untranslated region (3Ј-UTR) of mRNAs is the AU-rich element (ARE) (for a review, see Ref.2). 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 3Ј-UTR that plays a role in regulating its stability (3, 4). The ARE bcl-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 destabili...

RNA-binding protein nucleolin in disease

“…34 In HL-60 leukemia cells, nucleolin and the RBP HuR bound the BCL2 ARE concurrently and had a synergistic effect on Bcl-2 expression, as both RBPs stabilized the BCL2 mRNA, while HuR also enhanced its translation; 35 by contrast, AUF1 antagonized nucleolin binding to the BCL2 ARE and triggered BCL2 mRNA decay. 36 Treatments with all-trans retinoic acid, taxol or okadaic acid enhanced nucleolin degradation and promoted BCL2 mRNA decay. 32,37 APP mRNA.…”

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells

“…Both of these anti-apoptotic proteins have been shown to play a role in beta cell survival [22,40]. The effect of AUF1 on BCL2 may be mediated, at least in part, by direct association of the RNA-binding protein to the AU-rich elements present in the 3′-UTR of Bcl2 mRNA, resulting in messenger destabilisation [21,41]. Indeed, we observed that silencing of AUF1 results in a small but significant increase in Bcl2 mRNA stability.…”

Modulation of Gene Expression by RNA Binding Proteins: mRNA Stability and Translation

“…For instance HuR, AUF1, and nucleolin bind BCL-2 mRNA. While nucleolin and HuR promote the stability, AUF1 enhances the degradation of BCL-2 mRNA [63,[86][87][88][89]. This implies that HuR and nucleolin have a cooperative effect which is antagonized by AUF1.…”

“…Unlike HuR, AUF1 promotes the degradation of the vast majority of its known targets through the recruitment of the mRNA to the exosome and the proteasome [23,24], for example: cell cycle related genes such as cyclin D1, p21, p27 and p16INK4a [59][60][61][62], apoptosis regulators such as B cell leukemia (BCL-2) and growth arrest and DNA-damageinducible protein alpha (GADD45α) [63,64]; inflammatory related genes such as granulocyte-macrophage colony-stimulating factor (GM-CSF): and interleukin 6 (IL6) and human inducible nitric oxide synthase (NOS) [65][66][67] and DNA replication and repair related genes such as thymidylate synthase (TYMS), jun D proto-oncogene (JUND and c-fos (FOS) [68][69][70]. However, AUF1 can promote the stability and translation of some target transcripts.…”