Abstract
Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3'-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to bcl-2 mRNA in vivo. We also observed an RNA-dependent coprecipitation of HuR and nucleolin, suggesting that the two proteins are present in common mRNP complexes. Moreover, nucleolin and HuR bind concurrently to bcl-2 AU-rich element (ARE) RNA in vitro, suggesting separate binding sites for these proteins on bcl-2 mRNA. Knockdown of HuR in A431 cells leads to down-regulation of bcl-2 mRNA and protein levels. Observation of a decreased ratio of bcl-2 mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating bcl-2 stability. Recombinant HuR retards exosome-mediated decay of bcl-2 ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of bcl-2 mRNA stability in HL60 cells, as well as in A431 cells. Addition of nucleolin and HuR to HL60 cell extracts produced a synergistic protective effect on decay of bcl-2 ARE RNA. HuR knockdown also leads to redistribution of bcl-2 mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of bcl-2 mRNA translation and mRNA stability.
Highlights
Bcl-2 protein belongs to a family of proteins that are fundamentally important in the regulation of apoptosis and tumorigenesis
Identification of HuR in AREbcl-2 RNA Complexes We have previously showed that nucleolin binds to the AREbcl-2 and plays a role in the regulation of bcl-2 mRNA stability [23, 24]
This suggests that the association of HuR and nucleolin is dependent on the presence of RNA, consistent with the concept that nucleolin and HuR are present in common messenger ribonucleoprotein complexes
Summary
Bcl-2 protein belongs to a family of proteins that are fundamentally important in the regulation of apoptosis and tumorigenesis. Bcl-2 was one of the first proto-oncogenes found to promote carcinogenesis by prolonging cell survival rather than by increasing the rate of cell proliferation [1]. Bcl-2 protein seems to exert its antiapoptotic activity by heterodimerizing with its proapoptotic partners BAX and BAK [2, 3], whereas its activity can be down-regulated by interaction with other proteins, such as the proapoptotic BAD protein [4]. It has been suggested that the balance of the levels of Bcl-2 family members is an important factor in determining cell fate [5]. The level of bcl-2 expression is a critical determinant of the sensitivity of cells to apoptotic stimuli. Not surpri‐ singly, a number of cancers have been described to have high levels of Bcl-2 protein [6,7,8], and elevated bcl-2 expression has been found to contribute to resistance to chemotherapeutic agents of some hematologic malignancies [9, 10]
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