Data from Regulation of Bcl-2 Expression by HuR in HL60 Leukemia Cells and A431 Carcinoma Cells

Abstract

<div>Abstract<p>Overexpression of the proto-oncogene <i>bcl-2</i> promotes abnormal cell survival by inhibiting apoptosis. Expression of <i>bcl-2</i> is determined, in part, by regulatory mechanisms that control the stability of <i>bcl-2</i> mRNA. Elements in the 3′-untranslated region of <i>bcl-2</i> 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 <i>bcl-2</i> mRNA in HL60 cells. Here, we have identified HuR as a component of <i>bcl-2</i> messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to <i>bcl-2</i> mRNA <i>in vivo</i>. 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 <i>bcl-2</i> AU-rich element (ARE) RNA <i>in vitro</i>, suggesting separate binding sites for these proteins on <i>bcl-2</i> mRNA. Knockdown of HuR in A431 cells leads to down-regulation of <i>bcl-2</i> mRNA and protein levels. Observation of a decreased ratio of <i>bcl-2</i> mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating <i>bcl-2</i> stability. Recombinant HuR retards exosome-mediated decay of <i>bcl-2</i> ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of <i>bcl-2</i> 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 <i>bcl-2</i> ARE RNA. HuR knockdown also leads to redistribution of <i>bcl-2</i> mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of <i>bcl-2</i> mRNA translation and mRNA stability. (Mol Cancer Res 2009;7(8):1354–66)</p></div>