Regulation of Bcl-2 expression by HuR in HL60 leukemia cells and A431 carcinoma cells.
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.
Ishimaru
Daniella
D
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
Ramalingam
Sivakumar
S
Sengupta
Tapas K
TK
Bandyopadhyay
Sumita
S
Dellis
Stephanie
S
Tholanikunnel
Baby G
BG
Fernandes
Daniel J
DJ
Spicer
Eleanor K
EK
D000954:Antigens, Surface / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000473:pathology*; D002499:Centrifugation, Density Gradient; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D018922:HL-60 Cells; D006801:Humans; D047468:Immunoprecipitation; D007938:Leukemia / Q000235:genetics / Q000473:pathology*; D010750:Phosphoproteins / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D011485:Protein Binding; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism*; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics; D000097796:Nucleolin
D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D010750:Phosphoproteins; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D000097796:Nucleolin; C098770:ELAVL1 protein, human; C000715470:MAS1 protein, human