Mechanism of regulation of bcl-2 mRNA by nucleolin and A+U-rich element-binding factor 1 (AUF1).
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 (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.
Ishimaru
Daniella
D
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425.
Zuraw
Lisa
L
Department of Chemistry, The Citadel, Charleston, South Carolina 29409.
Ramalingam
Sivakumar
S
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425.
Sengupta
Tapas K
TK
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425.
Bandyopadhyay
Sumita
S
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425.
Reuben
Adrian
A
Division of Gastroenterology and Hepatology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425.
Fernandes
Daniel J
DJ
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425.
Spicer
Eleanor K
EK
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425. Electronic address: spicer@musc.edu.
D020413:3' Untranslated Regions; D052157:Aptamers, Nucleotide; D006367:HeLa Cells; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007938:Leukemia / Q000235:genetics / Q000401:mortality; D008954:Models, Biological; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000235:genetics; D020871:RNA Stability / Q000187:drug effects / Q000502:physiology*; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000097796:Nucleolin
D020413:3' Untranslated Regions; D052157:Aptamers, Nucleotide; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012334:RNA, Neoplasm; D016601:RNA-Binding Proteins; D000097796:Nucleolin; C513936:AGRO 100; C000630986:HNRNPD protein, human