Abstract
bcl-2 mRNA contains an AU-rich element (ARE) that functions in regulating bcl-2 stability. Our earlier studies indicated that taxol- or okadaic acid-induced bcl-2 mRNA destabilization in HL-60 cells is associated with decreased binding of trans-acting factors to the ARE. To identify factors that play a role in the regulation of bcl-2 mRNA stability, bcl-2 ARE-binding proteins were purified from HL-60 cells. Three polypeptides of 100, 70, and 32 kDa were isolated from a bcl-2 ARE affinity matrix. Matrix-assisted laser desorption ionization mass spectroscopy analysis identified these proteins as full-length nucleolin and proteolytic fragments of nucleolin. RNA gel shifts assays indicated that recombinant nucleolin (residues 284-707) binds specifically to bcl-2 ARE RNA. In addition, recombinant nucleolin decreases the rate of decay of mRNA in HL-60 cell extracts in an ARE-dependent manner. Taxol or okadaic acid treatment of HL-60 cells results in proteolysis of nucleolin in a similar time frame as drug-induced bcl-2 mRNA down-regulation. These findings suggest that nucleolin functions as a bcl-2-stabilizing factor and that taxol and okadaic acid treatment induces apoptosis in HL-60 cells through a process that involves down-regulation of nucleolin and destabilization of bcl-2 mRNA.
Highlights
Many of the elements that regulate mRNA stability are located in the 3Ј-untranslated region (3Ј-UTR) of the mRNA
We have recently reported that HL-60 cell extracts contain proteins that bind to the first AU-rich element (ARE) in bcl-2 mRNA (ARE 1) (nucleotides 921–1057 of bcl-2 cDNA [1]) and that these proteins are inactive or decreased in HL-60 cells treated with taxol or okadaic acid for 32 h [8]
These studies suggested that the 70-kDa protein and possibly others binds and stabilizes bcl-2 mRNA in HL-60 cells
Summary
Many of the elements that regulate mRNA stability are located in the 3Ј-untranslated region (3Ј-UTR) of the mRNA (for review, see Ref. 9). To identify factors that play a role in the regulation of bcl-2 mRNA stability, bcl-2 ARE-binding proteins were purified from HL-60 cells. Taxol or okadaic acid treatment of HL-60 cells results in proteolysis of nucleolin in a similar time frame as drug-induced bcl-2 mRNA down-regulation.
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