Functional dissection of the c-myc mRNA coding region instability determinant
The c-myc oncogene encodes a transcription factor that promotes cell proliferation and whose overexpression leads to cancer. As seen with other tightly regulated genes, c-myc mRNA contains AU-rich elements in its 3'-untranslated region (UTR) that maintain its expression at a low level by destabilizing the mRNA. In addition, the c-myc mRNA is unique in that it also contains a coding region destabilizing element that functions independently from typical 3'-UTR instability elements. This 249 nt coding region instability determinant (CRD) is bound by the IMP-1 RNA-binding protein and contains endonuclease cleavage sites identified in vitro. In order to characterize the cis-acting sequences responsible for the destabilizing function of this element, we produced a number of CRD deletions for studying the sequences required for both the in vitro protein-binding activity of IMP-1 and for the ability of the CRD to function as a destabilizing element in cells. The decay of a β-globin reporter mRNA containing the CRD was measured during HL-60 and K562 cell differentiation and arsenite-induced cell stress in an attempt to identify cell systems where the CRD was specifically targeted. The role of the IMP family as trans-acting factors on the destabilizing activity of the CRD was analyzed by measuring CRD-mediated decay following IMP overexpression or siRNA-mediated knock-down. Based on UV-crosslinking data with cell extracts, we also determined that IMP-1 is not the only protein to bind this region. We identify several of the additional RNA-binding proteins as AUF1, nucleolin, and UNR. We verified the ability of these proteins to interact with the CRD using recombinant proteins in RNA electromobility shift assays as well as examining the potential role of these proteins in CRD-mediated instability following siRNA-mediated knockdown in HL-60 cells. We conclude that while IMP-1, AUF1, nucleolin, and UNR can all bind the CRD, only AUF1 is directly involved in the destabilizing mechanism of the CRD. ^
Biology, Molecular|Biology, Cell
John Joseph Castorino,
"Functional dissection of the c-myc mRNA coding region instability determinant"
(January 1, 2008).
ETD Collection for Thomas Jefferson University.