Nucleic Acids Research Advance Access published online on January 30, 2007
Nucleic Acids Research, doi:10.1093/nar/gkl1148
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CRD-BP shields c-myc and MDR-1 RNA from endonucleolytic attack by a mammalian endoribonuclease
Chemistry Program, University of Northern British Columbia, 3333 University Way, Prince George, BC V2N 4Z9, Canada
*To whom correspondence should be addressed. Tel: +250 960 5413; Fax: +250 960 5170; Email: leec{at}unbc.ca
Received October 9, 2006. Revised November 29, 2006. Accepted December 17, 2006.
The c-myc mRNA coding region determinant-binding protein (CRD-BP) has high affinity for the coding region determinant (CRD) of c-myc mRNA. Such affinity is believed to protect c-myc CRD from endonucleolytic attack. We have recently purified a mammalian endoribonuclease which can cleave within the c-myc CRD in vitro. The availability of this purified endonuclease has made it possible to directly test the interaction between CRD-BP and the endonuclease in regulating c-myc CRD RNA cleavage. In this study, we have identified the coding region of MDR-1 RNA as a new target for CRD-BP. CRD-BP has the same affinity for c-myc CRD nts 1705–1886 and MDR-1 RNA nts 746–962 with Kd of 500 nM. The concentration-dependent affinity of CRD-BP to these transcripts correlated with the concentration-dependent blocking of endonuclease-mediated cleavage by CRD-BP. In contrast, three other recombinant proteins tested which had no affinity for c-myc CRD did not block endonuclease-mediated cleavage. Finally, we have identified RNA sequences required for CRD-BP binding. These results provide the first direct evidence that CRD-BP can indeed protect c-myc CRD cleavage initiated by an endoribonuclease, and the framework for further investigation into the interactions between CRD-BP, c-myc mRNA, MDR-1 mRNA and the endoribonuclease in cells.