Specific trans-acting proteins interact with auxiliary RNA polyadenylation elements in the COX-2 3′-UTR
- Department of Biochemistry and Molecular Biology, New Jersey Medical School and Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA
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↵1 These authors contributed equally to this work.
Abstract
Two cyclooxygenase (COX) enzymes, COX-1 and COX-2, are present in human cells. While COX-1 is constitutively expressed, COX-2 is inducible and up-regulated in response to many signals. Since increased transcriptional activity accounts for only part of COX-2 up-regulation, we chose to explore other RNA processing mechanisms in the regulation of this gene. Previously, we showed that COX-2 is regulated by alternative polyadenylation, and that the COX-2 proximal polyadenylation signal contains auxiliary upstream sequence elements (USEs) that are very important in efficient polyadenylation. To explore trans-acting protein factors interacting with these cis-acting RNA elements, we performed pull-down assays with HeLa nuclear extract and biotinylated RNA oligonucleotides representing COX-2 USEs. We identified PSF, p54nrb, PTB, and U1A as proteins specifically bound to the COX-2 USEs. We further explored their participation in polyadenylation using MS2 phage coat protein-MS2 RNA binding site tethering assays, and found that tethering any of these four proteins to the COX-2 USE mutant RNA can compensate for these cis-acting elements. Finally, we suggest that these proteins (p54nrb, PTB, PSF, and U1A) may interact as a complex since immunoprecipitations of the transfected MS2 fusion proteins coprecipitate the other proteins.
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Footnotes
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Reprint requests to: Carol S. Lutz, Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, MSB E671, Newark, NJ 07103, USA; e-mail: lutzcs{at}umdnj.edu; fax: (973) 972-5594.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.577707.
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- Received March 22, 2007.
- Accepted April 5, 2007.
- Copyright © 2007 RNA Society