Novel cis-trans interactions are involved in post-transcriptional regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 mRNA
1 Department of Internal Medicine, Wayne State University and John D. Dingell VA Medical Center, Room B4325, 4646 John R, Detroit, MI 48201, USA
2 Department of Surgery and Karmanos Cancer Institute, Wayne State University, and John D. Dingell VA Medical Center, Room B4245, 4646 John R, Detroit, MI 48201, USA
3 Department of Internal Medicine, Wayne State University and John D. Dingell VA Medical Center, Room B4324, 4646 John R, Detroit, MI 48201, USA
4 Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA
5 Karmanos Cancer Institute, Department of Internal Medicine, Wayne State University and John D. Dingell VA Medical Center, Room B4325, 4646 John R, Detroit, MI 48201, USA
6 Departments of Obstetrics & Gynecology, and Biochemistry University of Western Ontario Schulich School of Medicine and Dentistry, 4th Floor Victoria Research Labs, A4-130a 800 Commissioners Road East, ON N6C 2V5, London
Journal of Molecular Signaling 2010, 5:12 doi:10.1186/1750-2187-5-12Published: 12 August 2010
A variety of pathways target CDKI p21WAF1/CIP1 expression at transcriptional, post-transcriptional as well as translational levels. We previously found that cell growth suppressing retinoid CD437 enhanced expression of p21WAF1/CIP1 and DNA damage inducible GADD45 proteins in part by elevating their mRNA stability.
Here, we investigated molecular mechanisms of CD437-dependent post-transcriptional regulation of p21WAF1/CIP1 expression. By utilizing MDA-MB-468 HBC cells expressing chimeric rabbit β-globin-p21WAF1/CIP1 transcripts we mapped multiple CD437-responsive sequences located within positions 1195 to 1795 of the 3'-untranslated region of p21WAF1/CIP1 mRNA. Several cytoplasmic proteins present in MDA-MB-468, MCF-7 HBC as well as HL-60R leukemia cells bound specifically, in vitro, with these CD437-responsive sequences. CD437 treatment of cells resulted in elevated binding of ~85 kD and ~55 kD cytoplasmic proteins with putative CD437-responsive sequences. A 12 nt RNA sequence (5'-UGUGGUGGCACA-3') present within CD437-responsive region of p21WAF1/CIP1 mRNA displayed specific and elevated binding with the above noted proteins. Treatment of cells with ActD or CHX prior to CD437 exposure did not abrogate RNA-protein interactions. However, treatment of cytoplasmic protein extracts with proteinase K or alkaline phosphatase resulted in loss of RNA-protein interactions.
CD437 regulates cell growth in part by regulating stability of p21WAF1/CIP1 mRNA that involves specific RNA-protein interactions that are phosphorylation-dependent, while not requiring nascent transcription or protein synthesis.