Novel cis-trans interactions are involved in post-transcriptional regulation of cyclin-dependent kinase inhibitor p21(WAF1/CIP1 )mRNA

BACKGROUND: A variety of pathways target CDKI p21(WAF1/CIP1 )expression at transcriptional, post-transcriptional as well as translational levels. We previously found that cell growth suppressing retinoid CD437 enhanced expression of p21(WAF1/CIP1 )and DNA damage inducible GADD45 proteins in part by elevating their mRNA stability. RESULTS: Here, we investigated molecular mechanisms of CD437-dependent post-transcriptional regulation of p21(WAF1/CIP1 )expression. By utilizing MDA-MB-468 HBC cells expressing chimeric rabbit β-globin-p21(WAF1/CIP1 )transcripts we mapped multiple CD437-responsive sequences located within positions 1195 to 1795 of the 3'-untranslated region of p21(WAF1/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 p21(WAF1/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. CONCLUSIONS: CD437 regulates cell growth in part by regulating stability of p21(WAF1/CIP1 )mRNA that involves specific RNA-protein interactions that are phosphorylation-dependent, while not requiring nascent transcription or protein synthesis.