Novel cis-trans interactions are involved in post-transcriptional regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 mRNA

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.CDKI p21WAF1/CIP1, an important cell-cycle regulatory molecule, was originally identified as a gene regulated by the tumor suppressor protein p53 [1]. Subsequent studies also demonstrated induction of p21WAF1/CIP1 via mechanisms independent of p53 [[2], refs within]. Indeed, exposure of cells to a wide variety of stress agents leads to induction of p21WAF1/CIP1, which, in turn, participates in mediating cell-cycle arrest. It is thought that cell-cycle arrest due to induction of p21WAF1/CIP1 following exposure of cells to stress stimuli, functions as a protective factor in determining the survival of the cell. The mechanisms underlying stress-induced expression of p21WAF1/CIP1 have been found to