Destabilising properties of the uuauuuauu element in the 3′ utr of plasminogen activator inhibitor type 2 (PAI-2) mRNA

Plasminogen activator inhibitor type 1 (PAJ-2) is a serine protease inhibitor involved in the modulation of urokinase-dependent proteolysis during cell differentiation and tissue remodelling In most human cells and tissues, PAI-2 gene remains silent under constitutive conditions but can be markedly induced by cytokines and growth factors. These agents strongly induce PAI-2 gene transcription, but their influence on post-transcriptionai events is still poorly understood. We have previously shown that the 3'UTR of PAI-2 significantly decreased the steady state level of chimeric β-globin/PAI-2 mRNAs in stably transfected cells This effect could be partially reversed when a UUAUUUAUU sequence in the 3'UTR of PAI-2 was disrupted (Maurer and Medcalf J. Biol. Chem. 271, pp 26074-26080, 1996). Here, we demonstrate that this AU-rich element (ARE) can independently decrease β-globin mRNA steady state level. We then analysed the decaying properties of the PAI-2 3 'UTR in a transiently inducible mRNA model, using the human growth hormone (HGH) reporter transcript placed under the control of the serum responsive c-fos promoter. In stably transfected NIH-3T3 cells, the Pc./ driven HGH mRNA has a half life of at least 16 hours, but insertion of PAI-2 3'UTR in its 3'UTR accelerates this decay to 4.5 hours. Mutagenesis of the nonameric ARE substantially stabilises the chimeric transcript (approx. 9 hrs). In another series of experiments using a constitutively expressed PAI-2 mRNA, quantitative analyses show that disruption of the UUAUUUAUU nonamer in the 3'UTR is sufficient to produce a two-fold increase of both PAI-2 mRNA and antigen levels. Finally, using an in vitro transcribed RNA encompassing the AU-rich motif as a probe and cytoplasmic extracts from both untreated or PMA-treated HT-1080 cells, we identify three specific mRNA-protein complexes in electromobility shift assays. Taken together, these observations underscore the important role of both the nonameric AU-rich element and frans-acting cytoplasmic factors in the posttranscriptional control of PAI-2 gene expression.