An important mechanism in the up-regulation of cytochrome P-450 2A5 (CYP2A5, coumarin hydroxylase, Coh) is the stabilization of the corresponding mRNA; some evidence suggests that proteins binding to CYP2A5 mRNA may be involved in this stabilization. Here we report that pyrazole, a well known inducer of CYP2A5 and stabilizer of its message, enhances the binding of a set of proteins to 32P-labelled 3´-untranslated region (3´UTR) of CYP2A5 to give 32P-labelled bands of apparent molecular mass 37/39, 45/48 and 70/72 kDa after UV cross-linking/RNase cleavage; in addition, we found different proteins binding to other parts of CYP2A5 mRNA. The 70/72 kDa bands are also formed with the 3´UTR of c-jun. The inducible proteins are found in different cellular subfractions at different concentrations, with a maximum of five-fold induction of binding activity in microsomes. When a gel-mobility-shift assay was combined with UV cross-linking to resolve different pyrazole-inducible RNA–protein complexes into single RNA-binding protein bands, the smallest complex contained a double band of 37/39 kDa, 45/48 kDa bands, 70/72 kDa bands, and additional weaker bands at higher molecular masses (around 120 kDa). This composition was found also for all other complexes detected by gel-mobility-shift assay; occasionally, bands at higher molecular masses were also observed. The proteins of the smallest complex might therefore represent a core with which other proteins interact to build up larger complexes. Binding of proteins 37/39 kDa and 70/72 kDa was located to a 20-base loop and adjacent sequences in a 70 nt AU-rich region of the 3´UTR of the CYP2A5. Based on our previous evidence, this 70-nt sequence may play an important role in the stabilization and processing of the message.

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