Pyrazole-inducible proteins in DBA/2 mouse liver bind with high affinity to the 3'-untranslated regions of the mRNAs of coumarin hydroxylase (CYP2A5) and c-jun.
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.
Thulke-Gross
M
M
Division of Toxicology and Cancer Risk Factors, Deutsches Krebsforschungszentrum (DKFZ), D-69120 Heidelberg, Germany.
Hergenhahn
M
M
Tilloy-Ellul
A
A
Lang
M
M
Bartsch
H
H
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D003432:Cross-Linking Reagents; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072467:Cytochrome P450 Family 2; D004591:Electrophoresis, Polyacrylamide Gel; D016758:Genes, jun / Q000235:genetics*; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008811:Mice, Inbred DBA; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics*; D011132:Polyribosomes / Q000201:enzymology; D011720:Pyrazoles / Q000494:pharmacology*; D015347:RNA Probes; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D011720:Pyrazoles; D015347:RNA Probes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C031280:pyrazole; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585520:Cyp2a5 protein, mouse; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2