Analysis of RNA-protein interactions of moose liver cytochrome P4502A5 mRNA

In our previous studies we have identified a 37/39 kDa, pyrazole inducible, cytochrome P4502A5 (CYP2A5) mRNA binding protein and provided evidence tha t it may play a role in the stabilization and processing of the RNA [Genest e, Rafalli and Lang (1996) Biochem. J. 313, 1029-1037; Thulke-Gross, Hergen hahn, Tilloy-Ellul, Lang and Bartsch (1998) Biochem. J. 331, 473-481]. Det ails of the RNA-protein interactions are, however, not known. In this repor t we have performed an analysis of the interaction between the CYP2A5 mRNA and the 37/39 kDa protein. With UV-cross linking experiments, using RNA pro ber; corresponding to various parts of the CYP2A5 mRNA, and with antisense oligonucleotides complementary to certain areas of the 3'-untranslated regi on (3'UTR), we could map the primary binding site to the tip of a 71 nt hai r-pin loop at the 3'-UTR. This analysis also showed that the protein may ha ve more than one site of interaction with the RNA and/or that, within the b inding region, there could be more than one protein molecule binding to the RNA. Analysis of the probable conformations of the various probes used in the UV cross-linking experiments, in combination with the estimated binding affinities of the protein to the different probes, suggests that important factors in the high-affinity binding are the UAG triplet flanked by GA-ric h sequences at the tip of the hair-pin loop, in addition to the conformatio n of the loop itself. Within the binding region, similarities with known bi nding sites of heterogeneous nuclear ribonucleoprotein (hnRNP)Al in other R NA molecules were revealed by sequence alignment analysis. Moreover, compet ition experiments with an oligoribonucleotide corresponding to a known high -affinity binding site of hnRNP Al, and immunoprecipitation of the UV cross -linked 37/39 kDa complex showed that the protein binding to the CYP2A5 mRN A could be hnRNP Al or its close analogue. It was also shown that the 37/39 kDa protein binds with less affinity to CYP2A4 mRNA than to CYP2A5 mRNA. T his is in accordance with experiments characterizing the binding site, sinc e these two otherwise highly homologous genes are kown to have a three nucl eotide difference within the region important for the high binding affinity . Since the response of CYP2A4 to pyrazole is known to be weak, as compared with CYP2A5, this observation provides further evidence for a regulatory r ole of the 37/39 kDa protein in CYP2A5 mRNA metabolism.