Rat liver cholesterol 7 alpha-hydroxylase. Pretranslational regulation for circadian rhythm

A complete cDNA encoding cholesterol 7 alpha-monooxygenase (EC 1.14.13.17) which had been isolated from rat liver cDNA libraries by using specific antibodies to the enzyme (Noshiro, M., Nishimoto, M., and Okuda, K. (1989) FEBS Lett. 257, 97-100) was totally sequenced. The cDNA contained a 1,509-base pair open reading frame encoding 503 amino acid residues (Mr = 56,880) and an unusually long 3'-untranslated region rich in AT sequence in the total length of 3,545 base pairs. The predicted amino acid sequence displays less than 30% similarity to other sequenced cytochrome P-450s indicating that the 7 alpha-hydroxylase constitutes a novel family of cytochrome P-450. The AT-rich region often contained ATTTA motifs, 5'-AAT-3' or 5'-TAA-3' trinucleotides which were reported to be involved in rapidly degrading mRNA. Employing the specific antibodies and the cDNA as probes, a diurnal variation of the levels of the three factors, i.e. enzyme protein, mRNA, and enzyme activity, was studied on rat livers prepared at various times of the day. In normal animals, all three factors exhibited maximum level at 10:00 p.m. and minimum at 10:00 a.m. No significant sexual difference was observed. Cholestyramine feeding increased all three factors at 10:00 a.m. close to the maximum levels of the normal rats, but did not show a significant increase at 10:00 p.m. On the contrary, starvation markedly decreased all three factors either at 10:00 a.m. or at 10:00 p.m., while maintaining still the diurnal variation. A good correlation of the levels of mRNA to the enzyme activities and the protein levels demonstrates that pretranslational regulation is most likely a mechanism for the circadian rhythm of 7 alpha-hydroxylase. The marked diurnal fluctuation of the amount of protein and the level of mRNA also indicates their rapid turnover. The short half-life of mRNA could be correlated with the structure of the 3'-untranslated region of the mRNA characteristic of rapidly degrading mRNA, i.e. abundance of motif, AUUUA, and existence of 5'-AAU-3' or 5'-UAA-3' trinucleotides in single-stranded regions of the secondary structure.