IDENTIFICATION AND CHARACTERIZATION OF A 44-KDA PROTEIN THAT BINDS SPECIFICALLY TO THE 3'-UNTRANSLATED REGION OF CYP2A5 MESSENGER-RNA - INDUCIBILITY, SUBCELLULAR-DISTRIBUTION AND POSSIBLE ROLE IN MESSENGER-RNASTABILIZATION

Stabilization of mRNA is important in the regulation of CYP2a5 express ion but the factors involved in the process are not known [Aida and Ne gishi (1991) Biochemistry 30, 8041-8045]. In this paper, we describe, for the first time, a protein that binds specifically to the 3'-untran slated region of CYP2a5 mRNA and which is inducible by pyrazole, a com pound known to increase the half-life of CYP2a5 mRNA. We also demonstr ate that pyrazole treatment causes an elongation of the CYP2a5 mRNA po ly(A) tail, and that phenobarbital, which is transcriptional activator of the CYP2a5 gene that does not affect the mRNA half-life, neither i nduces the RNA-binding protein nor affects the poly(A) tail size. SDS/ PAGE of the UV-cross-linked RNA-protein complex demonstrated that the RNA-binding protein has an apparent molecular mass of 44 kDa. The prot ein-binding site was localized to a 70-nucleotide region between bases 1585 and 1655. Treatment of cytoplasmic extracts with an SH-oxidizing agent, diamide, an SH-blocking agent, N-ethylmaleimide or potato acid phosphatase abolished complex-formation, suggesting that the CYP2a5 m RNA-binding protein is subject to post-translational regulation. Subce llular fractionation showed that the 44 kDa protein is present in poly ribosomes and nuclei, and that its apparent induction is much stronger in polyribosomes than in nuclear extracts. We propose that this 44 kD A RNA-binding protein is involved in the stabilization of CYP2a5 mRNA by controlling the length of the poly(A) tail.