Identification and characterization of a 44 kDa protein that binds specifically to the 3'-untranslated region of CYP2a5 mRNA: inducibility, subcellular distribution and possible role in mRNA stabilization.
Stabilization of mRNA is important in the regulation of CYP2a5 expression but the factors involved in the process are not known [Aida and Negishi (1991) Biochemistry 30, 8041-8045]. In this paper, we describe, for the first time, a protein that binds specifically to the 3'-untranslated region of CYP2a5 mRNA and which is inducible by pyrazole, a compound known to increase the half-life of CYP2a5 mRNA. We also demonstrate that pyrazole treatment causes an elongation of the CYP2a5 mRNA poly(A) tail, and that phenobarbital, which is transcriptional activator of the CYP2a5 gene that does not affect the mRNA half-life, neither induces 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 protein-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 mRNA-binding protein is subject to post-translational regulation. Subcellular fractionation showed that the 44 kDa protein is present in polyribosomes and nuclei, and that its apparent induction is much stronger in polyribosomes than in nuclear extracts. We propose that this 44 kDa RNA-binding protein is involved in the stabilization of CYP2a5 mRNA by controlling the length of the poly(A) tail.
Geneste
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Program of Molecular Toxicology, International Agency for Research on Cancer, Lyon, France.
Raffalli
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Lang
M A
MA
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072467:Cytochrome P450 Family 2; D018076:DNA, Complementary / Q000235:genetics; D004355:Drug Stability; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008811:Mice, Inbred DBA; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010634:Phenobarbital / Q000494:pharmacology; D011061:Poly A / Q000737:chemistry; D011720:Pyrazoles / Q000494:pharmacology; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D018076:DNA, Complementary; D011720:Pyrazoles; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011061:Poly A; 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; D010634:Phenobarbital