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Identification of a Novel cis-Element in the 3'-Untranslated Region of Mammalian Peptidylglycine -Amidating Monooxygenase Messenger Ribonucleic Acid

INSERM U-297, Faculté de Médecine Nord, IFR Jean Roche, 13916 Marseille, France

Address all correspondence and requests for reprints to: Dr. L’Houcine Ouafik, INSERM U-297, Faculté de Médecine Nord, IFR Jean Roche, boulevard Pierre Dramard, 13916 Marseille Cedex 20, France. E-mail: ouafik.h{at}jean-roche.univ-mrs.fr

Peptidylglycine -amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal -amidation of peptidylglycine substrates, yielding amidated products. Growing evidence suggests that the metabolism of PAM messenger RNAs (mRNAs) can be regulated within the cytoplasm. To understand the mechanisms controlling the metabolism of PAM mRNAs, we sought to identify cis elements of the 3'-untranslated region (3'-UTR) of PAM mRNA that are recognized by cytoplasmic factors. From gel retardation assays, one sequence element is shown to form a specific RNA-protein complex. The protein-binding site of the complex was determined by ribonuclease T1 mapping, by blocking the putative binding site with antisense oligonucleotide, and by competition assays. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 20-nucleotide cis element that interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) to form a 60-kDa PAM mRNA-binding protein complex. The binding activity was redox sensitive. Tissue distribution of the protein in the rat showed a marked tissue-specific expression, with ovary, testis, lung, heart septum, anterior pituitary and hypothalamus containing large amounts compared with liver, ventricle, atrium, and neurointermediate lobe. No binding activity was detectable in pancreas, intestine, or kidney extracts. Northwestern blot analysis of AtT-20 (mouse corticotrope tumor cell line) cytoplasmic extracts revealed a protein of 46 kDa. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 3'-UTR of PAM mRNA from many animal species. Although these data suggest that cis element-binding activity could be a cytoplasmic regulator of PAM mRNA metabolism, the functional consequences of this binding remain to be determined.

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