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J. Biol. Chem., Vol. 278, Issue 42, 40647-40657, October 17, 2003

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The Upstream Open Reading Frame Mediates Constitutive Effects on Translation of Cytochrome P-450c27 from the Seventh In-frame AUG Codon in Rat Liver^*

Khalid M. Lodhi  , Mehmet H. Ozdener  ¶ and Rass M. Shayiq ||

From the Division of Clinical Pharmacology, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

The 2.3-kb mRNA that codes for cytochrome P-450c27 (CYP27) has an unexpectedly long 5'-untranslated region (UTR) that holds six AUGs, leading to several upstream open reading frames (uORFs). The initiation of translation from the seventh AUG forms a putative 55-kDa precursor, which is processed in mitochondria to form a 52-kDa mature protein. The first three AUGs form fully overlapping uORF1, uORF2, and uORF3 that are in-frame with the seventh AUG and next two form fully overlapping uORF4 and uORF5 that are out-of-frame with the seventh AUG. Although not recognized by the scanning ribosomes under normal conditions, the sixth in-frame AUG forms a putative 57-kDa extension of the main open reading frame. The purpose of this study was to identify the elements in the 5'-UTR that direct CYP27 mRNA translation exclusively from the seventh AUG. Expression of 5' deletion mutants in COS cells reveal that the intact 5'-UTR not only directs the initiation of translation from the seventh AUG but also acts as a negative regulator. A 2-kb deletion mutant that lacks uORF1 initiates translation equally from the sixth and the seventh AUGs, forming both 57- and 55-kDa precursor proteins with a 2-fold increase in rate of translation. However, induction in translation does not affect the levels of the mature 52-kDa form in mitochondria but causes accumulation of the precursor form in cytosol not seen in COS cells transfected with wild-type cDNA. Mutation of the stop codon that terminates uORF1 completely shifts the initiation of translation from the seventh to the first AUG, forming a 67-kDa precursor that is processed into a 52-kDa mature protein in mitochondria. Confirmation of the bicistronic nature of CYP27 mRNA by epitope mapping of uORF1 suggests that translation of CYP27 mRNA from the seventh AUG is directed and regulated by uORF1 expression.

Received for publication, February 27, 2003 , and in revised form, July 30, 2003.

^* This work was supported in part by United States Public Health Service Grant GM50290. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Indianapolis-Marion County Forensic Service Agency, 40 S. Alabama St., Indianapolis, IN 46204.

These authors contributed equally to this work.

^¶ Present address: Monell Chemical Senses Center, 3500 Market St., Philadelphia, PA 19104-3308.

^|| To whom correspondence should be addressed: E-mail: rmshayiq{at}hotmail.com.

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