Lodhi Khalid M, Ozdener Mehmet H, Shayiq Rass M
Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 2003 Oct 17;278(42):40647-57. doi: 10.1074/jbc.M302081200. Epub 2003 Aug 7.
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.
编码细胞色素P - 450c27（CYP27）的2.3kb mRNA具有一个出乎意料的长5'非翻译区（UTR），其中包含六个AUG，导致多个上游开放阅读框（uORF）。从第七个AUG开始翻译形成一个推定的55kDa前体，该前体在线粒体中加工形成一个52kDa的成熟蛋白。前三个AUG形成与第七个AUG读码框相同的完全重叠的uORF1、uORF2和uORF3，接下来的两个形成与第七个AUG读码框不同的完全重叠的uORF4和uORF5。尽管在正常条件下不被扫描核糖体识别，但第六个读码框相同的AUG形成了主要开放阅读框的一个推定的57kDa延伸。本研究的目的是确定5'UTR中仅指导CYP27 mRNA从第七个AUG进行翻译的元件。在COS细胞中表达5'缺失突变体表明，完整的5'UTR不仅指导从第七个AUG开始的翻译起始，还作为一个负调节因子。一个缺少uORF1的2kb缺失突变体从第六个和第七个AUG同等程度地起始翻译，形成57kDa和55kDa的前体蛋白，翻译速率增加2倍。然而，翻译的诱导并不影响线粒体中成熟的52kDa形式的水平，但会导致胞质溶胶中前体形式的积累，这在转染野生型cDNA的COS细胞中未见。终止uORF1的终止密码子的突变完全将翻译起始从第七个AUG转移到第一个AUG，形成一个67kDa的前体，该前体在线粒体中加工成一个52kDa的成熟蛋白。通过uORF1的表位作图对CYP27 mRNA的双顺反子性质的确认表明，CYP27 mRNA从第七个AUG的翻译是由uORF1的表达指导和调节的。
