1. Translational repression of the McKusick-Kaufman syndrome transcript by unique upstream open reading frames encoding mitochondrial proteins with alternative polyadenylation sites.
- Author
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Akimoto C, Sakashita E, Kasashima K, Kuroiwa K, Tominaga K, Hamamoto T, and Endo H
- Subjects
- Abnormalities, Multiple metabolism, Abnormalities, Multiple pathology, Amino Acid Sequence, Animals, Cell Line, Tumor, Gene Library, Genes, Reporter, Haplorhini, Heart Defects, Congenital metabolism, Heart Defects, Congenital pathology, Humans, Hydrocolpos metabolism, Hydrocolpos pathology, Luciferases, Mice, Mitochondria genetics, Mitochondria metabolism, Mitochondrial Proteins metabolism, Molecular Sequence Data, Polyadenylation, Polydactyly metabolism, Polydactyly pathology, Protein Biosynthesis, RNA, Messenger metabolism, Rats, Sequence Alignment, Uterine Diseases metabolism, Uterine Diseases pathology, 5' Untranslated Regions, Abnormalities, Multiple genetics, Alternative Splicing, Heart Defects, Congenital genetics, Hydrocolpos genetics, Mitochondrial Proteins genetics, Open Reading Frames, Polydactyly genetics, RNA, Messenger genetics, Uterine Diseases genetics
- Abstract
Background: Upstream open reading frames (uORFs) are commonly found in the 5'-untranslated region (UTR) of many genes and function in translational control. However, little is known about the existence of the proteins encoded by uORFs, and the role of the proteins except translational control. There was no report about uORFs of the McKusick-Kaufman syndrome (MKKS) gene that causes a genetic disorder., Methods: Northern blotting, 3'-RACE, and bioinformatics were used for determining the length of transcripts and their 3' ends. Luciferase assay and in vitro translation were used for evaluation of translational regulatory activity of uORFs. Immunoblotting and immunocytochemical analyses were used for detection of uORF-derived protein products and their subcellular localization., Results: The MKKS gene generates two types of transcripts: a canonical long transcript that encodes both uORFs and MKKS, and a short transcript that encodes only uORFs by using alternative polyadenylation sites at the 5'-UTR. The simultaneous disruption of the uORF initiation codons increased the translation of the downstream ORF. Furthermore, both protein products from the two longest uORFs were detected in the mitochondrial membrane fraction of HeLa cells. Database searches indicated that such uORFs with active alternative polyadenylation sites at the 5'-UTR are atypical but surely exist in human transcripts., Conclusions: Multiple uORFs at the 5'-UTR of the MKKS long transcript function as translational repressor for MKKS. Two uORFs are translated in vivo and imported onto the mitochondrial membrane., General Significance: Our findings provide unique insights into production of uORF-derived peptides and functions of uORFs.
- Published
- 2013
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