Translational repression of the McKusick–Kaufman syndrome transcript by unique upstream open reading frames encoding mitochondrial proteins with alternative polyadenylation sites

article i nfo Background: Upstream openreadingframes(uORFs)arecommonlyfoundinthe5'-untranslatedregion(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 wereused for determining thelength of transcripts and their 3'ends. Luciferase assay and invitrotranslation 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 mem- brane fractionof HeLacells.Databasesearchesindicated that such uORFs with activealternative 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-derivedpeptidesand functions of uORFs.