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

• Published in: Biochimica et biophysica acta Vol. 1830; no. 3; pp. 2728 - 2738
• Main Authors: Akimoto, Chizuru, Sakashita, Eiji, Kasashima, Katsumi, Kuroiwa, Kenji, Tominaga, Kaoru, Hamamoto, Toshiro, Endo, Hitoshi
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2013
• Subjects: 5' Untranslated Regions; Abnormalities, Multiple - genetics; Abnormalities, Multiple - metabolism; Abnormalities, Multiple - pathology; Alternative Splicing; Amino Acid Sequence; Animals; bioinformatics; Cell Line, Tumor; codons; Gene Library; genes; Genes, Reporter; genetic disorders; Haplorhini; Heart Defects, Congenital - genetics; Heart Defects, Congenital - metabolism; Heart Defects, Congenital - pathology; Humans; Hydrocolpos - genetics; Hydrocolpos - metabolism; Hydrocolpos - pathology; immunoblotting; in vitro studies; luciferase; Luciferases; McKusick–Kaufman syndrome; membranes; messenger RNA; Mice; Mitochondria - genetics; Mitochondria - metabolism; mitochondrial membrane; Mitochondrial Proteins - genetics; Mitochondrial Proteins - metabolism; Mitochondrion; Molecular Sequence Data; mRNA processing; Northern blotting; Open Reading Frames; peptides; Polyadenylation; Polyadenylation signal; Polydactyly - genetics; Polydactyly - metabolism; Polydactyly - pathology; Protein Biosynthesis; protein products; rapid amplification of cDNA ends; Rats; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Alignment; translation (genetics); Translational regulation; Upstream open reading frame; Uterine Diseases - genetics; Uterine Diseases - metabolism; Uterine Diseases - pathology; MKKS; PS; mRNA processing; McKusick–Kaufman syndrome; Polyadenylation signal; Translational regulation; ESTs; Mitochondrion; UTR; IRES; RACE; uORF; Upstream open reading frame; BBS6; WT
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2012.12.010

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. 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. 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. 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. Our findings provide unique insights into production of uORF-derived peptides and functions of uORFs. ► MKKS mRNA has multiple uORFs and alternative polyadenylation sites at the 5′-UTR. ► The presence of uORFs at the MKKS 5′-UTR represses the translation of the main ORF. ► Two uORFs of the MKKS mRNA are translated as mitochondrial proteins in human cells.