Tissue-specific translation of murine branched-chain alpha-ketoacid dehydrogenase kinase mRNA is dependent upon an upstream open reading frame in the 5'-untranslated region.
The committed step in the pathway for leucine, isoleucine, and valine catabolism is catalyzed by branched-chain alpha-ketoacid dehydrogenase (BCKD). This multienzyme complex is itself regulated through reversible subunit phosphorylation by a specific kinase (BCKD-kinase). Although BCKD is present in the mitochondria of all mammalian cells, BCKD-kinase has a tissue-specific pattern of expression. Various experimental, nutritional, and hormonal conditions have been used to alter the expression of BCKD-kinase, yet little is known regarding the regulation of basal BCKD-kinase expression under normal conditions including the mechanism of its tissue specificity in any organism. Here we use tissue-derived cultured cells to explore the mechanisms used to control BCKD-kinase expression. Whereas the amount of BCKD-kinase protein is significantly higher in mitochondria from C2C12 myotubes than in BNL Cl.2 liver cells, gene transcription and stability of BCKD-kinase mRNA share similar properties in these two cell types. Our results show that the amount of protein synthesized is regulated at the level of translation of BCKD-kinase mRNA and that an upstream open reading frame in the 5'-untranslated region of this transcript controls its translation. The location and putative 19-residue peptide are conserved in the mouse, rat, chimpanzee, and human genes. Likewise, gene structure of mouse, chimpanzee, and human BCKD-kinase is conserved, whereas the rat gene has lost intron 9.
Graduate Program in Genetics and Molecular Biology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Eric A
EA
Muller
Dean J
DJ
Danner
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism; D003609:Dactinomycin / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007438:Introns; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D016366:Open Reading Frames; D002679:Pan troglodytes; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000096:biosynthesis* / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013997:Time Factors; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014162:Transfection
D020121:5' Untranslated Regions; D010455:Peptides; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D003609:Dactinomycin; D012313:RNA; D008156:Luciferases; D011494:Protein Kinases; C035627:(3-methyl-2-oxobutanoate dehydrogenase (lipoamide)) kinase; D003850:Deoxyribonuclease I