Abstract
A rare form of human ACAT1 mRNA, containing the optional long 5'-untranslated region, is produced as a 4.3-kelonucleotide chimeric mRNA through a novel interchromosomal trans-splicing of two discontinuous RNAs transcribed from chromosomes 1 and 7. To investigate its function, we express the chimeric ACAT1 mRNA in Chinese hamster ovary cells and show that it can produce a larger ACAT1 protein, with an apparent molecular mass of 56 kDa on SDS-PAGE, in addition to the normal, 50-kDa ACAT1 protein, which is produced from the ACAT1 mRNAs without the optional long 5'-untranslated repeat. To produce the 56-kDa ACAT1, acat1 sequences located at both chromosomes 7 and 1 are required. The 56-kDa ACAT1 can be recognized by specific antibodies prepared against the predicted additional amino acid sequence located upstream of the N-terminal of the ACAT1(ORF). The translation initiation codon for the 56-kDa protein is GGC, which encodes for glycine, as deduced by mutation analysis and mass spectrometry. Similar to the 50-kDa protein, when expressed alone, the 56-kDa ACAT1 is located in the endoplasmic reticulum and is enzymatically active. The 56-kDa ACAT1 is present in native human cells, including human monocyte-derived macrophages. Our current results show that the function of the chimeric ACAT1 mRNA is to increase the ACAT enzyme diversity by producing a novel isoenzyme. To our knowledge, our result provides the first mammalian example that a trans-spliced mRNA produces a functional protein.
Highlights
Acyl-coenzyme A:cholesterol acyltransferase (ACAT)1 is an intracellular enzyme that plays important roles in lipid metabolism
Two Human ACAT1 Proteins of Different Molecular Mass Can Be Produced from the Full-length ACAT1 cDNA K1—In our previous work, we had only examined the translation product of the 1.7-kb ACAT1 cDNA, which is composed of exons 1–16
We had previously reported that one of the four ACAT1 mRNAs, the 4.3-knt mRNA, contains an optional long 5Ј-UTR, and is probably produced by interchromosomal trans-splicing of two discontinuous pre-mRNAs [15]
Summary
Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that plays important roles in lipid metabolism. Human ACAT1 message and protein are present in many tissues and various cell types examined, including adrenal, kidney, hepatocytes, Kupffer cells, intestinal enterocytes, fibroblasts, macrophages, and neurons in the brain (5, 9 –12). The functional significance of finding both ACAT enzymes in the same cell types (i.e. hepatocytes, intestinal enterocytes, and macrophages) is not clear and is currently under investigation. Northern analyses have revealed the presence of four ACAT1 mRNAs (7.0, 4.3, 3.6, and 2.8-knt), present in almost all of the human tissues and cells examined. These messages share the same coding sequence. The results demonstrate the existence of a novel ACAT1 isoenzyme in transfected cells, as well as in human macrophage cells
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