Human Acyl-CoA:Cholesterol Acyltransferase-1 (ACAT-1) Gene Organization and Evidence That the 4.3-Kilobase ACAT-1 mRNA Is Produced from Two Different Chromosomes

Abstract

. The human ACAT-1 cDNA was cloned by a somatic cell and molecular genetic approach. Chinese hamster ovary (CHO) cell mutants lacking ACAT activity (including clone AC29) were isolated (8); subsequent stable transfection experiments showed that human genomic DNAs complemented the ACAT deficiency in AC29 cells (9). A 1.2-kb human genomic DNA fragment was cloned from the stable transfectants. This fragment (designated as G2 DNA) led to the eventual cloning of a full-length human ACAT cDNA K1 (4011 bp in length). Expression of this cDNA, designated as ACAT-1, in AC29 cells complemented the ACAT deficiency of the mutant (10). Additional results showed that expressing this cDNA in insect cells, which do not contain endogenous ACAT-like activity, produced high levels of ACAT activity in vitro, confirming that this cDNA encodes the catalytic component of ACAT enzyme (11). The coding region of the ACAT gene has been mapped to chromosome 1q 25 (12). Protein sequence analysis revealed the ACAT-1 protein as a hydrophobic protein containing multiple transmembrane domains and sharing several peptide regions in common with other acyltransferases (10). Recombinant human ACAT-1 protein expressed in CHO cells has been purified to homogeneity; the homogeneous ACAT-1 protein remains catalytically active and uses cholesterol as a substrate in a highly cooperative manner (13). Homologues of human ACAT-1 cDNA have also been cloned from other species (reviewed in Ref. 1), including two yeast homologues (14,15). Disruption of the ACAT-1 gene in mice has been reported (16); the ACAT-1 gene-deficient mice exhibit marked reduction in cholesteryl ester levels in only selective tissues and not in all the tissues examined. These and other results led to the molecular cloning of ACAT-2 cDNA (17-19). The predicted amino acid sequence of ACAT-2 is homologous but distinct from that of ACAT-1. The physiological roles of ACAT-1 and ACAT-2 in various tissues of different species are currently under intense investigation by several laboratories. In humans, immunodepletion experiments suggest that the ACAT-1 protein plays major catalytic roles in hepatocytes, adrenal glands, macrophages, and kidneys, but not in the intestines (20).The 4.0-kb human ACAT-1 cDNA contains a single open reading frame of 1.65 kb. It also contains an unusually long 5Ј-untranslated region (5Ј-UTR; 1396 bp) and 965 bp of 3Ј-untranslated region. Using the coding region as probe, North-

Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing

“…Trans-splicing refers to the recombination of sequences from two independently transcribed pre-mRNAs to form a composite mRNA. [1][2][3][4][5][6][7][8][9] We are developing a technology called spliceosome mediated RNA trans-splicing (SMaRT) to take advantage of the cell's endogenous splicing machinery as a strategy for modifying pre-mRNA. 10 The SMaRT process uses pretherapeutic RNA molecules (PTMs) that are designed to base pair with the intron of a targeted pre-mRNA to suppress target cis-splicing while enhancing trans-splicing between the PTM and target.…”

Modulation in vitro of H-ras oncogene expression by trans-splicing

“…It has been shown that a newly developed, natural splicing reaction (that is thought to involve unconventional splicing) might play a role in the expression of some mammalian genes, when performed in combination with the cis-splicing process (Akopian et al, 1999;Breen and Ashcroft, 1997;Caudevilla et al, 1998;Kawasaki et al, 1999;Li et al, 1999;Shimizu and Honjo, 1993;Sullivan et al, 1991;Vellard et al, 1992;Zaphiropoulos, 1999;Frantz et al, 1999). One example is rat carnitine octanoyltransferase (COT) pre-mRNA, which undergoes a natural trans-splicing process that yields three COT mRNAs: one with a duplication of the COT E2 exon (....E2-E2...); a second with a duplication of the COT E2-E3 exons (...E2-E3-E2-E3...) and a third with no exon duplication.…”

Exo-endo trans splicing: a new way to link

“…In contrast to the canonical SL-type trans splicing, which is a prerequisite for generating all mature mRNAs in trypanosomes, different variations of trans splicing patterns have been described in higher eukaryotes ( Figure 1): (1) intragenic trans splicing, in which two identical pre-mRNA molecules from the same gene are spliced together to generate an mRNA with duplicated exon sequences; (2) intergenic trans splicing, whereby an mRNA is generated from two pre-mRNAs that are derived from different genes, which can be located even on different chromosomes (interchromosomal trans splicing). A relatively well-characterized example for the latter case is the human ACAT1 gene, as previously described by Bo-Liang Li and colleagues [8]; importantly, characterization of trans splicing has not only been performed at the mRNA level, as in many other studies, but also been extended to the protein level and the effects on biochemical activities. In this case, exons of the ACAT1 mRNA are transcribed from two different chromosomes, namely 1 and 7, and the resulting 4.3-kb human ACAT1 mRNA encodes two functional protein isoforms (50-and 56-kDa in size) with differential enzymatic activities [8].…”

“…A relatively well-characterized example for the latter case is the human ACAT1 gene, as previously described by Bo-Liang Li and colleagues [8]; importantly, characterization of trans splicing has not only been performed at the mRNA level, as in many other studies, but also been extended to the protein level and the effects on biochemical activities. In this case, exons of the ACAT1 mRNA are transcribed from two different chromosomes, namely 1 and 7, and the resulting 4.3-kb human ACAT1 mRNA encodes two functional protein isoforms (50-and 56-kDa in size) with differential enzymatic activities [8]. Now, Hu et al [1] continued their molecular analyses of ACAT1 trans splicing en detail by combining the characterization of both mRNA and protein products of ACAT1.…”

“…Previously the same group has demonstrated that the 4.3-kb ACAT1 mRNA is produced through interchromosomal trans splicing [8]. Now they npg show that the chimeric mRNA derived from chromosomes 1 and 7 is required for the exo-endo trans splicing event (Figure 1).…”