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

Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis. Four human ACAT-1 mRNAs (7.0, 4.3, 3.6, and 2.8 kilobases (kb)) share the same short 5'-untranslated region (exon 1) and coding sequence (exons 2-15). The 4.3-kb mRNA contains an additional 5'-untranslated region (1289 nucleotides in length; exons Xa and Xb) immediately upstream from the exon 1 sequence. One ACAT-1 genomic DNA insert covers exons 1-16 and a promoter (the P1 promoter). A separate insert covers exon Xa (1277 base pairs) and a different promoter (the P7 promoter). Gene mapping shows that exons 1-16 and the P1 promoter sequences are located in chromosome 1, while exon Xa and the P7 promoter sequence are located in chromosome 7. RNase protection assays demonstrate three different protected fragments, corresponding to the 4.3-kb mRNA and the two other mRNAs transcribed from the two promoters. These results are consistent with the interpretation that the 4.3-kb mRNA is produced from two different chromosomes, by a novel RNA recombination mechanism involving trans-splicing of two discontinuous precursor RNAs.     

Regulator of G protein signaling 5 is highly expressed in parathyroid tumors and inhibits signaling by the calcium-sensing receptor

The molecular mechanisms responsible for aberrant calcium signaling in parathyroid disease are poorly understood. The loss of appropriate calcium-responsive modulation of PTH secretion observed in parathyroid disease is commonly attributed to decreased expression of the calcium-sensing receptor (CaSR), a G protein-coupled receptor. However, CaSR expression is highly variable in parathyroid adenomas, and the lack of correlation between CaSR abundance and calcium-responsive PTH kinetics indicates that mechanisms independent of CaSR expression may contribute to aberrant calcium sensing in parathyroid disease. To gain a better understanding of parathyroid tumors and the molecular determinants that drive parathyroid adenoma development, we performed gene expression profiling on a panel of 64 normal and neoplastic parathyroid tissues. The microarray data revealed high-level expression of genes known to be involved in parathyroid biology (PTH, VDR, CGA, CaSR, and GCM2). Moreover, our screen identified regulator of G protein signaling 5 (RGS5) as a candidate inhibitor of CaSR signaling. We confirmed RGS5 to be highly expressed in parathyroid adenomas relative to matched-pair normal glands. Transient expression of RGS5 in cells stably expressing CaSR resulted in dose-dependent abrogation of calcium-stimulated inositol trisphosphate production and ERK1/2 phosphorylation. Furthermore, we found that RGS5-nullizygous mice display reduced plasma PTH levels, an outcome consistent with attenuated opposition to CaSR activity. Collectively, these data suggest that RGS5 can act as a physiological regulator of calcium sensing by CaSR in the parathyroid gland. The abnormally elevated expression of RGS5 observed in parathyroid adenomas could thus represent a novel mechanism of CaSR desensitization in patients with primary hyperparathyroidism.     

A novel human insulin-like growth factor I messenger RNA is expressed in normal and tumor cells

We have identified and characterized a novel human insulin-like growth factor I (IGF-I) precursor from the transplantable T61 human breast cancer xenograft and from normal liver. The mRNA encoding this precursor contains a 5'-untranslated region that is 83% identical to the corresponding region of a previously described variant rat IGF-I. The nucleotide sequence of the cloned cDNA predicts an IGF-IA protein precursor of 137 amino acids, including a 32 residue signal peptide, 70 amino acid IGF-I, and a 35 residue COOH-terminal extension or E peptide. The exon encoding this variant maps in the genome between IGF-I exons 1 and 2, in a similar location to the homologous rat exon 1a. The rat and human exons 1a are 59% identical over 1443 nucleotides, with DNA sequence conservation occurring in a mosaic pattern. Human IGF-I mRNAs encoding this novel exon are expressed in liver, T61 tumor cells, and in an ovarian carcinoma cell line, NIH OVCAR3. These studies demonstrate that as in the rat, the human IGF-I gene contains six exons that are variably processed into multiple IGF-I mRNAs. The mechanisms responsible for generating different IGF-I mRNAs thus appear to be conserved among mammalian species.     

Nonconservative utilization of aldolase A alternative promoters

Recently, analysis of the sequence and expression of the human aldolase A gene revealed the unique arrangement of three tandem promoters and exons preceding a common coding sequence. A muscle-specific promoter (M) and two flanking widely used promoters (N and H) produce mRNA species which, in their mature forms, differ only in the sequence of their 5'-untranslated regions. We have isolated and investigated the expression of a mouse aldolase A gene. This mouse gene represents a functional gene by sequence analysis, recombinational screening, and by transfection into C2C12 cells. Although there is a high degree of sequence similarity between the mouse and the human gene in the region of the alternative first exons, we have been unable to detect a functional utilization of the 5'-most promoter (N) in the mouse. Steady state mRNAs isolated from a variety of adult tissues and cultured cells were analyzed by RNase protection and primer extension to identify first exon utilization. Consistent with previous reports, exon M is found only in skeletal muscle and exon H, the "housekeeping" exon, is utilized in every tissue where aldolase A is expressed. Under identical conditions we fail to see any evidence of the N exon. Therefore, although sequence homology exists between rodents and primates in the N region, the absence of selective pressure to preserve its primate pattern of expression may have resulted in functional promoter extinction.