Elements of the rat tropoelastin gene associated with alternative splicing

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.     

Biochemical and genetic aspects of 7-dehydrocholesterol reductase and Smith-Lemli-Opitz syndrome

In recent years, several inherited human disorders caused by defects in cholesterol biosynthesis have been identified. These are characterized by malformations, multiple congenital anomalies, mental and growth retardation and/or skeletal and skin abnormalities indicating a pivotal role of cholesterol in morphogenesis and embryonic development. The first recognized and most common of these developmental disorders is Smith-Lemli-Opitz syndrome, an autosomal recessive trait caused by mutations in the DHCR7 gene resulting in a deficiency of the encoded sterol Delta(7)-reductase, alternatively called 7-dehydrocholesterol reductase (EC 1.3.1.21). This enzyme catalyzes the final step in cholesterol biosynthesis, which is the reduction of the Delta(7) double bond of 7-dehydrocholesterol to produce cholesterol.     

Multiple sites of alternative splicing of the rat fibronectin gene transcript

We describe analyses of the structure and expression of the rat fibronectin gene with particular attention to the 40-kb stretch from the center of the gene which encodes 17 type-III repeating units. Each repeat is precisely separated from its neighbors by introns and most are encoded by pairs of exons. Three repeats are encoded precisely by single exons and two of these (EIIIA and EIIIB) are alternatively spliced in a cell type-specific fashion. A third site of alternative splicing (EIIIB) reported here is similar in expression to the previously described EIIIA segment. Both are excluded from mRNA in liver cells and are, therefore, absent from plasma fibronectin. These two alternative splices, plus a third one (V) reported previously, can occur in all possible combinations giving 12 fibronectin mRNAs from a single gene. These splicing variations account for most but not all of the known fibronectin subunit variants. We report investigations designed to detect other regions of alternative splicing. We also show that the pattern of alternative splicing is somewhat altered on oncogenic transformation.     

Effects of Tween 80 on liver microsomal 7-dehydrocholesterol reductase

Enzymatic conversion of 7-dehydrocholesterol to cholesterol by liver microsomes was increased by addition of Tween 80. This increase was proportional to Tween 80 concentration, and reached its maximum of 250% of baseline activity after addition of 300 μg/ml of Tween 80. This enhancement was comparable to that achieved by addition of 7.4 mg/ml of cytosol protein. No additive effect was observed with Tween 80 combined with cytosol protein. These data suggest that Tween 80 can substitute for sterol carrier protein 2 in the conversion of 7-dehydrocholesterol to cholesterol.     

A novel mutation of the human 7-dehydrocholesterol reductase gene reduces enzyme activity in patients with holoprosencephaly

Defects in cholesterol biosynthesis genes are recognized as a leading cause for holoprosencephaly (HPE). Previous reports suggest that mutations of human 7-dehydrocholesterol reductase (Dhcr7), which catalyzes the final step of cholesterol biosynthesis, may cause HPE [Clin. Genet. 53 (1998) 155]. To determine whether Dhcr7 mutations are involved in HPE pathogenesis, we analyzed the sequence of exon 9, which contains both a catalytic domain and a mutational hot spot. We examined 36 prematurely terminated fetuses with HPE at their gestation ages in the range from 21 to 33 weeks by single strand conformation polymorphism analysis and DNA sequencing. A novel missense mutation was identified: G344D. Dhcr7 enzyme assays using overexpressed recombinant mutant proteins revealed altered enzyme activity. Mutant G344D harbored less than 50% of enzyme activity compared with the control. Two previously reported mutations, R404C and G410S, abolished enzyme activity. These results suggest that mutation of the Dhcr7 gene is involved in HPE pathogenesis.     

Negative regulation of Hedgehog signaling by the cholesterogenic enzyme 7-dehydrocholesterol reductase

Cholesterol regulates Hedgehog (Hh) signaling during early vertebrate development. Smith-Lemli-Opitz syndrome (SLOS) is caused by defects in 7-dehydrocholesterol reductase (DHCR7), an enzyme catalyzing the final step of cholesterol biosynthesis. Many developmental malformations attributed to SLOS occur in tissues and organs where Hh signaling is required for development, but the precise role of DHCR7 deficiency in this disease remains murky. We report that DHCR7 and Sonic Hedgehog (Shh) are co-expressed during midline development in Xenopus embryos. DHCR7 has previously been implicated to function as a positive regulator of Hh signaling that acts to regulate the cholesterol adduction of Hh ligand or to affect Hh signaling in the responding cell. We present gain- and loss-of-function analyses suggesting that DHCR7 functions as a negative regulator of Hh signaling at the level or downstream of Smoothened (Smo) and affects intracellular Hh signaling. Our analysis also raises the possibility that the human condition SLOS is caused not only by disruption of the enzymatic role of DHCR7 as a reductase in cholesterol biosynthesis, but may also involve defects in DHCR7 resulting in derepression of Shh signaling.     

Light regulates alternative splicing of hydroxypyruvate reductase in pumpkin

Hydroxypyruvate reductase (HPR) is a leaf peroxisomal enzyme that functions in the glycolate pathway of photorespiration in plants. We have obtained two highly similar cDNAs for pumpkin HPR (HPR1 and HPR2). It has been revealed that two HPR mRNAs might be produced by alternative splicing from a single type of pre-mRNA. The HPR1 protein, but not the HPR2 protein, was found to have a targeting sequence into leaf peroxisomes at the C-terminus, suggesting that alternative splicing controls the subcellular localization of the two HPR proteins. Immunoblot analysis and subcellular fractionation experiments showed that HPR1 and HPR2 proteins are localized in leaf peroxisomes and the cytosol, respectively. Moreover, indirect fluorescence microscopy and analyses of transgenic tobacco cultured cells and Arabidopsis thaliana expressing fusion proteins with green fluorescent protein (GFP) revealed the different subcellular localizations of the two HPR proteins. Both mRNAs were induced developmentally and by light, but with quantitative differences. Almost equal amounts of the mRNAs were detected in pumpkin cotyledons grown in darkness, but treatment with light greatly enhanced the production of HPR2 mRNA. These findings indicate that light regulates alternative splicing of HPR mRNA, suggesting the presence of a novel mechanism of mRNA maturation, namely light-regulated alternative splicing, in higher plants.     

Neonatal urinary steroids in Smith-Lemli-Opitz syndrome associated with 7-dehydrocholesterol reductase deficiency.

The biosynthetic abnormality in Smith-Lemli-Opitz syndrome (SLOS) is a deficiency of 7-dehydrocholesterol (7DHC) reductase, the enzyme responsible for catalyzing the final step in the Kandutsch-Russell pathway for cholesterol synthesis. Because the disposition of 7DHC and 8-dehydrocholesterol [8DHC; cholesta-5,8(9)-dien-3beta-ol] produced in this syndrome is little understood, we have analyzed urine from three young infants by gas chromatography/mass spectrometry to characterize its steroid metabolites. All steroid metabolites of adrenal origin found in normal infant urine were also found in urine from the patients with SLOS but in reduced amount. Quantitatively, the major steroids in these SLOS patients were identified by mass spectrometry as homologs of normal neonatal steroids possessing an additional double bond. Generally, two forms of each steroid were present in a similar amount. Because of the markedly increased levels of 7DHC and 8DHC in SLOS, these almost certainly represented the 5,7 and 5,8(9) unsaturated forms of each metabolite. The most abundant steroids were tentatively identified as 3beta,16alpha-dihydroxy-5,7-pregnadien-20-one and 3beta,16alpha-dihydroxy-5,8(9)-pregnadien-20-one, although similar 21-hydroxylated steroids and homologs of 16alpha-hydroxy-DHEA were also found. This study shows that all enzymatic steps used by cholesterol in the DHEA synthetic pathway are also functional for 7DHC and 8DHC.