Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo

Alternative splicing of pre-mRNA is a commonly used mechanism to regulate gene expression in higher eukaryotes. However, with the exception of regulated cascades in Drosophila, the cis-acting elements and the trans-acting factors that control tissue- and/or developmentally regulated splicing remain largely unidentified. Cis-acting elements that control smooth muscle-specific repression of exon 3 of alpha-tropomyosin (alpha-TM) have been identified recently and consist of two regions that flank this exon. Deletion of either element causes misregulated splicing of alpha-TM in transfected smooth muscle cells. In experiments designed to characterize essential sequences within each element and the factors that interact with these sequences, we have identified two overlapping sequences within the downstream regulatory element (DRE) that are identical to binding sites for polypyrimidine tract binding protein (PTB) that were identified using iterative selection techniques. Mutation of these sites caused aberrant splicing regulation in transfected smooth muscle cells. In addition, sequences identical to high-affinity PTB binding sites were also detected upstream of exon 3 and mutation of these sites also resulted in misregulation of splicing in vivo, suggesting that PTB binding to specific sequences flanking exon 3 is responsible, in part, for the repression of exon 3. Consistent with this hypothesis, UV crosslinking and equilibrium binding assays confirm that the same mutations that cause misregulated splicing also disrupt PTB binding to RNA.     

An improved method for the production of antibodies to lipophilic carboxylic hapten using small amount of hapten-carrier conjugate

Normal human diploid cells have a limited proliferative lifespan in in vitro cultures. Changes in gene expression have been examined for understanding control mechanisms of limited proliferative lifespan. and enhanced expression of growth suppressing genes such as p21 was reported in late-passaged cells. We screened genes which were expressed preferentially in mid-passaged cells by the differential plaque screening of the subtracted cDNA libraries prepared from young, life-extended, and immortalized SV40-transformed human fibroblasts. Among isolated clones, ASF/SF2, which was known to affect alternative splicing, was expressed in normal fibroblasts with a peak at mid-passage. Relative expression levels of SC35 and hnRNPA1, which are also known to affect alternative splicing, was also highest at mid-passage. Changes in alternative splicing at mid-passage, if it occurred, may play a crucial role in the process of cellular senescence.     

Caldesmon

Caldesmon is a protein that is found in smooth muscle and in non-muscle cells. Two isoform classes produced by alternative splicing of one gene have been characterized. The smooth muscle, high molecular weight (89-93 kDa), caldesmon isoforms are exclusively found in adult and fully differentiated smooth muscle cells. The non-muscle, low molecular weight (59-63 kDa), caldesmon isoforms are found in non-muscle and in de-differentiated smooth muscle cells. The conserved regions of all isoforms contain caldesmon's properties such as binding to actin, tropomyosin, Ca(2+)-calmodulin, myosin and phospholipids. All isoforms are also very potent inhibitors of the actin-tropomyosin activated myosin MgATPase. Non-muscle and smooth muscle isoforms of caldesmon perform different roles in vivo. This may be reflected by the distinct cellular distribution of these isoform classes. Non-muscle caldesmon is a regulatory factor in the microfilament network and is thus involved in the assembly and stabilization of microfilaments. Smooth muscle caldesmon together with tropomyosin is a mediating factor for Ca(2+)-dependent inhibition of smooth muscle contraction.     

Autoxidation of pyridoxalated hemoglobin polyoxyethylene conjugate

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10(-5) M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.     

Splicing of 5' introns dictates alternative splice selection of acetylcholinesterase pre-mRNA and specific expression during myogenesis

Splicing of alternative exon 6 to invariant exons 2, 3, and 4 in acetylcholinesterase (AChE) pre-mRNA results in expression of the prevailing enzyme species in the nervous system and at the neuromuscular junction of skeletal muscle. The structural determinants controlling splice selection are examined in differentiating C2-C12 muscle cells by selective intron deletion from and site-directed mutagenesis in the Ache gene. Transfection of a plasmid lacking two invariant introns (introns II and III) within the open reading frame of the Ache gene, located 5' of the alternative splice region, resulted in alternatively spliced mRNAs encoding enzyme forms not found endogenously in myotubes. Retention of either intron II or III is sufficient to control the tissue-specific pre-mRNA splicing pattern prevalent in situ. Further deletions and branch point mutations revealed that upstream splicing, but not the secondary structure of AChE pre-mRNA, is the determining factor in the splice selection. In addition, deletion of the alternative intron between the splice donor site and alternative acceptor sites resulted in aberrant upstream splicing. Thus, selective splicing of AChE pre-mRNA during myogenesis occurs in an ordered recognition sequence in which the alternative intron influences the fidelity of correct upstream splicing, which, in turn, determines the downstream splice selection of alternative exons.     

Protein conformational changes determined by matrix-assisted laser desorption mass spectrometry

Caldesmon is an actin/calmodulin/tropomyosin protein located in the thin filaments of smooth muscle cells and microfilaments of nonmuscle cells. Two isoforms of caldesmon, h- and l-types, shown to exist in vertebrate smooth and nonmuscle cells respectively, are produced by alternative splicing of the caldesmon mRNA encoded by a single gene. To study the expression of smooth muscle specific h-caldesmon during the differentiation of mesenchymal cells into smooth muscle cells, soluble protein and total RNA from the gizzard primordium in the gut region of 5-day and gizzards of 7-, 9-, 13-, 17- and 21-day embryos and 2-days post-hatch chicks were extracted and analyzed for caldesmon expression at both protein and mRNA levels. Western blot analysis of proteins and immunofluorescence microscopy of tissue section were carried out using an antibody specific for h-caldesmon. Total RNA was analyzed by Northern blotting using a caldesmon cDNA probe, and h- and l-caldesmon cDNAs were identified due to the difference in their molecular sizes (4.8 and 4.1 kb respectively). The mRNA was also analyzed by reverse transcribed-polymerase chain reaction (RT-PCR) and Southern blot analysis. Our results show that the I-caldesmon mRNA was expressed at higher levels in the gizzard primordium during the early stages of development, and decreased gradually during growth. The h-caldesmon protein and mRNA, not expressed at day 5, is minimally expressed at day 7 and is fully turned on by day 9. Additionally, sequence analyses of the RT-PCR products of I-caldesmon showed that it lacked the spacer region, as predicted. RT-PCR analysis of total RNA gave two h-caldesmon fragments. These two fragments were identified as two different isoforms of h-caldesmon since they both contained the spacer region. They also showed homology in the region of exon 4 had differences in the region of exon 3b.     

Serum deprivation alters the expression and the splicing at exons 7, 8 and 15 of the beta-amyloid precursor protein in the C6 glioma cell line

Amyloid deposition characterizes the pathological lesions of Alzheimer's disease. We investigated the effect of serum deprivation on the regulation of beta-amyloid precursor protein (APP) mRNA expression in C6 glioma cells. Serum deprivation increased APP mRNA levels approximately 4-fold over controls. This increase was accompanied by changes in the pattern of alternative splicing, including the novel alternatively spliced site at exon 15. The proportion of isoforms containing exons 7 and 8 significantly increased from 61% to 68%, while isoforms lacking these exons decreased from 14% to 8%. The proportion of leukocyte-derived APP, which is a novel alternatively spliced isoform lacking exon 15, significantly increased from 19% to 40%. Among the six major isoforms produced by the two independent splicing sites, L-APP752 which contains exons 7 and 8, but lacks exon 15, increased the most (approximately 10-fold). Our findings provide evidence linking APP expression to alterations in alternative splicing at exon 15. These results demonstrate that in glial cells, APP mRNA regulation involves the alteration in alternative splicing at exons 7, 8 and 15, suggesting that not only increased expression but also an imbalance in the relative abundance of the six APP isoforms in stressed condition might affect the amyloidogenesis in Alzheimer's disease.     

Regulation of the expression of the tissue transglutaminase gene by DNA methylation

We have investigated the role of DNA methylation in the regulation of the expression of the human tissue transglutaminase gene. Studies on the methylation of the transglutaminase promoter in normal and neoplastic human cells demonstrated that the promoter is methylated in vivo and hypomethylation of the promoter is correlated with constitutive gene expression. Demethylation of the promoter in vivo by treatment of the cells with 5-azacytidine increased transglutaminase expression and hypermethylation of the promoter in vitro suppressed its activity. These studies suggest that alternations in DNA methylation may be one of the mechanisms regulating the tissue-specific expression of the tissue transglutaminase gene.