Reduction In mRNA Abundance Research Articles

Differential regulation of human α1-adrenoceptor subtypes

We have compared the agonist-induced down-regulation of human alpha1A-, alpha1B- and alpha1D-adrenoceptors upon stable expression in rat-1 fibroblasts. During a 24-h incubation the agonist phenylephrine downregulated alpha1A- and alpha1 -adrenoceptors in a concentration-dependent manner. While maximum downregulation was similar for both subtypes, the threshold concentration for significant reductions was markedly higher for alpha1A- than for alpha(1B-adrenoceptors (10 microM vs. 100 nM). The downregulation of both subtypes by 100 microM phenylephrine was time-dependent, and significant reductions were observed already after 2-4 h. In contrast, incubation of alpha1D-adrenoceptor-expressing cells with phenylephrine increased receptor number in a time- and concentration-dependent manner. The downregulation of alpha1B-adrenoceptors by 100 microM phenylephrine for 24 h was accompanied by a matching reduction in mRNA abundance, but no such reduction was seen for alpha-adrenoceptors. These treatment conditions also caused a functional desensitization of agonist-stimulated inositol phosphate formation for alpha1A- and alpha1B- but not for alpha1D-adrenoceptors. Treatment with the phorbol ester phorbol-12-myristate-13-acetate did not change receptor density or mRNA abundance and did not cause functional desensitization. We conclude that human alpha1-adrenoceptor subtypes are differentially regulated by agonist treatment even if they are expressed in the same cell line.

Cloning, Characterization, and Properties of Seven Triplet Repeat DNA Sequences

Several neuromuscular and neurodegenerative diseases are caused by genetically unstable triplet repeat sequences (CTG.CAG, CGG.CCG, or AAG.CTT) in or near the responsible genes. We implemented novel cloning strategies with chemically synthesized oligonucleotides to clone seven of the triplet repeat sequences (GTA.TAC, GAT.ATC, GTT.AAC, CAC.GTG, AGG.CCT, TCG.CGA, and AAG.CTT), and the adjoining paper (Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D.(1996) J. Biol. Chem. 271, 16784-16791) describes studies on TTA.TAA. This approach in conjunction with in vivo expansion studies in Escherichia coli enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of approximately 16 up to 158 triplets in both orientations with varying extents of polymorphisms. The inserts were characterized by DNA sequencing as well as DNA polymerase pausings, two-dimensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA conformations. AAG.CTT and AGG.CCT form intramolecular triplexes, and the other five repeat sequences do not form any previously characterized non-B structures. However, long tracts of TCG.CGA showed strong inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG.CAG and CGG.CCG (Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., and Wells, R. D.(1995) J. Biol. Chem. 270, 27014-27021). This work along with other studies (Wells, R. D.(1996) J. Biol. Chem. 271, 2875-2878) on CTG.CAG, CGG.CCG, and TTA.TAA makes available long inserts of all 10 triplet repeat sequences for a variety of physical, molecular biological, genetic, and medical investigations. A model to explain the reduction in mRNA abundance in Friedreich's ataxia based on intermolecular triplex formation is proposed.

Cyclic AMP regulates basement membrane heparan sulfate proteoglycan, perlecan, metabolism in rat glomerular epithelial cells.

Perlecan, the basement membrane heparan sulfate proteoglycan (HSPG), has been fully cloned from mouse and human tissues. When a cRNA probe of murine perlecan cDNA was employed in RNase protection assay to test whether rat glomerular epithelial cells (GEC) constitutively express perlecan, several bands of hybridization were seen, suggesting that sequences between rat and murine perlecan may not be identical. Using primers based on published cDNA sequences of murine and human perlecan and poly A+ RNA of rat GEC, we synthesized a 497 bp product (RPD-I) by RT-PCR. The deduced aminoacid sequence showed an 85% and 88% homology with domain I of murine and human perlecan, respectively. The three putative sites containing the consensus sequence SGD for attachment of heparan sulfate chains were fully conserved in the rat perlecan as was a site (NFT) for attachment of N-linked oligosaccharide. RPD-I detected a > 9.5 kb transcript of perlecan in RNA of GEC, similar in size to that present in rat glomeruli. Employing a riboprobe synthesized from RPD-I in RNase protection assay we examined whether dbcAMP regulated perlecan expression in the GEC. At 1, 6, 24 and 48 h of incubation, 1 mM dbcAMP caused 43%, 32%, 47% and 40% reduction in mRNA abundance of perlecan, respectively. Immunoprecipitation showed a corresponding reduction of 61%, 70% and 65% in the synthesis of 35SO4 labeled basement membrane HSPG by the GEC following 12, 24 and 48 h of incubation with dbcAMP. Following incubation for 1 and 24 h prostaglandins, PGE1 and PGE2 (1 uM), known activators of glomerular adenylate cyclase, reduced perlecan mRNA abundance to a similar extent as dbcAMP on northern analysis. Our results show that glomerular basement membrane HSPG synthesized by the GEC belongs to the perlecan family. Decrease of GEC perlecan gene expression and synthesis by cAMP and prostaglandins may be of relevance to proteinuric states characterized by activation of these mediators.

Low cytoplasmic mRNA levels of immunoglobulin kappa light chain genes containing nonsense codons correlate with inefficient splicing.

We have previously reported down-regulation of mRNA expression of some of the kappa light chain transgenes in a hybridoma derived from a secondary immune response. Of the five heavily mutated transgene copies present in that hybridoma, three included premature stop codons and were poorly represented at the mRNA level. Here we show that the nonsense mutations are the cause of the low mRNA levels. While we found no evidence that the reduction in mRNA abundance was attributable to an increased rate of cytoplasmic mRNA decay, the amount of cytoplasmic mRNA correlated with the accumulation of unspliced transcripts in the nucleus. Similar results were obtained with a chimeric immunoglobulin gene containing a premature chain termination codon in the variable gene segment. We suggest that inhibition of splicing induced by in-frame premature stop codons is an important mechanism for down-regulation of undesirable immunoglobulin transcripts.

Development of insulin sensitivity in rat skeletal muscle Studies of glucose transporter and insulin receptor mRNA levels

Expression of GLUT-4 and insulin receptor mRNAs was investigated in rat skeletal muscle by Northern hybridization. GLUT-4 mRNA was barely detectable in foetal muscle, was expressed at low levels by 1–8 days and at 2–3-fold higher levels during and after weaning (18–40 days). In contrast there was little change in insulin receptor mRNA levels prior to weaning and a reduction in mRNA abundance between 18 and 40 days. Weaning rats on to a diet rich in fat prevented the increase in GLUT-4 abundance seen between 15 and 29 days in animals weaned on a high-carbohydrate diet.