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PubMed2001

Rna-binding protein Musashi2: developmentally regulated expression in neural precursor cells and subpopulations of neurons in mammalian CNS.

Musashi1 (Msi1) is a mammalian neural RNA-binding protein highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic and postnatal CNS development. Here, we identified Musashi2 (Msi2), a novel mammalian RNA-binding protein that exhibits high sequence similarity to Msi1. The Msi2 transcript appeared to be distributed ubiquitously in a wide variety of tissues, consistent with the mRNA distribution of its Xenopus homolog, xrp1. However, the present study revealed cell type-specific and developmentally regulated expression of Msi2 in the mammalian CNS. Interestingly, Msi2 was expressed prominently in precursor cells in the ventricular zone and subventricular zone with the same pattern as Msi1 throughout CNS development. In the postnatal and adult CNS, this concurrent expression of Msi2 and Msi1 was seen in cells of the astrocyte lineage, including ependymal cells, a possible source for postnatal CNS stem cells. During neurogenesis, the expression of both Msi2 and Msi1 was lost in most postmitotic neurons, whereas Msi2 expression persisted in a subset of neuronal lineage cells, such as parvalbumin-containing GABA neurons in the neocortex and neurons in several nuclei of the basal ganglia. Msi2 may have a unique role that is required for the generation and/or maintenance of specific neuronal lineages. Furthermore, in vitro studies showed that Msi2 and Msi1 have similar RNA-binding specificity. These two RNA-binding proteins may exert common functions in neural precursor cells by regulating gene expression at the post-transcriptional level.

Alternative Splicing↗
PubMed2001

Characterization of two evolutionarily conserved, alternatively spliced nuclear phosphoproteins, NFAR-1 and -2, that function in mRNA processing and interact with the double-stranded RNA-dependent protein kinase, PKR.

We report here the isolation and characterization of two proteins, NFAR-1 and -2, which were isolated through their ability to interact with the dsRNA-dependent protein kinase, PKR. The NFAR proteins, of 90 and 110 kDa, are derived from a single gene through alternative splicing and are evolutionarily conserved nuclear phosphoproteins that interact with double-stranded RNA. Both NFAR-1 and -2 are phosphorylated by PKR, reciprocally co-immunoprecipitate with PKR, and colocalize with the kinase in a diffuse nuclear pattern within the cell. Transfection studies indicate that the NFARs regulate gene expression at the level of transcription, probably during the processing of pre-mRNAs, an activity that was increased in fibroblasts lacking PKR. Subsequent functional analyses indicated that amino acids important for NFAR's activity were localized to the C terminus of the protein, a region that was found to specifically interact with FUS and SMN, proteins also known as regulators of RNA processing. Accordingly, both NFARs were found to associate with both pre-mRNAs and spliced mRNAs in post-transcriptional studies, similar to the known splicing factor ASF/SF-2. Collectively, our data indicate that the NFARs may facilitate double-stranded RNA-regulated gene expression at the level of post-transcription and possibly contribute to host defense-related mechanisms in the cell.

Alternative Splicing↗
PubMedDate not supplied

Possible participation of pICln in the regulation of angiogenesis through alternative splicing of vascular endothelial growth factor receptor mRNAs.

In this study, the authors applied a modified Antisense Display method to human vascular endothelial cells (ECs) in culture to isolate new angiostatic genes. Screening of a 10mer antisense oligodeoxyribonucleotide (oligo) repertoire identified a subpool that consistently stimulated EC growth. Subsequent screening of oligos with increasing chain length led to the isolation of a unique growth-stimulatory 14mer, 5'-TTCCACATCATATT-3'. cDNA/EST data-base search and expression analyses in ECs indicated pICln as the corresponding gene. A longer unique antisense oligo against a different region of pICln mRNA was found to also enhance EC growth and tube formation and to decrease mRNAs for soluble Flt-1 and neuropilin-1 vascular endothelial growth factor (VEGF) receptors, the angiostatic factors that are generated by alternative RNA splicing. Conversely,pICln overexpression suppressed EC growth and increased the mRNAs for both soluble Flt-1 and soluble neuropilin-1. The present findings thus suggest that pICln plays a role in autocrine regulation of angiogenesis, probably through alternative splicing.

Alternative Splicing↗
PubMed2002

Inhibition of system A amino acid transport activity by ethanol in BeWo choriocarcinoma cells.

OBJECTIVE: Our purpose was to investigate the influence of ethanol on system A amino acid transporter in BeWo cells. STUDY DESIGN: BeWo cells were cultured in the absence or presence of ethanol. The function of system A was monitored by the transport of alpha-(methylamino)isobutyric acid. Messenger RNA levels for system A were assessed by Northern analysis. RESULTS: Treatment of BeWo cells with ethanol reduced the activity of system A. The effect was dose and treatment time dependent. The decrease in system A activity was 38% +/- 3% at 0.75% ethanol with a 16-hour treatment time. The activities of several other transporters tested were not affected. The effect on system A activity was associated with a decrease in the maximal velocity of the transport system without affecting the substrate affinity. Ethanol did not alter the messenger RNA levels for system A. CONCLUSION: Exposure of BeWo cells to ethanol significantly reduces the function of system A. This finding has potential implications that may be relevant to the pathogenesis of the fetal alcohol syndrome.

Amino Acid Transport System A↗
PubMed2005

ENaC proteins are required for NGF-induced neurite growth.

Neurite growth is required for nervous system development and repair. Multiple signals, including neurotrophic factors and intact mechanosensing mechanisms, interact to regulate neurite growth. Degenerin/epithelial Na(+) channel (DEG/ENaC) proteins have been identified as putative mechanosensors in sensory neurons. Recently, others have shown that the neurotrophic factor NGF stimulates expression of acid-sensing ion channel molecules, which are members of the DEG/ENaC family. However, it is unknown whether NGF regulates ENaC expression or whether ENaC expression is required for neurite formation. Therefore, the aims of the present study were to determine whether ENaC expression is 1) regulated by NGF and 2) required for NGF-induced neurite growth in pheochromocytoma PC-12 cells. We found NGF-induced expression of beta- and gamma-subunits of ENaC, but not alpha-ENaC. Tyrosine kinase A (TrkA) receptor blockade abolished NGF-induced beta- and gamma-ENaC expression and neurite formation. NGF-induced neurite formation was inhibited by disruption of ENaC expression using 1) pharmacological blockade with benzamil, a specific ENaC inhibitor; 2) small interfering RNA; and 3) dominant-negative ENaC molecules. These data indicate NGF-TrkA regulation of ENaC expression may be required for neurite growth and may suggest a novel role for DEG/ENaC proteins in neuronal remodeling and differentiation.

Amiloride↗
PubMed1994

Differential regulation of slow-skeletal and cardiac troponin I mRNA during development and by thyroid hormone in rat heart.

We have examined mRNA levels for the cardiac troponin I (cTnI) and slow-skeletal (ssTnI) in perinatal rat hearts. Northern blots showed that hypothyroidism was associated with a delay in the expected isoform switching. RNA slot blots showed a six-fold increase in cTnI mRNA from day 3 to day 21 in hearts from postnatal euthyroid rats compared to a three-fold increase in cTnI for the same period in the hypothyroid hearts. On the other hand, ssTnI mRNA levels were higher after 3 days in hearts from the hypothyroid animals and fell to undetectable levels after 21 days. In euthyroid hearts ssTnI was not detectable after 14 days and was not re-expressed in adult hearts. In the ventricles from 28-day-old animals the most significant differences in cTnI mRNA levels were between the euthyroid and T3-treated hypothyroid preparations and in the 87 to 125-day-old group between euthyroid and hypothyroid ventricles. T3 treatment of the 87 to 125-day-old hypothyroid animals did not increase the cTnI mRNA above euthyroid levels despite elevated serum T3. These results show that thyroid hormone influences expression of the cTnI isoform in postnatal and young adult rats, but not to the same extent in animals greater than 28 days of age.

Animals↗
PubMed2003

A Hox gene mutation that triggers nonsense-mediated RNA decay and affects alternative splicing during Drosophila development.

Nonsense mutations are usually assumed to affect protein function by generating truncated protein products. Nonetheless, it is now clear that these mutations affect not just protein synthesis but also messenger RNA stability. The surveillance mechanism responsible for the detection and degradation of 'nonsense' RNA messages is termed nonsense-mediated RNA decay (NMD). Essential biochemical components of the NMD machinery have been defined in several species. Here we identify the Drosophila orthologue of one of these factors, Upf1, and document its expression during embryogenesis. To test whether NMD acts during Drosophila development, we make use of a mutation that introduces a stop codon into a variably spliced exon of the Hox gene Ultrabithorax (Ubx). Using real-time quantitative RT-PCR we demonstrate that Ubx transcripts containing the premature stop codon are expressed at lower levels than their wild type counterpart. Unexpectedly, we also find that the same mutation significantly increases the levels of a Ubx splicing isoform that lacks the exon containing the premature termination codon. These findings indicate that NMD is operational during Drosophila development and suggest that nonsense mutations may affect development by altering the spectrum of splicing products formed, as well as by reducing or eliminating protein synthesis.

Alternative Splicing↗
PubMed2006

Expression of p53 and p53/47 are controlled by alternative mechanisms of messenger RNA translation initiation.

P53 controls the growth and survival of cells by acting in response to a multitude of cellular stresses. It is, however, not yet fully understood how different p53 activation pathways result in either cell cycle arrest or apoptosis. We and others have described an N-terminally truncated p53 protein (p53/47) originating from a second translation initiation site in the p53 messenger RNA (mRNA), which can interact with p53 and impose altered stability and transactivation properties to p53 complexes. Here we show that cap-dependent and cap-independent mechanisms of initiation govern the translation of the p53 mRNA. Changes in synthesis of full-length p53 or p53/47 are regulated through distinct cell stress-induced pathways acting through separate regions of the p53 mRNA. We also show that some cytotoxic drugs require the presence of full-length p53 to induce apoptosis, whereas for others p53/47 is sufficient. This indicates that by harbouring alternative translation initiation sites, the p53 mRNA gives rise to different levels of the p53 isoforms which help to orchestrate the cell biological outcome of p53 activation in response to different types of cell stress. This sheds new light into the way p53 can integrate and differentiate a large multiplicity of changes in the cellular environment.

5' Untranslated Regions↗
PubMed2006

Molecular cloning of multiple forms of the ovine B7-2 (CD86) costimulatory molecule.

To facilitate analysis of the role of costimulatory molecules in the ovine immune system, we cloned and sequenced eight putative alternatively spliced transcripts of the sheep CD86 (B7-2) costimulatory molecule. Using RT-PCR and rapid amplification of cDNA ends (RACE), we cloned the sheep CD86 (B7-2) molecule that encodes eight forms, which differ in the length of the signal peptide, the presence or absence of a transmembrane region and in their cytoplasmic tails. Comparison of the deduced amino acid sequence of the largest ovine CD86 TM form (CD86-2) with the sequence of cattle, pig, human and mouse CD86 indicated that the deduced protein had a higher degree of similarity to cattle (85% of amino acid identity) than to pig (77%), human (59%), and mouse sequence (45% of identity). Our results indicate that mRNA transcripts encoding different CD86 protein forms are expressed in sheep, like in other mammals, and suggest that the expression of this gene may be regulated at the transcriptional or RNA splicing level, which could give rise to tissue-specific expression of CD86. It is possible that, in the sheep, these CD86 mRNA variants could play different regulatory roles in T cell activation.

Alternative Splicing↗
PubMed2004

Zinc and cadmium specifically interfere with RNA-binding activity of human iron regulatory protein 1.

The cellular pro-oxidative stress induced by high zinc concentrations or cadmium is most likely mediated by disruption of redox (mainly thiol) homeostasis or by mishandling of redox-active transition metals. The impact of zinc and cadmium on the main regulators of iron homeostasis in metazoans, the iron regulatory proteins (IRP) 1 and 2, has been probed with the human recombinant proteins. Using purified proteins or extracts of yeast producing human IRP, zinc and cadmium were shown to interfere with the IRE-binding activity of IRP1, but not with that of IRP2 or the aconitase activity of IRP1. The IRP1 active site cysteines in positions 437, 503 and 506 were not directly involved in the effects of zinc and cadmium. The loss of RNA-binding activity is due to the reversible and specific aggregation of the IRP1 apoprotein with zinc and cadmium, since precipitation did not occur with other divalent metals such as manganese, cobalt or magnesium. The reported data suggest a new mechanism for the biological toxicity of cadmium and high zinc concentrations by interference with iron metabolism.

Cadmium↗
PubMed2006

Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy.

RNA-mediated pathogenesis is a recently developed disease model that proposes that certain types of mutant genes produce toxic transcripts that inhibit the activities of specific proteins. This pathogenesis model was proposed first for the neuromuscular disease myotonic dystrophy (DM), which is associated with the expansion of structurally related (CTG)(n) and (CCTG)(n) microsatellites in two unrelated genes. At the RNA level, these expansions form stable hairpins that alter the pre-mRNA splicing activities of two antagonistic factor families, the MBNL and CELF proteins. It is unclear which altered activity is primarily responsible for disease pathogenesis and whether other factors and biochemical pathways are involved. Here, we show that overexpression of Mbnl1 in vivo mediated by transduction of skeletal muscle with a recombinant adeno-associated viral vector rescues disease-associated muscle hyperexcitability, or myotonia, in the HSA(LR) poly(CUG) mouse model for DM. Myotonia reversal occurs concurrently with restoration of the normal adult-splicing patterns of four pre-mRNAs that are misspliced during postnatal development in DM muscle. Our results support the hypothesis that the loss of MBNL1 activity is a primary pathogenic event in the development of RNA missplicing and myotonia in DM and provide a rationale for therapeutic strategies designed either to overexpress MBNL1 or inhibit MBNL1 interactions with CUG and CCUG repeat expansions.

Alternative Splicing↗
PubMed2000

Identification of GRY-RBP as an apolipoprotein B RNA-binding protein that interacts with both apobec-1 and apobec-1 complementation factor to modulate C to U editing.

C to U editing of apolipoprotein B (apoB) mRNA involves the interaction of a multicomponent editing enzyme complex with a requisite RNA sequence embedded within an AU-rich context. This enzyme complex includes apobec-1, an RNA-specific cytidine deaminase, and apobec-1 complementation factor (ACF), a novel 65-kDa RNA-binding protein, that together represent the minimal core of the editing enzyme complex. The precise composition of the holo-enzyme, however, remains unknown. We have previously isolated an enriched fraction of S100 extracts, prepared from chicken intestinal cells, that displays apoB RNA binding and which, following supplementation with apobec-1, permits efficient C to U editing. Peptide sequencing of this most active fraction reveals the presence of ACF as well as GRY-RBP, an RNA-binding protein with approximately 50% homology to ACF. GRY-RBP was independently isolated from a two-hybrid screen of chicken intestinal cDNA. GRY-RBP binds to ACF, to apobec-1, and also binds apoB RNA. Experiments using recombinant proteins demonstrate that GRY-RBP binds to ACF and inhibits both the binding of ACF to apoB RNA and C to U RNA editing. This competitive inhibition is rescued by addition of ACF, suggesting that GRY-RBP binds to and sequesters ACF. As further evidence of the role of GRY-RBP, rat hepatoma cells treated with an antisense oligonucleotide to GRY-RBP demonstrated an increase in C to U editing of endogenous apoB RNA. ACF and GRY-RBP colocalize in the nucleus of transfected cells and, in cotransfection experiments with apobec-1, each appears to colocalize in a predominantly nuclear distribution. Taken together, the results indicate that GRY-RBP is a member of the ACF gene family that may function to modulate C to U RNA editing through binding either to ACF or to apobec-1 or, alternatively, to the target RNA itself.

APOBEC-1 Deaminase↗
PubMed2005

Corticotropin-releasing hormone (CRH) and urocortin act through type 1 CRH receptors to stimulate dehydroepiandrosterone sulfate production in human fetal adrenal cells.

CONTEXT: Near term, the human fetal adrenal increases the production of cortisol and dehydroepiandrosterone sulfate (DHEAS). DHEAS, which acts as substrate for placental estrogen production, induces key changes involved in parturition. OBJECTIVE: The objective of this study was to determine quantitatively the effect of CRH on mRNA levels of enzymes needed for DHEAS production (steroidogenic acute regulatory protein, CYP11A, CYP17, and SULT2A1), to determine the CRH receptor (CRH-R) subtype(s) responsible for CRH action, and to determine the effect of CRH on CRH-R mRNA expression in human adrenal fetal zone (FZ) cells. DESIGN: Human adrenal FZ cells were treated with CRH, ACTH, urocortin (Unc), and CRH antagonists, and RNA was analyzed by microarray and real-time RT-PCR. SETTING: This study was performed at an academic research laboratory. MAIN OUTCOME MEASURE: The main outcome measure was the expression of steroidogenic enzymes and CRH-R. RESULTS: Microarray analysis of human FZ cells treated for 24 h with CRH or ACTH showed increased mRNA expression levels of the genes needed for DHEAS production. Real-time RT-PCR analysis confirmed these data. Induction was lost in the presence of CRH-R1 antagonists, but not CRH-R2 antagonists. Stimulation was reproduced by Unc. The CRH-R1alpha mRNA splice variant was the only type 1 receptor isoform expressed in the fetal adrenal, and treatment with CRH up-regulates its mRNA levels. CONCLUSIONS: CRH, Unc, and ACTH stimulate all elements of the DHEAS synthetic pathway and activate CRH-R1 as well. The resulting increased DHEAS levels can be used for placental estrogen synthesis and contribute to the process leading to parturition in humans.

Adrenal Glands↗
PubMed2000

Heart-specific splice-variant of a human mitochondrial ribosomal protein (mRNA processing; tissue specific splicing).

It has been proposed that splice-variants of proteins involved in mitochondrial RNA processing and translation may be involved in the tissue specificity of mitochondrial DNA disease mutations (Fischel-Ghodsian, 1998. Mol. Genet. Metab. 65, 97-104). To identify and characterize the structural components of mitochondrial RNA processing and translation, the Mammalian Mitochondrial Ribosomal Consortium has been formed. The 338 amino acid (aa) residues long MRP-L5 was identified (O'Brien et al., 1999. J. Biol. Chem. 274, 36043-36051), and its transcript was screened for tissue specific splice-variants. Screening of the EST databases revealed a single putative splice-variant, due to the insertion of an exon consisting of 89 nucleotides prior to the last exon. Screening of multiple cDNA libraries revealed this inserted exon to be present only in heart tissue, in addition to the predominant MRP-L5 transcript. Sequencing of this region confirmed the EST sequence, and showed in the splice-variant a termination triplet at the beginning of the last exon. Thus the inserted exon replaces the coding sequence of the regular last exon, and creates a new 353 aa long protein (MRP-L5V1). Sequence analysis and 3D modeling reveal similarity between MRP-L5 and threonyl-t-RNA synthetases, and a likely RNA binding site within MRP-L5, with the C-terminus in proximity to the RNA binding site. Sequence analysis of MRP-L5V1 also suggests a likely transmembrane domain at the C-terminus. Thus it is possible that the MRP-L5V1 C-terminus could interfere with RNA binding and may have gained a transmembrane domain. Further studies will be required to elucidate the functional significance of MRP-L5V1.

Amino Acid Sequence↗
PubMed2004

Blotting analysis of native IRP1: a novel approach to distinguish the different forms of IRP1 in cells and tissues.

Iron regulatory protein 1 (IRP1) is a bifunctional protein, which either has aconitase activity or binds to specific mRNA structures to regulate the expression of iron proteins. Using recombinant human IRP1, we found that the two functional forms are resolved by nondenaturing polyacrylamide gel electrophoresis and that they are distinguished from IRP1/RNA complexes. This allowed us to use specific antibodies to develop a blotting system that recognized the iron-free and iron-containing IRP1 forms in the soluble fraction and the RNA-bound IRP1 in the high-speed precipitate fraction of cell extracts. The system was used to study IRP1 in HeLa, K562 cells, and monocytes/macrophages before and after treatment with iron salts, iron chelators, or hydrogen peroxide, as well as in stomach and duodenum biopsies. The results showed that iron-bound aconitase IRP1 is by far the prevalent form in most cells and that the major effect of cellular iron modifications is a shift between free and RNA-bound IRP1. The fraction of RNA-bound IRP1 was highly variable among different cells and was often a minor one. Furthermore, blotting showed that electrophoretic mobility shift assay, as commonly used, tends to under-evaluate the amount of total IRP1 and to over-evaluate the actual RNA-binding activity of IRP1. In conclusion, blotting analysis of IRP1 is a new, useful, and convenient method to analyze the amount and conformations of the protein that reveals previously undetected differences in IRP1 compartmentalization among various cell types.

Blotting, Western↗
PubMed1998

Expression patterns of voltage-dependent calcium channel alpha 1 subunits (alpha 1A-alpha 1E) mRNA in rat retina.

The transcript levels of the genes encoding for the different alpha1 (alpha1A-alpha1E) subunits of voltage-dependent calcium channels (VDCCs) were studied in the retina of the rat using RT-PCR, Northern blotting, and in situ hybridization. Abundant expression of alpha1A and alpha1B was found with RT-PCR and on Northern blots of total retina RNA, corresponding with high expression levels in all nuclear layers (outer and inner nuclear layers and the ganglion cell layer) of the retina. VDCC alpha1D mRNA was also present in all nuclear layers of the retina but at less abundant levels than alpha1A or alpha1B. Expression level of alpha1C in the retina was low as deduced from a faint Northern blot signal and a moderate yield after PCR amplification. VDCC alpha1E specific amplification of retinal cDNA yielded a longer product (designated alpha1E-L) than obtained from the hippocampus. Nucleotide sequencing of this PCR product revealed a 129 bp insert which is largely homologous (97%) with a previously described insert in the same position in human alpha1E cDNA. In situ hybridization in rat brain showed a differential expression pattern of the long and short variants of alpha1E mRNA. Northern blotting of retinal RNA confirmed the absence of the short variant (alpha1E-S), while alpha1E-L was present at low levels. In situ hybridization detected a significant level of expression of alpha1E-L in the inner nuclear layer. The prevalent expression of alpha1A and alpha1B, and to a lesser extent, of alpha1D, indicates that P/Q-, N-, and L-type calcium currents play a prominent role in the various cell types involved in the retinal signal-transduction pathway. The absence of alpha1C transcript in the retina suggests that the slowly inactivating L-type calcium currents involved in neurotransmitter release from the terminals of photoreceptors and bipolar cells may be encoded by the alpha1D isoform.

Alternative Splicing↗
PubMed2002

Prolonged hypoxia upregulates vascular endothelial growth factor messenger RNA expression in ovine fetal membranes and placenta.

OBJECTIVE: In ovine fetuses, 4 days of hypoxia resulted in a large increase in urine flow, without the development of polyhydramnios, which suggests that intramembranous absorption of the amniotic fluid was enhanced. Because vascular endothelial growth factor is speculated to be a regulator of intramembranous absorption through increases of membrane vascularity and fluid transport, we hypothesized that hypoxia upregulated vascular endothelial growth factor gene expression in the fetal membranes. STUDY DESIGN: Five near-term ovine fetuses that were subjected to 4 days of hypoxia and 5 age-matched time controls were studied. On day 4, the amnion, chorion, and placenta were collected for cellular localization and quantification of vascular endothelial growth factor messenger RNA and for the determination of vascular endothelial growth factor molecular forms that were expressed. The data were analyzed statistically with the use of t tests and 2-factor analyses of variance. RESULTS: Vascular endothelial growth factor messenger RNA was expressed in the fetal membranes localized to the amniotic epithelium and chorionic cytotrophoblast, and to the villous cytotrophoblast of the placenta. In hypoxic fetuses, vascular endothelial growth factor messenger RNA levels in these cell layers were significantly increased compared with the controls. Five vascular endothelial growth factor molecular forms were identified with vascular endothelial growth factor(164) being the most abundant form expressed. The pattern of expression of the forms was not altered by hypoxia. CONCLUSION: In the near-term ovine fetus, hypoxia induced vascular endothelial growth factor messenger RNA expression in the amnion, chorion, and placenta. This was associated with an increase in intramembranous absorption of amniotic fluid. We speculate that the increased intramembranous absorption was mediated by a vascular endothelial growth factor-induced increase in the transport of amniotic fluid into the fetal membranes.

Animals↗
PubMed2004

Translational control of the rat angiotensin type 1a receptor by alternative splicing.

The rat angiotensin type 1a receptor (AT1aR) is comprised of three exons. Two transcripts are possible due to alternative splicing of exon 2 (E1,3 and E1,2,3). Both transcripts code for identical AT1aR proteins since they differ only in the length of their 5' leader sequence (5'LS). We investigated the functional differences of these two transcripts in stably transfected Chinese hamster ovary (CHO) cells and also determined the splice variant composition in rat tissues. E1,3 expressing cells exhibited 1.8-fold higher AT1R densities and five-fold higher levels of Ang II-stimulated inositol phosphate production compared to E1,2,3 expressing cells. No differences in E1,3 and E1,2,3 mRNA levels or mRNA stability were seen. In vitro translation assays revealed 1.8-fold higher AT1aR protein levels from E1,3 compared to E1,2,3 transcripts, suggesting exon 2 reduces functional AT1R expression by inhibiting translation. Deletion of 10 nucleotides in exon 2 increased translation of the mutated E1,2,3 transcript to levels which were indistinguishable from E1,3, suggesting that this loop region of a predicted hairpin contributes to the inhibitory RNA cis element within exon 2. Comparison of AT1aR exonic composition and AT1R densities in rat tissues suggests alternative splicing is regulated in a tissue-specific manner and contributes to tissue-specific differences in AT1R density.

Adrenal Cortex↗