Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins

SR proteins are a family of essential splicing factors required for early recognition of splice sites during spliceosome assembly. They also function as alternative RNA splicing factors when overexpressed in vivo or added in excess to extracts in vitro. SR proteins are highly phosphorylated in vivo, a modification that is required for their function in spliceosome assembly and splicing catalysis. Here we show that SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation. We further show that the virus-encoded protein E4-ORF4 activates dephosphorylation by protein phosphatase 2A of HeLa SR proteins and converts their splicing properties into that of SR proteins purified from late adenovirus-infected cells. Taken together, our results suggest that E4-ORF4 is an important factor controlling the temporal shift in adenovirus alternative RNA splicing. We conclude that alternative pre-mRNA splicing, like many other biological processes, is regulated by reversible protein phosphorylation.     

Prostanoid receptor heterogeneity through alternative mRNA splicing

Prostaglandin (PG) and thromboxane (TX) receptors are G-protein coupled receptors that mediate the physiological actions of the five principal prostanoid metabolites: PGD2, PGE2, PGF2alpha, PGI2 (prostacyclin) and TXA2. Five major subdivisions of the prostanoid receptor family have been defined pharmacologically which correspond to each of the metabolites as follows: DP, EP, FP IP and TP. The EP receptors have been further classified pharmacologically into the EP1, EP2, EP3 and EP4 subtypes. Molecular biological studies have resulted in the cloning of cDNA's encoding all of these prostanoid receptors. In addition, the cloning of these receptors has revealed further heterogeneity through the use of alternative mRNA splicing. Specifically, mRNA splice variants have been identified for the EP1, EP3, FP and TP receptors. Interestingly, except for the EP1 receptors, the mechanisms giving rise to these receptor isoforms involves the use of splice sites located in the cytoplasmic carboxyl termini of these receptors. Thus, the eight human EP3 isoforms that have been identified are otherwise identical except for their carboxyl termini. Similarly, the optional use of a potential splice site encoding the carboxyl terminus gives rise to each of the two FP and TP receptor isoforms. Because the carboxyl termini of G-protein coupled receptors are generally implicated in interactions with G-proteins, it is not surprising that these receptor isoforms differ mainly with respect to their activation of second messenger pathways and not in their pharmacological characteristics. Differences also exist with respect to their levels of constitutive activity (e.g., in the absence of agonist) and in their desensitization.     

Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin

The N-methyl-D-aspartate receptor (NMDA-R) and brain spectrin, a protein that links membrane proteins to the actin cytoskeleton, are major components of post-synaptic densities (PSDs). Since the activity of the NMDA-R channel is dependent on the integrity of actin and leads to calpain-mediated spectrin breakdown, we have investigated whether the actin-binding spectrin may interact directly with NMDA-Rs. Spectrin is reported here to interact selectively in vitro with the C-terminal cytoplasmic domains of the NR1a, NR2A and NR2B subunits of the NMDA-R but not with that of the AMPA receptor GluR1. Spectrin binds at NR2B sites distinct from those of alpha-actinin-2 and members of the PSD95/SAP90 family. The spectrin-NR2B interactions are antagonized by Ca2+ and fyn-mediated NR2B phosphorylation, but not by Ca2+/calmodulin (CaM) or by Ca2+/CaM-dependent protein kinase II-mediated NR2B phosphorylation. The spectrin-NR1 interactions are unaffected by Ca2+ but inhibited by CaM and by protein kinase A- and C-mediated phosphorylations of NR1. Finally, in rat synaptosomes, both spectrin and NR2B are loosened from membranes upon addition of physiological concentrations of calcium ions. The highly regulated linkage of the NMDA-R to spectrin may underlie the morphological changes that occur in neuronal dendrites concurrently with synaptic activity and plasticity.     

Truncated human endothelin receptor A produced by alternative splicing and its expression in melanoma

PURPOSE: Traumatic sphincter disruption frequently is associated with a rectovaginal fistula, but the effect of a persistent sphincter defect on the outcome of rectovaginal fistula repair is poorly documented. We analyzed the outcome of rectovaginal fistula repairs based on preoperative sphincter status. PATIENTS AND METHODS: We identified 52 women who underwent 62 repairs of simple obstetrical rectovaginal fistulas between 1992 and 1995. Fourteen patients (27 percent) had preoperative endoanal ultrasound studies and 25 (48 percent) had anal manometry studies. Follow-up was by mailed questionnaire in 36 patients (69 percent) and by telephone interview in 12 (23 percent), for a total response rate of 92 percent. Median age was 30.5 (range, 18-70) years, and median follow-up was 15 (range, 0.5-123) months. Twenty-five patients (48 percent) complained of varying degrees of fecal incontinence before surgery. There were 27 endorectal advancement flaps and 35 sphincteroplasties (28 with and 8 without levatoroplasty). RESULTS: Success rates were 41 percent with endorectal advancement flaps and 80 percent with sphincteroplasties (96 percent success with and 33 percent without levatoroplasty; P = 0.0001). Endorectal advancement flap was successful in 50 percent of patients with normal sphincter function but in only 33 percent of patients with abnormal sphincter function (P = not significant). For sphincteroplasties, success rates were 73 vs. 84 percent for normal and abnormal sphincter function, respectively (P = not significant). Results were better after sphincteroplasties vs. endorectal advancement flaps in patients with sphincter defects identified by endoanal ultrasound (88 vs. 33 percent; P = not significant) and by manometry (86 vs. 33 percent; P = not significant). Poor results correlated with prior surgery in patients undergoing endorectal advancement flaps (45 percent vs. 25 percent; P = not significant) but not sphincteroplasties (80 vs. 75 percent; P = not significant). CONCLUSIONS: All patients with rectovaginal fistula should undergo preoperative evaluation for occult sphincter defects by endoanal ultrasound or anal manometry or both procedures. Local tissues are inadequate for endorectal advancement flap repairs in patients with sphincter defects and a history of previous repairs. Patients with clinical or anatomic sphincter defects should be treated by sphincteroplasty with levatoroplasty.     

A novel short isoform of the D3 dopamine receptor generated by alternative splicing in the third cytoplasmic loop

The mouse D3 dopamine receptor has been cloned from olfactory tubercle cDNA using polymerase chain reaction and has been found to exist in two alternatively spliced forms. These two mRNA isoforms differ by the presence or absence of 63 base pairs (bp), which encode 21 amino acids in the putative third cytoplasmic loop of the receptor. The longer form corresponds to the previously reported rat D3 dopamine receptor, to which it bears sequence homology of 94%. Northern blot analysis shows the mouse D3 receptor to be most abundant in the olfactory tubercle. Expression studies show the novel short D3 isoform to bind dopaminergic ligands with a D3-like pharmacological profile. Polymerase chain reaction analysis on different mouse brain regions shows the long and short D3 receptors to be present in the same tissues, the longer form invariably being the predominant one. Analysis of the gene for the mouse D3 dopamine receptor shows that no separate exon encodes the 63-bp stretch and reveals the presence of a consensus sequence for an acceptor site at the 3' end of the 63-bp stretch. This suggests that an internal acceptor site in the exon coding for the distal part of the third cytoplasmic loop directs alternative splicing of the D3 dopamine receptor.     

Tyrosine phosphorylation of NMDA receptor in rat striatum: effects of 6-OH-dopamine lesions

It is technically demanding to make intracellular measurements of mechanoreception in intact arthropod cuticular receptors. Here we introduce a method for recording mechanically induced electrical events in a class of spider mechanoreceptors using single electrode voltage- or current-clamp. A concave piece of cuticle containing a mechanosensitive lyriform slit organ was dissected free and fixed with wax onto a specially designed holder. This holder-cuticle complex, filled with spider saline, allowed displacement of the slit membrane from below while simultaneously recording intracellularly from neurons of the organ through a thin saline film. Extracellular ion concentrations could be changed and ion channel blockers could be applied to the bath. The method promises to allow the investigation of the ion channels responsible for mechanically transduced receptor signals and spike encoding.     

Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo

The definitive function of pancreatic polypeptide in mammalian physiology remains unknown. The identification of specific PP target tissues should be helpful to further investigations into the possible regulatory actions of this peptide. An in vivo radioreceptor assay was used in the rat to locate potential binding sites of I(125) bovine PP. In vitro, high concentrations of unlabeled hormone competitively inhibit binding to receptors by low concentrations of labeled hormone. In vivo studies showed that, in the presence of concentrated unlabeled pancreatic polypeptide, labeled PP distributes between the plasma and interstitial fluid. When excess unlabeled PP is replaced with saline in the companion animals, the labeled peptide appears to distribute in a volume that exceeds the combined plasma volume and interstitial fluid volume of the tissue. Using this in vivo receptor assay, the distribution volume that exceeds the anatomic extracellular volume has been identified as the receptor compartment. With this assay we demonstrated in the rat specific and displaceable PP binding to the ductus choledochus, duodenum, ileum, and adrenal gland. The NVV determined in the adrenal gland of experimental animals was 3.9 times greater than that found in the control group. Binding was rapid and was displaced only by excess unlabeled pancreatic polypeptide. Neither excess insulin nor excess neuropeptide Y significantly reduced this binding.     

Induction of alternative splicing of HLA-B27 by bacterial invasion

This study introduces an attributional processing approach to study age differences in dispositional attributions. Dispositional attributions made in the context of relationship vignettes were examined among younger and older adults in 2 conditions (immediate and delayed rating conditions). By using a direct assessment of a 2-step process for making dispositional attributions, it was inferred that a spontaneous adjustment stage occurred following an initial characterization stage as a function of age group and content of vignettes. Older adults made lower dispositional ratings if they were given more time to think about the situations than if asked to make an immediate judgment. By contrast, younger adults made higher dispositional ratings if they were given more time to make the judgments. Qualitative analyses of schemas elicited by a subsample of participants for each vignette suggested a relationship between dispositional attributional ratings and content-evoked schemas.     

Mouse prolactin receptor gene: genomic organization reveals alternative promoter usage and generation of isoforms via alternative 3'-exon splicing

In rodents, the prolactin receptor is expressed as multiple isoforms with identical extracellular and membrane-proximal region sequences but with different 3' sequences, encoding different cytoplasmic regions, and different 5' untranslated region (UTR) sequences. These divergent sequences could be the result of multiple prolactin receptor genes or of a single gene which displays alternative promoter usage and 3'-exon splicing. To investigate the molecular basis for these observations, we have cloned and determined the organization of the mouse prolactin receptor gene. Genomic DNA cloning allowed the arrangement of promoters 1A, 1B, and 1C to be determined. 5'-RACE-PCR from mouse liver identified two novel 5' prolactin receptor sequences, indicating that the gene has at least five different promoters, four of which are active in liver. The remaining nonvariable 5' UTR is encoded by a separate exon (exon 2), while a further 11 coding exons follow, the last 4 of which are alternatively spliced to produce the four isoforms of the receptor. Functional units were found to be exon specific. Thus, the multiple prolactin receptor isoforms are the product of a single gene of >120 kb which displays multiple promoter usage and 3'-exon splicing.     

Regulation of ganglioside metabolism by phosphorylation and dephosphorylation

Cell differentiation is frequently accompanied by alterations in the composition of gangliosides in the plasma membrane resulting from a regulation of the enzyme activities involved. The regulation of CMP-NeuAc:GM1 alpha2-3-sialyltransferase (ST-IV) and UDP-GalNAc:GM3 N-acetylgalactosaminyltransferase (Gal-NAc-T) by the degree of enzyme phosphorylation was analyzed by determination of the enzyme activity on incubation of NG108-15 cells with various protein phosphatase inhibitors (okadaic acid and orthovanadate) or protein kinase activators (phorbol ester and forskolin). Incubation with okadaic acid, but not with orthovanadate, inhibited the ST-IV activity to 45% of that of control cells with t(1/2) = 60 min for the inactivation reaction. This indicates a rapid hyperphosphorylation of ST-IV due to the inhibition of a serine/threonine-specific phosphatase. A similar rate of inactivation was found on stimulation of protein kinase C with phorbol ester. In contrast to ST-IV, the activity of GalNAc-T was increased on stimulation of intracellular phosphorylation systems. The fastest activation of GalNAc-T was achieved with forskolin, yielding up to 160% of the initial activity within 30 min of effector incubation. Up-regulation of GalNAc-T in conjunction with down-regulation of ST-IV by stimulation of phosphorylation is suggested to serve as a physiological mechanism to increase the concentration of GM1, which was found to be elevated in correlation with the cell density. This assumption was corroborated by metabolic labeling studies with radioactive ganglioside precursors indicating an enhancement of the relative amount of a-series gangliosides subsequent to GM3 on phosphorylation stimulation. In particular, the biosynthesis of GM1 was specifically elevated within 2 h of incubation with forskolin. We conclude from the overall data that the ganglioside composition during the cell differentiation of NG108-15 cells can be specifically regulated by both protein kinase A- and protein kinase C-related phosphorylation systems.