RNA-editing of the 5-HT(2C) receptor alters agonist-receptor-effector coupling specificity.

1. The serotonin(2C) (5-HT(2C)) receptor couples to both phospholipase C (PLC)-inositol phosphate (IP) and phospholipase A(2) (PLA(2))-arachidonic acid (AA) signalling cascades. Agonists can differentially activate these effectors (i.e. agonist-directed trafficking of receptor stimulus) perhaps due to agonist-specific receptor conformations which differentially couple to/activate transducer molecules (e.g. G proteins). Since editing of RNA transcripts of the human 5-HT(2C) receptor leads to substitution of amino acids at positions 156, 158 and 160 of the putative second intracellular loop, a region important for G protein coupling, we examined the capacity of agonists to activate both the PLC-IP and PLA(2)-AA pathways in CHO cells stably expressing two major, fully RNA-edited isoforms (5-HT(2C-VSV), 5-HT(2C-VGV)) of the h5-HT(2C) receptor. 2. 5-HT increased AA release and IP accumulation in both 5-HT(2C-VSV) and 5-HT(2C-VGV) expressing cells. As expected, the potency of 5-HT for both RNA-edited isoforms for both responses was 10 fold lower relative to that of the non-edited receptor (5-HT(2C-INI)) when receptors were expressed at similar levels. 3. Consistent with our previous report, the efficacy order of two 5-HT receptor agonists (TFMPP and bufotenin) was reversed for AA release and IP accumulation at the non-edited receptor thus demonstrating agonist trafficking of receptor stimulus. However, with the RNA-edited receptor isoforms there was no difference in the relative efficacies of TFMPP or bufotenin for AA release and IP accumulation suggesting that the capacity for 5-HT(2C) agonists to traffic receptor stimulus is lost as a result of RNA editing. 4. These results suggest an important role for the second intracellular loop in transmitting agonist-specific information to signalling molecules.

RNA editing of the human serotonin 5-HT2C receptor. alterations in suicide and implications for serotonergic pharmacotherapy.

RNA encoding the human serotonin 5-HT2C receptor (5-HT(2C)R) undergoes adenosine-to-inosine RNA editing events at five positions, resulting in an alteration of amino acids in the second intracellular loop. Several edited 5-HT(2C)Rs possess a reduced G-protein coupling efficiency compared to the completely non-edited isoform. The current studies show that the efficacy of the hallucinogenic drug lysergic acid diethylamide and of antipsychotic drugs is regulated by RNA editing, suggesting that alterations in editing efficiencies or patterns might result in the generation of a 5-HT(2C)R population differentially responsive to serotonergic drugs. An examination of the efficiencies of RNA editing of the 5-HT(2C)R in prefrontal cortex of control individuals vs. subjects diagnosed with schizophrenia or major depressive disorder revealed no significant differences in RNA editing among the three populations. However, subjects who had committed suicide (regardless of diagnosis) exhibited a statistically significant elevation of editing at the A-site, which is predicted to change the amino acid sequence in the second intracellular loop of the 5-HT(2C)R. These findings suggest that alterations in RNA editing may contribute to or complicate therapy in certain psychiatric disorders.

A signal sequence is sufficient for green fluorescent protein to be routed to regulated secretory granules.

To investigate trafficking in neuroendocrine cells, green fluorescent protein (GFP) tags were fused to various portions of the preproneuropeptide Y (NPY) precursor. Two neuroendocrine cell lines, AtT-20 corticotrope tumor cells and PC-12 pheochromocytoma cells, along with primary anterior pituitary cells, were examined. Expression of chimeric constructs did not disrupt trafficking or regulated secretion of endogenous ACTH and prohormone convertase 1 in AtT-20 cells. Western blot and immunocytochemical analyses demonstrated that the chimeric constructs remained intact, as long as the Lys-Arg cleavage site within preproNPY was deleted. GFP was stored in, and released from, regulated granules in cells expressing half of the NPY precursor fused to GFP, and also in cells in which only the signal sequence of preproNPY was fused to GFP. Thus, in neuroendocrine cells, entering the lumen of the secretory pathway is sufficient to target GFP to regulated secretory granules.

Functions and mechanisms of RNA editing.

RNA editing can be broadly defined as any site-specific alteration in an RNA sequence that could have been copied from the template, excluding changes due to processes such as RNA splicing and polyadenylation. Changes in gene expression attributed to editing have been described in organisms from unicellular protozoa to man, and can affect the mRNAs, tRNAs, and rRNAs present in all cellular compartments. These sequence revisions, which include both the insertion and deletion of nucleotides, and the conversion of one base to another, involve a wide range of largely unrelated mechanisms. Recent advances in the development of in vitro editing and transgenic systems for these varied modifications have provided a better understanding of similarities and differences between the biochemical strategies, regulatory sequences, and cellular factors responsible for such RNA processing events.

Mice lacking alpha-calcitonin gene-related peptide exhibit normal cardiovascular regulation and neuromuscular development.

alpha-Calcitonin gene-related peptide (alphaCGRP) is a pleiotropic peptide neuromodulator that is widely expressed throughout the Central and peripheral nervous systems. CGRP has been implicated in a variety of physiological processes including peripheral vasodilation, cardiac acceleration nicotinic acetylcholine receptor (AChR) synthesis and function, testicular descent, nociception, carbohydrate metabolism, gastrointestinal motility, neurogenic inflammation, and gastric acid secretion. To provide a better understanding of the physiological role(s) mediated by this peptide neurotransmitter, we have generated alphaCGRP-null mice by targeted modification in embryonic stem cells. Mice lacking alpha CGRP expression demonstrate no obvious phenotypic differences from their wild-type littermates. Detailed analysis of systemic cardiovascular function revealed no differences between control and mutant mice regarding heart rate and blood pressure under basal or exercise-induced conditions and subsequent to pharmacological manipulation. Characterization of neuromuscular junction in morphology including nicotinic receptor localization, terminal sprouting in response to denervation, developmental regulation of AChR subunit expression, and synapse elimination also revealed no differences in alphaCGRP-deficient animals. These results suggest that alphaCGRP is not required for the systemic regulation of cardiovascular hemodynamics or development of the neuromuscular junction.

Serotonin-2C receptor pre-mRNA editing in rat brain and in vitro by splice site variants of the interferon-inducible double-stranded RNA-specific adenosine deaminase ADAR1.

The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA editing enzyme implicated in the site-selective deamination of adenosine to inosine in cellular pre-mRNAs. The pre-mRNA for the rat serotonin-2C receptor (5-HT2CR) possesses four editing sites (A, B, C, and D), which undergo A-to-I nucleotide conversions that alter the signaling function of the encoded G-protein-coupled receptor. Measurements of 5-HT2CR pre-mRNA editing in vitro revealed site-specific deamination catalyzed by ADAR1. Three splice site variants, ADAR1-a, -b, and -c, all efficiently edited the A site of 5-HT2CR pre-mRNA, but the D site did not serve as an efficient substrate for any of the ADAR1 variants. Mutational analysis of the three double-stranded (ds) RNA binding motifs present in ADAR1 revealed a different relative importance of the individual dsRNA binding motifs for deamination of the A site of 5-HT2CR and synthetic dsRNA substrates. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that the 5-HT2CR pre-mRNA was most highly expressed in the choroid plexus of rat brain. However, ADAR1 and the related deaminase ADAR2 showed significant expression in all regions of the brain examined, including cortex, hippocampus, olfactory bulb, and striatum, where the 5-HT2CR pre-mRNA was extensively edited.

Regulation of alternative splicing by RNA editing.

The enzyme ADAR2 is a double-stranded RNA-specific adenosine deaminase which is involved in the editing of mammalian messenger RNAs by the site-specific conversion of adenosine to inosine. Here we identify several rat ADAR2 mRNAs produced as a result of two distinct alternative splicing events. One such splicing event uses a proximal 3' acceptor site, adding 47 nucleotides to the ADAR2 coding region, changing the predicted reading frame of the mature ADAR2 transcript. Nucleotide-sequence analysis of ADAR2 genomic DNA revealed the presence of adenosine-adenosine (AA) and adenosine-guanosine (AG) dinucleotides at these proximal and distal alternative 3' acceptor sites, respectively. Use of the proximal 3' acceptor depends upon the ability of ADAR2 to edit its own pre-mRNA, converting the intronic AA to an adenosine-inosine (AI) dinucleotide which effectively mimics the highly conserved AG sequence normally found at 3' splice junctions. Our observations indicate that RNA editing can serve as a mechanism for regulating alternative splicing and they suggest a novel strategy by which ADAR2 can modulate its own expression.

RNA editing of the human serotonin 5-hydroxytryptamine 2C receptor silences constitutive activity.

RNA transcripts encoding the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor (5-HT2CR) undergo adenosine-to-inosine RNA editing events at up to five specific sites. Compared with rat brain, human brain samples expressed higher levels of RNA transcripts encoding the amino acids valine-serine-valine (5-HT2C-VSV) and valine-glycine-valine (5-HT2C-VGV) at positions 156, 158, and 160, respectively. Agonist stimulation of the nonedited human receptor (5-HT2C-INI) and the edited 5-HT2C-VSV and 5-HT2C-VGV receptor variants stably expressed in NIH-3T3 fibroblasts demonstrated that serotonergic agonists were less potent at the edited receptors. Competition binding experiments revealed a guanine nucleotide-sensitive serotonin high affinity state only for the 5-HT2C-INI receptor; the loss of high affinity agonist binding to the edited receptor demonstrates that RNA editing generates unique 5-HT2CRs that couple less efficiently to G proteins. This reduced G protein coupling for the edited isoforms is primarily due to silencing of the constitutive activity of the nonedited 5-HT2CR. The distinctions in agonist potency and constitutive activity suggest that different edited 5-HT2CRs exhibit distinct responses to serotonergic ligands and further imply that RNA editing represents a novel mechanism for controlling physiological signaling at serotonergic synapses.

Identification and characterization of RNA editing events within the 5-HT2C receptor.

RNA editing is a post-transcriptional modification that generates an RNA transcript with a nucleotide sequence different from its gene. We have recently discovered RNA editing events, involving the conversion of adenosine bases to inosine residues, within the RNA encoding the serotonin 2C (5-HT2C) receptor. Editing events at four major positions, termed A, B, C and D, as well as one minor site termed C', are predicted to alter amino acids within the second intracellular loop of the G-protein coupled 5-HT2C receptor. Editing is mediated by at least two members of a family of adenosine deaminases and is contingent upon the presence of an extensive RNA duplex structure formed by exonic and intronic sequences of 5-HT2C receptor precursor messenger RNA (pre-mRNA). This critical secondary structure has been observed within brain pre-mRNA derived from four species; the isolation of edited 5-HT2C receptor transcripts from these samples further confirms the evolutionary conservation of this RNA processing event. Among members of the 5-HT2 receptor family, editing within second intracellular loop RNA is unique to the 5-HT2C receptor. Editing within the 5-HT2C receptor generates receptor isoforms that differ in their ability to interact with the phospholipase C signaling cascade in a transfected cell line, suggesting that this RNA processing event may contribute to the modulation of serotonergic neurotransmission in the central nervous system.

Regulation of serotonin-2C receptor G-protein coupling by RNA editing.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) elicits a wide array of physiological effects by binding to several receptor subtypes. The 5-HT2 family of receptors belongs to a large group of seven-transmembrane-spanning G-protein-coupled receptors and includes three receptor subtypes (5-HT2A, 5-HT(2B) and 5-HT(2C)) which are linked to phospholipase C, promoting the hydrolysis of membrane phospholipids and a subsequent increase in the intracellular levels of inositol phosphates and diacylglycerol. Here we show that transcripts encoding the 2C subtype of serotonin receptor (5-HT(2C)R) undergo RNA editing events in which genomically encoded adenosine residues are converted to inosines by the action of double-stranded RNA adenosine deaminase(s). Sequence analysis of complementary DNA isolates from dissected brain regions have indicated the tissue-specific expression of seven major 5-HT(2C) receptor isoforms encoded by eleven distinct RNA species. Editing of 5-HT(2C)R messenger RNAs alters the amino-acid coding potential of the predicted second intracellular loop of the receptor and can lead to a 10-15-fold reduction in the efficacy of the interaction between receptors and their G proteins. These observations indicate that RNA editing is a new mechanism for regulating serotonergic signal transduction and suggest that this post-transcriptional modification may be critical for modulating the different cellular functions that are mediated by other members of the G-protein-coupled receptor superfamily.

Identification, molecular cloning, and distribution of a short variant of the 5-hydroxytryptamine2C receptor produced by alternative splicing.

The actions of the neurotransmitter 5-hydroxytryptamine (5-HT) (serotonin) are mediated by multiple receptor subtypes. One of the prominent serotonin receptors in the brain is the 5-HT2C receptor (5-HT2C-R). We report the occurrence of a second 5-HT2C-R transcript, first identified using S1 nuclease protection of total RNA isolated from the choroid plexus. Analyses of the distribution of these two RNAs revealed that the short form is expressed in the same structures as the 5-HT2C-R mRNA, including choroid plexus, striatum, hippocampus, hypothalamus, olfactory tubercles, and spinal cord. Cloning and sequence analyses revealed a second cDNA with a 95-nt deletion in the region coding for the putative second intracellular loop and the fourth transmembrane domain of the 5-HT2C-R. This deletion leads to a frameshift in the coding sequence and the introduction of a premature stop codon. The predicted truncated protein (5-HT2C-tr) contains 172 amino acids, with 153 residues at the amino terminus, identical to the 5-HT2C-R, and 19 carboxyl-terminal amino acids that are unique. Although antibodies specific to the 5-HT2C-tr protein showed that the truncated form is expressed in a transfected fibroblast cell model system, there was no serotonergic ligand binding activity or phosphoinositide hydrolysis. Analyses of the 5-HT2C-R gene revealed that the two transcripts arise from a single gene by differential splicing using alternative donor sites and a common 3'-splice acceptor. Polymerase chain reaction amplification of mouse and human brain cDNAs demonstrated the occurrence of the same splicing patterns in these species. Although this study demonstrates tissue-specific expression of two 5-HT2C mRNA splice variants in rat, mouse, and human, the significance of the truncated form in these three species remains to be established.

RNA editing.

RNA editing is a term describing a variety of novel mechanisms for the modification of nucleotide sequences of RNA transcripts in different organisms. These editing events include (a) the U-insertion and -deletion type of editing found in the mitochondrion of kinetoplastid protozoa, (b) the C-insertion editing found in the mitochondrion of Physarum, (c) the C-to-U substitution editing of the mammalian apoB mRNA, (d) a similar C-to-U substitution editing of mRNAs in higher plant mitochondria and chloroplasts and in tRNAs of marsupials and rats, (e) a diverse nucleotide substitution editing of tRNAs in Acanthomoeba mitochondria, and (f) the A-to-I type of editing found in the mammalian glutamate receptor subunits. These diverse phenomena involve several different enzymatic mechanisms. In several cases, duplex RNAs with internal or external guide sequences help determine the site specificity of editing. The A-to-I editing observed in RNAs encoding non-NMDA glutamate receptor subunits may be due to the actions of a double-stranded RNA-specific adenosine deaminase that is widespread in higher organisms. Although the function of many RNA editing events is unclear, the biological importance of RNA editing in other systems may prove as significant as the nucleotide modifications regulating the cation selectivity and electrophysiological profiles elaborated by non-NMDA glutamate receptors in the mammalian brain.

Glutamate receptor RNA editing in vitro by enzymatic conversion of adenosine to inosine.

RNA encoding the B subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptor (GluR-B) undergoes a posttranscriptional modification in which a genomically encoded adenosine is represented as a guanosine in the GluR-B complementary DNA. In vitro editing of GluR-B RNA transcripts with HeLa cell nuclear extracts was found to result from an activity that converts adenosine to inosine in regions of double-stranded RNA by enzymatic base modification. This activity is consistent with that of a double-stranded RNA-specific adenosine deaminase previously described in Xenopus oocytes and widely distributed in mammalian tissues.

Alternative splicing generates multiple isoforms of a rabbit prostaglandin E2 receptor.

Four cDNA clones homologous with a murine prostaglandin E2 receptor have been isolated from a rabbit kidney cortex cDNA library. These cDNAs encode related proteins that differ only in their COOH-terminal sequences. Southern blot analysis of rabbit genomic DNA indicates that these receptor cDNAs represent alternatively spliced variants derived from a single gene. This was confirmed by isolation and sequence analysis of genomic clones containing common region exons and unique 3'-coding exons, which contained intron/exon boundaries at the predicted splice junctions. Transient expression of a novel full-length cDNA in COS1 cells confirmed the ligand binding profile typical of an EP3 receptor subtype. Ribonuclease protection assays indicate that the gene encoding these receptors is most highly expressed in kidney, adrenal, and stomach with lower but significant expression in uterus, lung, heart, ileum, spleen, and brain. Moreover, each of the cloned isoforms is expressed in the kidney. In situ hybridization analyses of rabbit kidney demonstrated that the level of expression is highest in the outer medulla with lesser expression in the cortex and no detectable expression in the inner medulla. The ligand binding profile and tissue distribution of these receptors is consistent with a functional role for this family of EP3 receptors in mediating the renal actions of prostaglandins as well as the effects of prostaglandins on gastric acid secretion and adrenal function.

Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements.

The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery.

Alternative production of calcitonin and CGRP mRNA is regulated at the calcitonin-specific splice acceptor.

Alternative splicing of eukaryotic messenger RNA precursors represents a common mechanism for generating multiple transcripts from a single gene. Although there has been increasing information concerning the sequence requirements and the biochemical mechanisms involved in the constitutive splicing of primary RNA transcripts, very little is known about the sequences or mechanisms which determine alternative RNA-processing events in complex transcription units. The calcitonin/calcitonin gene-related peptide (CGRP) primary RNA transcript undergoes tissue-specific alternative processing, resulting in the differential production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons of the central and peripheral nervous systems. To elucidate the molecular mechanisms underlying these alternative RNA processing events, we have examined the nucleotide sequences involved in the production of calcitonin and CGRP mRNAs. Analyses of HeLa and F9 cell lines transfected with a variety of mutant calcitonin/CGRP transcription units have demonstrated that alternative splice-site selection is primarily regulated by cis-active element(s) near the calcitonin-specific 3'-splice junction. We suggest that the tissue-specific pattern of alternative RNA processing is conferred by sequence information at the calcitonin-specific acceptor which serves to inhibit the production of calcitonin transcripts in CGRP-producing cells.