Alternative Splicing and Extensive RNA Editing of Human TPH2 Transcripts

Brain serotonin (5-HT) neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2) in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1) is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide) and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour.

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title: A unique central tryptophan hydroxylase isoform.
abstract: Serotonin (5-hydroxytryptophan, 5-HT) is a neurotransmitter synthesized in the raphe nuclei of the brain stem and involved in the central control of food intake, sleep, and mood. Accordingly, dysfunction of the serotonin system has been implicated in the pathogenesis of psychiatric diseases. At the same time, serotonin is a peripheral hormone produced mainly by enterochromaffin cells in the intestine and stored in platelets, where it is involved in vasoconstriction, haemostasis, and the control of immune responses. Moreover, serotonin is a precursor for melatonin and is therefore synthesized in high amounts in the pineal gland. Tryptophan hydroxylase (TPH) catalyzes the rate limiting step in 5-HT synthesis. Until recently, only one gene encoding TPH was described for vertebrates. By gene targeting, we functionally ablated this gene in mice. To our surprise, the resulting animals, although being deficient for serotonin in the periphery and in the pineal gland, exhibited close to normal levels of 5-HT in the brain stem. This led us to the detection of a second TPH gene in the genome of humans, mice, and rats, called TPH2. This gene is predominantly expressed in the brain stem, while the classical TPH gene, now called TPH1, is expressed in the gut, pineal gland, spleen, and thymus. These findings clarify puzzling data, which have been collected over the last decades about partially purified TPH proteins with different characteristics and justify a new concept of the serotonin system. In fact, there are two serotonin systems in vertebrates, independently regulated and with distinct functions.

title: Tetrahydropterin-dependent amino acid hydroxylases.
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title: Animal studies of amygdala function in fear and uncertainty: Relevance to human research
abstract: This article reviews research in both animals and humans on the considerable progress made in elucidating a brain circuitry of fear, particularly the importance of the amygdala in fear conditioning. While there is considerable agreement about the participation of the amygdala in fear in both animals and humans, there are several issues about the function of the amygdala raised by the human research that have not been addressed by or may be answered by animal research. Three of these are addressed in this article: (1) is the amygdala involved in or necessary for both fear learning and unconditioned fear? (2) Does the amygdala code for intensity of fear? (3) Is the amygdala preferentially involved in fear, or is it also activated when there are no overt fear or aversive stimuli, but where the situation can be described as uncertain? We present evidence indicating that the rodent amygdala is involved in some types of fear (conditioned fear), but not all types (unconditioned fear), and may therefore have significance for a differential neurobiology of certain anxiety disorders in humans. Further, similar to the human amygdala, the rodent amygdala responds to varying intensities of aversive stimulation. Finally, it is suggested that, similar to humans, the rodent amygdala is involved in the evaluation of uncertainty. We conclude that progress on elucidating the role of the amygdala in fear is facilitated by corroboration of findings from both animal and human research.

title: The amygdala and reward
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title: Some assessments of the amygdala role in suprahypothalamic neuroendocrine regulation: a minireview.
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title: Synthesis of Serotonin by a Second Tryptophan Hydroxylase Isoform
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title: Serotonin regulates mammary gland development via an autocrine-paracrine loop.
abstract: Mammary gland development is controlled by a dynamic interplay between endocrine hormones and locally produced factors. Biogenic monoamines (serotonin, dopamine, norepinephrine, and others) are an important class of bioregulatory molecules that have not been shown to participate in mammary development. Here we show that mammary glands stimulated by prolactin (PRL) express genes essential for serotonin biosynthesis (tryptophan hydroxylase [TPH] and aromatic amine decarboxylase). TPH mRNA was elevated during pregnancy and lactation, and serotonin was detected in the mammary epithelium and in milk. TPH was induced by PRL in mammosphere cultures and by milk stasis in nursing dams, suggesting that the gene is controlled by milk filling in the alveoli. Serotonin suppressed beta-casein gene expression and caused shrinkage of mammary alveoli. Conversely, TPH1 gene disruption or antiserotonergic drugs resulted in enhanced secretory features and alveolar dilation. Thus, autocrine-paracrine serotonin signaling is an important regulator of mammary homeostasis and early involution.

title: Serotonin synthesis in murine embryonic stem cells.
abstract: Serotonin (5-HT) is a monoaminergic neurotransmitter involved in various processes in the mammalian nervous system with tryptophan hydroxylase (TPH) as the rate-limiting enzyme in its biosynthesis. Interestingly, there is accumulating evidence that neurotransmitters including 5-HT are directly involved in cleavage divisions and morphogenetic movements during early embryogenesis, even before neurons appear. Clonal cell models will be indispensable for investigating these pre-neuronal actions of neurotransmitter systems. Totipotent embryonic stem (ES) cells represent early embryonic stages, are amenable to genetic manipulations and can be easily induced to differentiate into cells with neuronal and glial properties enabling the recapacitation of neurulation. In this study, we used high-pressure liquid chromatography with fluorometric detection (HPLC-FD) to demonstrate the presence of 5-HT in ES cells. In addition, RNase protection assays and immunohistochemical methods detected TPH mRNA and protein, respectively, confirming the endogeneous production of 5-HT in these cells. Furthermore, TPH protein was detected in mouse zygotes after fertilization. These results indicate that ES cells may be useful for the investigation of neurotransmitters in pre-nervous embryos and their actions during ontogeny.

title: 7‐hydroxytryptophan, a novel, specific, cytotoxic agent for carcinoids and other serotonin‐producing tumors
abstract: Carcinoids and small cell lung carcinomas stimulate their growth in an autocrine manner by releasing serotonin, an effect that is blocked by selective serotonergic receptor antagonists that, unfortunately, exert undesirable side effects on serotonergic central nervous function. Moreover, conventional chemotherapeutic agents, such as streptozocin, fluorouracil, cyclophosphamide, and doxorubicin, which target tumor cells directly, have produced disappointing results in the treatment of patients with these tumors in the advanced stage. Therefore, there is still a need for more specific and potent chemotherapeutic agents in the fight against serotonin‐producing tumors.

title: Serotonylation of Small GTPases Is a Signal Transduction Pathway that Triggers Platelet α-Granule Release
abstract: Serotonin is a neurotransmitter in the central nervous system. In the periphery, serotonin functions as a ubiquitous hormone involved in vasoconstriction and platelet function. Serotonin is synthesized independently in peripheral tissues and neurons by two different rate-limiting tryptophan hydroxylase (TPH) isoenzymes. Here, we show that mice selectively deficient in peripheral TPH and serotonin exhibit impaired hemostasis, resulting in a reduced risk of thrombosis and thromboembolism, although the ultrastructure of the platelets is not affected. While the aggregation of serotonin-deficient platelets in vitro is apparently normal, their adhesion in vivo is reduced due to a blunted secretion of adhesive alpha-granular proteins. In elucidating the mechanism further, we demonstrate that serotonin is transamidated to small GTPases by transglutaminases during activation and aggregation of platelets, rendering these GTPases constitutively active. Our data provides evidence for a receptor-independent signaling mechanism, termed herein as "serotonylation," which leads to alpha-granule exocytosis from platelets.

title: Platelet-Derived Serotonin Mediates Liver Regeneration
abstract: The liver can regenerate its volume after major tissue loss. In a mouse model of liver regeneration, thrombocytopenia, or impaired platelet activity resulted in the failure to initiate cellular proliferation in the liver. Platelets are major carriers of serotonin in the blood. In thrombocytopenic mice, a serotonin agonist reconstituted liver proliferation. The expression of 5-HT2A and 2B subtype serotonin receptors in the liver increased after hepatectomy. Antagonists of 5-HT2A and 2B receptors inhibited liver regeneration. Liver regeneration was also blunted in mice lacking tryptophan hydroxylase 1, which is the rate-limiting enzyme for the synthesis of peripheral serotonin. This failure of regeneration was rescued by reloading serotonin-free platelets with a serotonin precursor molecule. These results suggest that platelet-derived serotonin is involved in the initiation of liver regeneration.

title: Intracellular Serotonin Modulates Insulin Secretion from Pancreatic β-Cells by Protein Serotonylation
abstract: Non-neuronal, peripheral serotonin deficiency causes diabetes mellitus and identifies an intracellular role for serotonin in the regulation of insulin secretion.

title: Effect of Tryptophan Hydroxylase 1 Deficiency on the Development of Hypoxia-Induced Pulmonary Hypertension
abstract: Tryptophan hydroxylase 1 catalyzes the rate-limiting step in the synthesis of serotonin in the periphery. Recently, it has been shown that expression of the tryptophan hydroxylase 1 gene is increased in lungs and pulmonary endothelial cells from patients with idiopathic pulmonary arterial hypertension. Here we investigated the effect of genetic deletion of tryptophan hydroxylase 1 on hypoxia-induced pulmonary arterial hypertension in mice by measuring pulmonary hemodynamics and pulmonary vascular remodeling before and after 2 weeks of hypoxia. In wild-type mice, hypoxia increased right ventricular pressure and pulmonary vascular remodeling. These effects of hypoxia were attenuated in the tryptophan hydroxylase 1−/−mice. Hypoxia increased right ventricular hypertrophy in both wild-type and tryptophan hydroxylase 1−/−mice suggesting that in vivo peripheral serotonin has a differential effect on the pulmonary vasculature and right ventricular hypertrophy. Contractile responses to serotonin were increased in pulmonary arteries from tryptophan hydroxylase 1−/−mice. Hypoxia increased serotonin-mediated contraction in vessels from the wild-type mice, but this was not further increased by hypoxia in the tryptophan hydroxylase 1−/−mice. In conclusion, these results indicate that tryptophan hydroxylase 1 and peripheral serotonin play an essential role in the development of hypoxia-induced elevations in pulmonary pressures and hypoxia-induced pulmonary vascular remodeling. In addition, the results suggest that, in mice, serotonin has differential effects on the pulmonary vasculature and right ventricular hypertrophy.

title: Genotyping of the G1463A (Arg441His) TPH2 polymorphism in a geriatric population of patients with major depression
abstract: Genotyping of the G1463A (Arg441His) TPH2 polymorphism in a geriatric population of patients with major depression

title: Support for the involvement of TPH2 gene in affective disorders
abstract: Disturbance of the serotonergic system has been suggested as a factor in the pathogenesis of affective disorders. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the serotonin biosynthetic pathway, and a new TPH isoform, TPH2, has been identified in the brain and localized to the long arm of chromosome 12 in a region that has been reported to contain bipolar and major depressive disorder susceptibility genes. An SNP-based association study on a case/control sample gave support to the existence of an affective disorder-associated haplotype in the TPH2 gene.

title: Transmission disequilibrium of polymorphic variants in the tryptophan hydroxylase-2 gene in attention-deficit/hyperactivity disorder
abstract: Attention-deficit/hyperactivity disorder (ADHD) is the most common behavioral disorder in childhood with substantial heritability. Pharmacological and molecular genetic studies as well as characterization of animal models have implicated serotonergic dysfunction in the pathophysiology of ADHD. Here, we investigated the effect of polymorphic variants in the gene of the tryptophan hydroxylase-2 (TPH2), the rate-limiting enzyme of serotonin (5-HT) synthesis in the brain, in children and adolescents with ADHD. We analyzed three single nucleotide polymorphisms (SNPs) in and downstream of the transcriptional control region of the TPH2 gene in 103 families with 225 affected children. Allelic association in families with more than one affected child was assessed using the pedigree disequilibrium test. Preferential transmissions were detected for the two SNPs in TPH2's regulatory region (rs4570625, P=0.049; rs11178997, P=0.034), but not for the third SNP in intron 2 (rs4565946, P=0.3517). Haplotype analysis revealed a strong trend of association between the regulatory region SNPs (rs4570625, rs11178997) and ADHD (P=0.064). Our results link potentially functional TPH2 variations to the pathophysiology of ADHD, and further support the relevance of 5-HT in disorders related to altered motor activity and cognitive processes.

title: Association between Tph2 gene polymorphism, brain tryptophan hydroxylase activity and aggressiveness in mouse strains
abstract: The brain neurotransmitter serotonin is involved in the regulation of aggressive behavior. The main factor determining the brain serotonin level is the activity of the rate‐limiting enzyme in the biosynthesis of the neurotransmitter – tryptophan hydroxylase isoform (TPH) 2 encoded by the Tph2 gene. Recently the C1473G single‐nucleotide polymorphism in the Tph2 gene was reported. Here we study the C1473G polymorphism in 10 inbred mouse strains (C57BL/6J, AKR/J, DD/He, C3H/HeJ, YT/Y, BALB/cJLac, CC57BR/Mv and A/He) and demonstrate the association of the polymorphism with brain TPH activity and intermale aggressiveness. TPH activity in the midbrain of mice homozygous for the 1473C allele was higher than that in mice carrying 1473G alleles. A close association of the 1473C allele with increased number of attacks towards another male was found. The results support a link between the C1473G polymorphism in Tph2 gene, trypthophan hydroxylase activity and intensity of intermale aggression.

title: Single nucleotide polymorphism and haplotype analysis of a novel tryptophan hydroxylase Isoform (TPH2) gene in suicide victims
abstract: Tryptophan hydroxylase, the rate-limiting enzyme in the biosynthesis of serotonin, represents a major candidate in numerous genetic association analyses of suicidal behavior; however, the results are so far inconclusive. Recently, a second tryptophan hydroxylase isoform (TPH2) was identified in mice, which was exclusively present in the brain. In a previous postmortem study of our own group, we could demonstrate that TPH2 is also expressed in the human brain but not in peripheral tissues.



We performed single nucleotide polymorphisms, haplotypes, and linkage disequilibrium studies on 263 suicide victims and 266 healthy control subjects with 10 single nucleotide polymorphisms in the TPH2 gene.



Significant association was detected between one single nucleotide polymorphism (p = .004, global p = .01) and suicide. Additionally, haplotype analysis also produced support for association (p < .0001, global p = .0001).



This is the first report about an association between TPH2 gene polymorphisms and completed suicide. Our findings provide evidence for an involvement of genetic variants in the TPH2 gene in suicidal behavior. These results might open up new research strategies for the analysis of the observed disturbances in the serotonergic system in several other psychiatric disorders.

title: Different properties of the central and peripheral forms of human tryptophan hydroxylase
abstract: Tryptophan hydroxylase (TPH) catalyses the rate‐limiting reaction in the biosynthesis of serotonin. In humans, two different TPH genes exist, located on chromosomes 11 and 12, respectively, and encoding two enzymes (TPH1 and TPH2) with an overall sequence identity of 71%. We have expressed both enzymes as various fusion proteins in Escherichia coli and using an in vitro transcription/translation system, and compared their solubility and kinetic properties. TPH2 is more soluble than TPH1, has a higher molecular weight and different kinetic properties, including a lower catalytic efficiency towards phenylalanine than TPH1. Both enzymes are phosphorylated by cAMP‐dependent protein kinase A. TPH2 was phosphorylated at Ser19, a phosphorylation site not present in TPH1. The differences between TPH1 and TPH2 have important implications for the regulation of serotonin production in the brain and the periphery and may provide an explanation for some of the diverging results reported for TPH from different sources in the past.

title: Analysis of tryptophan hydroxylase I and II mRNA expression in the human brain: a post-mortem study.
abstract: Dysregulation of brain serotonin transmission is an important contributing factor in many psychiatric disorders. Tryptophan hydroxylase (TPH), the rate limiting enzyme in the biosynthesis of serotonin plays a major role as candidate gene in several psychiatric disorders. Recently, a second TPH isoform (TPH2) was identified in mice which was exclusively expressed in the brain. Due to the lack of data about its anatomic expression in humans we performed a mRNA expression analysis comparing TPH1 and TPH2 mRNA in several regions of the human brain (cortex, thalamus, hypothalamus, hippocampus, amygdala, cerebellum and raphe nuclei). The study was performed with post-mortem specimens obtained from eight individuals with sudden deaths, not directly involving CNS diseases. Our results demonstrate that the mRNA of both genes is expressed in each investigated brain region with variations between the brain areas, as well as between the particular genes. The major finding of this study was the high expression level of TPH2 mRNA in the raphe nuclei ( approximately 4-fold more abundant than that of TPH1). The raphe nuclei showed the highest TPH2 mRNA levels at all, compared to the other regions (7-fold higher levels on average). To our knowledge, this is the fist study which demonstrates the localization of TPH1 and TPH2 mRNA in different regions of the human brain. Our findings provide further support for a duality of the serotonergic system and may open up new research strategies for the analysis of the repeatedly observed disturbances in the serotonergic system in patients suffering from several psychiatric disorders.

title: Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies
abstract: Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin. Once thought to be a single-gene product, TPH is now known to exist in two isoforms-TPH1 is found in the pineal and gut, and TPH2 is selectively expressed in brain. Heretofore, probes used for localization of TPH protein or mRNA could not distinguish between the TPH isoforms because of extensive homology shared by them at the nucleotide and amino acid level. We have produced monospecific polyclonal antibodies against TPH1 and TPH2 using peptide antigens from nonoverlapping sequences in the respective proteins. These antibodies allow the differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation, and immunocytochemical staining of tissue sections from brain and gut. TPH1 and TPH2 antibodies do not cross-react with either tyrosine hydroxylase or phenylalanine hydroxylase. Analysis of mouse tissues confirms that TPH1 is the predominant form expressed in pineal gland and in P815 mastocytoma cells with a molecular weight of 51 kDa. TPH2 is the predominant enzyme form expressed in brain extracts from mesencephalic tegmentum, striatum, and hippocampus with a molecular weight of 56 kDa. Antibody specificity against TPH1 and TPH2 is retained across mouse, rat, rabbit, primate, and human tissues. Antibodies that distinguish between the isoforms of TPH will allow studies of the differential regulation of their expression in brain and periphery.

title: Characterization of a Functional Promoter Polymorphism of the Human Tryptophan Hydroxylase 2 Gene in Serotonergic Raphe Neurons
abstract: Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in brain serotonin (5-HT) biosynthesis. Although dysfunction of 5-HT neurotransmission has been implicated in a variety of neuropsychiatric conditions, the human TPH2 promoter has not been characterized in vitro.



The functional relevance of TPH2 promoter polymorphisms was determined with luciferase assays in primary serotonergic neurons from rat raphe nuclei and in human small cell lung carcinoma cells (SHP-77 cells). We also investigated transcription factor binding to the variant promoter sequence with electrophoretic mobility shift assay (EMSA).



The polymorphism rs11178997 of the human TPH2 promoter significantly reduced TPH2 transcriptional activity by 22% and 7% in primary serotonergic neurons and in SHP-77 cells, respectively. In contrast, no significant differences in promoter activity were observed for the G- and T-alleles of rs4570625. The EMSA revealed reduced binding of the transcription factor POU3F2 (also known as Brn-2, N-Oct-3) to the A-allele of the polymorphism rs11178997. Overexpression of POU3F2 resulted in a robust activation of the TPH2 promoter (2.7-fold).



Our data suggest that the human TPH2 promoter polymorphism rs11178997 impacts on gene expression, which might have implications for the development and function of the serotonergic system in the brain.

title: A regulatory variant of the human tryptophan hydroxylase-2 gene biases amygdala reactivity
abstract: Recent studies have indicated that a newly identified second isoform of the tryptophan hydroxylase gene (TPH2) is preferentially involved in the rate-limiting synthesis of neuronal serotonin. Genetic variation in the human TPH2 gene (hTPH2) has been associated with altered in vitro enzyme activity as well as increased risk for mood disorders. Here, we provide the first in vivo evidence that a relatively frequent regulatory variant (G(−844)T) of hTPH2 biases the reactivity of the amygdala, a neural structure critical in the generation and regulation of emotional behaviors.

title: Amygdala responsiveness is modulated by tryptophan hydroxylase-2 gene variation
abstract: The tryptophan hydroxylase-2 gene (TPH2) codes for the enzyme of serotonin (5-HT) synthesis in the brain and variation of TPH2 has been implicated in disorders of emotion regulation. Here, we used functional magnetic resonance imaging (fMRI) to demonstrate that a potentially functional variant of TPH2 modulates amygdala responsiveness to emotional stimuli of both negative and positive valence.

title: Tryptophan Hydroxylase-2 Controls Brain Serotonin Synthesis
abstract: Dysregulation of brain serotonin contributes to many psychiatric disorders. Tryptophan hydroxylase-2 (Tph2), rather than Tph1, is preferentially expressed in the brain. We report a functional (C1473G) single-nucleotide polymorphism in mouse Tph2 that results in the substitution of Pro447 with Arg447 and leads to decreased serotonin levels in PC12 cells. Moreover, in BALB/cJ and DBA/2 mice that are homozygous for the 1473G allele, brain serotonin tissue content and synthesis are reduced in comparison to C57Bl/6 and 129X1/SvJ mice that are homozygous for the 1473C allele. Our data provide direct evidence for a fundamental role of Tph2 in brain serotonin synthesis.

title: A loss-of-function mutation in tryptophan hydroxylase 2 segregating with attention-deficit/hyperactivity disorder
abstract: A loss-of-function mutation in tryptophan hydroxylase 2 segregating with attention-deficit/hyperactivity disorder

title: Loss-of-Function Mutation in Tryptophan Hydroxylase-2 Identified in Unipolar Major Depression
abstract: Dysregulation of central serotonin neurotransmission has been widely suspected as an important contributor to major depression. Here, we identify a (G1463A) single nucleotide polymorphism (SNP) in the rate-limiting enzyme of neuronal serotonin synthesis, human tryptophan hydroxylase-2 (hTPH2). The functional SNP in hTPH2 replaces the highly conserved Arg441 with His, which results in approximately 80% loss of function in serotonin production when hTPH2 is expressed in PC12 cells. Strikingly, SNP analysis in a cohort of 87 patients with unipolar major depression revealed that nine patients carried the mutant (1463A) allele, while among 219 controls, three subjects carried this mutation. In addition, this functional SNP was not found in a cohort of 60 bipolar disorder patients. Identification of a loss-of-function mutation in hTPH2 suggests that defect in brain serotonin synthesis may represent an important risk factor for unipolar major depression.

title: Lack of association of TPH2 exon XI polymorphisms with major depression and treatment resistance
abstract: Lack of association of TPH2 exon XI polymorphisms with major depression and treatment resistance

title: Stress Upregulates TPH1 but not TPH2 mRNA in the Rat Dorsal Raphe Nucleus: Identification of Two TPH2 mRNA Splice Variants
abstract: Serotonin is implicated in stress-related psychopathologies. Two isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, TPH1 and TPH2, are known. We show here that in the rat dorsal raphe nucleus (DRN), the nucleus that contains the highest number of 5-HT neurons in the brain, TPH1 mRNA reveals a low level of expression but is detectable both by quantitative real-time PCR and in situ hybridization whereas in the pineal gland (PiG), TPH1 mRNA is strongly expressed. To examine effects of stress on TPH expression we exposed male Wistar rats to daily restraint stress for 1 week. As shown by quantitative real-time PCR, TPH1 mRNA is 2.5-fold upregulated by the stress in DRN but not in PiG. Using 3′-RACE, we identified two TPH2 mRNA splice variants in the rat DRN which differ in the length of their 3′-untranslated regions (UTRs). TPH2b (with a short 3′-UTR) is the predominant variant in the DRN, whereas TPH2a (with a longer 3′-UTR) shows a low abundance in this nucleus. In the PiG, only TPH2b is detectable revealing a low level of expression. Expression of both TPH2 splice variants is not affected by stress, neither in DRN nor in the PiG. These data indicate that TPH1 in the serotonergic neurons of the DRN might be relevant for stress-induced psychopathologies.

title: Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing.
abstract: GC-AG introns represent 0.7% of total human pre-mRNA introns. To study the function of GC-AG introns in splicing regulation, 196 cDNA-confirmed GC-AG introns were identified in Caenorhabditis elegans. These represent 0.6% of the cDNA- confirmed intron data set for this organism. Eleven of these GC-AG introns are involved in alternative splicing. In a comparison of the genomic sequences of homologous genes between C.elegans and Caenorhabditis briggsae for 26 GC-AG introns, the C at the +2 position is conserved in only five of these introns. A system to experimentally test the function of GC-AG introns in alternative splicing was developed. Results from these experiments indicate that the conserved C at the +2 position of the tenth intron of the let-2 gene is essential for developmentally regulated alternative splicing. This C allows the splice donor to function as a very weak splice site that works in balance with an alternative GT splice donor. A weak GT splice donor can functionally replace the GC splice donor and allow for splicing regulation. These results indicate that while the majority of GC-AG introns appear to be constitutively spliced and have no evolutionary constraints to prevent them from being GT-AG introns, a subset of GC-AG introns is involved in alternative splicing and the C at the +2 position of these introns can have an important role in splicing regulation.

title: Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions.
abstract: It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF(35)-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms.

title: The Pre-nervous Serotonergic System of Developing Sea Urchin Embryos and Larvae: Pharmacologic and Immunocytochemical Evidence
abstract: Forty serotonin-related neurochemicals were tested on embryos and larvae of Lytechinus variegatus and other sea urchin species. Some of these substances (agonists of 5-HT1 receptors, antagonists of 5-HT2, 5-HT3 or 5-HT4 receptors, and inhibitors of the serotonin transporter, SERT) perturbed post-blastulation development, eliciting changes in embryonic/larval phenotypes typical for each class of receptor ligand. These developmental malformations were prevented completely or partially by serotonin (5-HT) or 5-HT analogs (5-HTQ, AA-5-HT), providing evidence for the putative localization of cellular targets. Immunoreactive 5-HT, 5-HT receptors and SERT were found in pre-nervous embryos and larvae of both L. variegatus and Strongylocentrotus droebachiensis. During gastrulation, these components of the serotonergic system were localized to the archenteron (primary gut), mesenchyme-like cells, and often the apical ectoderm. These results provide evidence that pre-nervous 5-HT may regulate early events of sea urchin embryogenesis, mediated by 5-HT receptors or the 5-HT transporter.

title: Roles for serotonin in neuroembryogenesis.
abstract: Possible non-transmitter roles for 5-HT in different phases of early neuroembryogenesis have been discussed based upon experimental evidence from the rat and chick. Fluorescence histochemical studies have demonstrated sites of uptake and synthesis of 5-HT in the chick embryo during the first few days of incubation. These sites are located in discrete regions of the notochord and floor plate of the neural tube as well as in extra-neural regions such as the somites and primitive gut. The 5-HT patterns are distinctly different from those observed for the uptake and synthesis of norepinephrine in embryos of the same age. Spatio-temporal changes in the distribution of these sites during closure of the neural tube suggest a role for 5-HT in various aspects of neural tube development. Moreover, the non-overlapping localization of 5-HT and norepinephrine raises the possibility that these two amines may exert different and perhaps cooperative influences on early neurogenic processes in the chick. In the rat, autoradiographic and biochemical studies concerning the consequences of 5-HT depletion in the embryo for development of different brain regions have provided evidence that 5-HT acts as a "differentiation signal" regulating the time of neuronal genesis in those cell populations which will eventually receive 5-HT innervation. Although the details of this system are as yet unknown, these studies suggest that 5-HT (and possibly the other monoamine transmitters) may actually "mold" the construction of their own circuitry during neurogenesis. Further, the ability of drugs and stress to interact with this process during that period of gestation when the monoamines are required as humoral signals suggests that maternal influences can interfere with ontogeny of this circuitry during pre- and possibly postnatal development. It is not yet clear whether the data in chicks and rats can be directly analogized from the one species to the other. Nevertheless, the evidence that sites of 5-HT uptake and/or synthesis are present during the earliest phases of neurogenesis in the chick and the observation that 5-HT depletion can alter the time of genesis of 5-HT target cells in the rat provide a new context for the consideration of possible actions of 5-HT prior to its role as a neurotransmitter substance.

title: Functional Domains of Human Tryptophan Hydroxylase 2 (hTPH2)*
abstract: Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NΔ150 and NΔ150/CΔ24) using isopropyl β-d-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH2-terminal regulatory domain. The solubility of hTPH2, NΔ150, and NΔ150/CΔ24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t½ at 37 °C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NΔ150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH2-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH2-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase Vmax values. These data identify the NH2-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.

title: A Perfect Message RNA Surveillance and Nonsense-Mediated Decay
abstract: the disposal of nonfunctional by-products of normal The robustness of biological systems depends on the RNA processing. function of proofreading mechanisms against operating NMD—An Enigma in RNA Metabolism errors. Studies in yeast and of human genetic disorders NMD has captured much attention as one of the most have lead the way to a conserved surveillance mechaenigmatic processes in RNA metabolism. Basically, a cell nism that identifies faulty open reading frames (ORFs) must discriminate the normal stop codon from a premaand eliminates “imperfect messages” that contain preture one (discrimination phase). Subsequently, a PTCmature translation termination codons (PTCs) and code mutated mRNA is committed to a pathway (commitment for nonfunctional or even harmful polypeptides; this mechphase) that ultimately results in its degradation (degraanism has been termed nonsense-mediated mRNA decay dation phase). NMD in yeast is becoming increasingly (NMD). well understood (Jacobson and Peltz, 1996; Ruiz-EcheThe Biological and Medical Importance of NMD varria et al., 1998). By contrast, diverse models are conIt was noticed in 1979 that PTC mutations of the yeast sidered for mammalian NMD (Maquat, 1995; Li and Wilura3 gene destabilized the encoded mRNA (Losson and kinson, 1998). We will describe some key findings from Lacroute, 1979). In the same year, the level of PTCthe mammalian and yeast systems and will suggest that mutated b-globin mRNA from a thalassemia patient was most of the available evidence can be integrated into a reported to be very low (Chang and Kan, 1979), forecommon model. shadowing the broad phylogenetic distribution of NMD Mammalian NMD: Nuclear Aspects and its role in human genetic disorders. The vast majorThere is little doubt that mammalian NMD is a posttranity of b-thalassemia heterozygotes are phenotypically scriptional mechanism that involves the nucleus. Two healthy, whereas homozygotes suffer from a severe anetypes of alterations have been described: the abnormal mia. Normal erythropoiesis requires a/b-globin chain splicing of pre-mRNAs, and the degradation of spliced balance. In heterozygotes, the relative excess of insolumRNAs. Both alterations have been referred to as NMD. ble a-globin chains are degraded but accumulate in Regarding the first, examples of both nonsense-associhomozygotes to damage the erythroid precursors. PTC ated alternative splicing (e.g., fibrillin mRNA) and of inmutations in the two 59 proximal exons of the b-globin tron retention (e.g., Igk RNA) have been investigated. In gene are common, and the mRNA is subjected to NMD. these examples, PTCs are associated with an effect In contrast, a dominant form of inheritance is associated on splicing, for which we suggest the term “nonsensewith a rare nonsense mutation in the last exon. This associated altered splicing” (NAS). NAS is intriguing, PTC-mutated mRNA escapes NMD, is normally abunbecause it suggests that the sense of a pre-mRNA can dant, and is translated (Hall and Thein, 1994). This addibe interpreted by a nuclear reading frame scanner betional burden causes the proteolytic system of the eryfore splicing (Li and Wilkinson, 1998). Other alternative, throid precursor cells to fail, which results in the

title: A Conserved mRNA Export Machinery Coupled to pre-mRNA Splicing
abstract: Recent advances have led to a new understanding of how mRNAs are exported from the nucleus to the cytoplasm. This process requires a heterodimeric mRNA export receptor that is part of an elaborate machinery conserved from yeast to humans. Export of mRNAs is coupled to upstream steps in gene expression, such as pre-mRNA splicing, and to downstream events, including nonsense-mediated decay.

title: Control of human potassium channel inactivation by editing of a small mRNA hairpin
abstract: Genomic recoding by A→I RNA editing plays an important role in diversifying the proteins involved in electrical excitability. Here, we describe editing of an intronless potassium channel gene. A small region of human KV1.1 mRNA sequence directs efficient modification of one adenosine by human adenosine deaminase acting on RNA 2 (hADAR2). Mutational analysis shows that this region adopts a hairpin structure. Electrophysiological characterization reveals that the editing event (I/V) profoundly affects channel inactivation conferred by accessory β subunits. Drosophila melanogaster Shaker channels, mimicking this editing event through mutation, exhibit a similar effect. In addition, we demonstrate that mRNAs for the paralogous D. melanogaster Shab potassium channel are edited at the same position by fly ADAR—a clear example of convergent evolution driven by adenosine deamination. These results suggest an ancient and key regulatory role for this residue in KV channels.

title: Regulation of serotonin-2C receptor G-protein coupling by RNA editing
abstract: 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-HT2B and 5-HT2C) which are linked to phospholipase C, promoting the hydrolysis of membrane phospholipids and a subsequent increase in the intracellular levels of inositol phosphates and diacylglycerol1. Here we show that transcripts encoding the 2C subtype of serotonin receptor (5-HT2CR) 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-HT2C receptor iso-forms encoded by eleven distinct RNA species. Editing of 5-HT2CR 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.

title: RNA editing of AMPA receptor subunit GluR-B: A base-paired intron-exon structure determines position and efficiency
abstract: A functionally critical position (Q/R site) of the AMPA receptor subunit GluR-B is controlled by RNA editing that operates in the nucleus, since in brain and clonal cell lines of neural origin, unspliced GluR-B transcripts occur edited in the Q/R site CAG codon and, additionally, in intronic adenosines. Transfection of GluR-B gene constructs into PC12 cells revealed that the proximal part of the intron downstream of the unedited exonic site is required for Q/R site editing. This intron portion contains an imperfect inverted repeat preceding a 10 nt sequence with exact complementarity to the exon centered on the unedited codon. Single nucleotide substitutions in this short intronic sequence or its exonic complement curtailed Q/R site editing, which was recovered by restoring complementarity in the respective partner strand. Base conversion in the channel-coding region of GluR-B directed by base paired sequences may be executed by a ubiquitous nuclear adenosine deaminase specific for double-stranded RNA.

title: RNA editing produces glycine receptor alpha3(P185L), resulting in high agonist potency.
abstract: The function of supramedullary glycine receptors (GlyRs) is still unclear. Using Wistar rat collicular slices, we demonstrate GlyR-mediated inhibition of spike discharge elicited by low glycine (10 microM). Searching for the molecular basis of this phenomenon, we identified a new GlyR isoform. GlyR alpha3(P185L), a result of cytidine 554 deamination, confers high glycine sensitivity (EC50 approximately 5 microM) to neurons and thereby promotes the generation of sustained chloride conductances associated with tonic inhibition. The level of GlyR alpha3-C554U RNA editing is sensitive to experimentally induced brain lesion, inhibition of cytidine deamination by zebularine and inhibition of mRNA transcription by actinomycin D, but not to blockade of protein synthesis by cycloheximide. Conditional regulation of GlyR alpha3(P185L) is thus likely to be part of a post-transcriptional adaptive mechanism in neurons with enhanced excitability.

title: Editor meets silencer: crosstalk between RNA editing and RNA interference
abstract: The most prevalent type of RNA editing is mediated by ADAR (adenosine deaminase acting on RNA) enzymes, which convert adenosines to inosines (a process known as A→I RNA editing) in double-stranded (ds)RNA substrates. A→I RNA editing was long thought to affect only selected transcripts by altering the proteins they encode. However, genome-wide screening has revealed numerous editing sites within inverted Alu repeats in introns and untranslated regions. Also, recent evidence indicates that A→I RNA editing crosstalks with RNA-interference pathways, which, like A→I RNA editing, involve dsRNAs. A→I RNA editing therefore seems to have additional functions, including the regulation of retrotransposons and gene silencing, which adds a new urgency to the challenges of fully understanding ADAR functions.

title: C-to-U RNA Editing: Mechanisms Leading to Genetic Diversity*
abstract: Substitutional RNA Editing: Biochemical Mechanisms and Targets for C-to-U RNA Editing in Mammals RNA editing is an important mechanism for regulating genetic plasticity through the generation of alternative protein products from a single structural gene. Substitutional RNA editing employs a variety of genetic mechanisms, the biochemical basis of which has been elucidated following the development of in vitro assays that recapitulate important elements of this process. Two types of substitutional RNA exist in mammals, namely A-to-I and C-to-U RNA editing (1, 2). Important biochemical distinctions between these two processes provide an informative basis for understanding the mechanisms of C-to-U RNA editing and the adaptations that control target specificity. A-to-I RNA editing is mediated by a family of adenosine deaminases acting on double-stranded RNA (ADARs) with partially overlapping target specificity (1, 2). The absolute requirement for a double-stranded RNA template distinguishes A-to-I and C-to-U RNA editing because the former requires a pre-mRNA template containing intronic regions and is thus biochemically confined to unspliced transcripts. A further distinction biochemically is that ADAR enzymes do not require additional cofactors. ADARs contain both double-stranded RNA binding domains and a deaminase domain and function as modular editing enzymes (2, 3). The best characterized example of C-to-U RNA editing involves the nuclear transcript encoding intestinal apolipoprotein B (apoB) (4). ApoB RNA editing changes a CAA to a UAA stop codon, generating a truncated protein, apoB48 (4). ApoB RNA editing has important effects on lipoprotein metabolism, and its emergence defines distinct pathways for intestinal and hepatic lipid transport in mammals (4). C-to-U editing of apoB RNA requires a single-strand template  with well defined characteristics in the immediate vicinity of the edited base, as well as protein cofactors that assemble into a functional complex referred to as a holoenzyme or editosome. This functional complex includes a minimal core composed of apobec-1, the catalytic deaminase, and a competence factor, apobec-1 complementation factor (ACF), that functions as an adaptor protein by binding both the deaminase and the RNA substrate . The interaction of these protein components and their higher order interactions with the nuclear transcript illustrates the complexity of site-selectivity in C-to-U RNA editing. A second example of C-to-U RNA editing in mammals involves site-specific deamination of a CGA to UGA codon in the neurofibromatosis type 1 (NF1) mRNA (5). NF1 RNA editing generates a translational termination codon at position 3916 that is predicted to truncate the protein product neurofibromin at the 5 end of a critical domain (6) involved in GTPase activation . Unlike apoB RNA editing, there is no formal proof that a truncated protein is generated. This example of C-to-U RNA editing has been demonstrated in peripheral nerve sheath tumors from patients with NF1 and may share elements of the same machinery as apoB RNA editing, as discussed below. A third target for C-to-U editing, NAT1, was revealed following forced transgenic overexpression of apobec-1 in murine and rabbit hepatocytes (7). NAT1 is homologous to the translational repressor eIF4G and undergoes C-to-U editing at multiple sites, with the creation of stop codons that in turn reduce protein abundance (7).

title: The apolipoprotein B mRNA editing complex performs a multifunctional cycle and suppresses nonsense‐mediated decay
abstract: The C to U editing of apolipoprotein B (apoB) mRNA is mediated by a minimal complex composed of an RNA‐binding cytidine deaminase (APOBEC1) and a complementing specificity factor (ACF). This editing generates a premature termination codon and a truncated open reading frame. We demonstrate that the APOBEC1—ACF holoenzyme mediates a multifunctional cycle. The atypical APOBEC1 nuclear localization signal is involved in RNA binding and is used to import ACF into the nucleus as cargo. APOBEC1 alone induces nonsense‐mediated decay (NMD). The APOBEC1—ACF complex edits and remains associated with the edited RNA to protect it from NMD. The APOBEC1 nuclear export signal is involved in the export of ACF and the edited apoB mRNA together, to the site of translation.

title: Messenger RNA editing in mammals: new members of the APOBEC family seeking roles in the family business.
abstract: Alteration of mRNA sequence through base modification mRNA editing frequently generates protein diversity. Several proteins have been identified as being similar to C-to-U mRNA editing enzymes based on their structural domains and the occurrence of a catalytic domain characteristic of cytidine deaminases. In light of the hypothesis that these proteins might represent novel mRNA editing systems that could affect proteome diversity, we consider their structure, expression and relevance to biomedically significant processes or pathologies.

title: An Overview of Cytidine Deaminases
abstract: Enzymes that deaminate cytidine to uridine play an important role in a variety of pathways from bacteria to man.Ancestral members of this family were able to deaminate cytidine only in a mononucleotide or nucleoside context. Recently, a family of enzymes has been discovered with the ability to deaminate cytidines on RNA or DNA. The first member of this new family is APOBEC1, which deaminates apolipoprotein B messenger RNA to generate a premature stop codon. APOBEC1 has the conserved active site motif found in Escherichia coli cytidine deaminase. In addition, APOBEC1 has a unique motif containing 2 phenylalanine residues and an insert of 4 amino acid residues across the active site motif. This motif is present in APOBEC family members including activation-induced cytidine deaminase (AID), APOBEC2, and APOBEC3A through APOBEC3G. AID is essential for initiating class-switch recombination, somatic hypermutation, and gene conversion. The APOBEC3 family is unique to primates. APOBEC3G is able to protect cells from human immunodeficiency virus and other viral infections.This function is not unique to APOBEC3G; other APOBEC3 family members also have this ability. Overexpression of enzymes in this family can cause cancer, suggesting that the genes for the APOBEC family of proteins are proto-oncogenes. Recent advances in the understanding of the mechanism of action of this family are summarized in this review.

title: How Stress and Fluoxetine Modulate Serotonin 2C Receptor Pre-mRNA Editing
abstract: In two inbred strains of mice, C57BL/6 and 129Sv, the majority of forebrain neocortical pre-mRNA encoding the serotonin 2C (5-HT2C) receptor is altered by adenosine-to-inosine editing. As a result, >60% of all mRNAs encode receptors with reduced constitutive and agonist-stimulated activity. However, in the BALB/c strain, a genetically distinct inbred strain with lower forebrain serotonin levels, spontaneously elevated anxiety, and increased stress reactivity, the majority of 5-HT2C mRNA is nonedited and encodes receptors with the highest constitutive activity and the highest agonist affinity and potency. Neither acute stress (the forced swim test) nor chronic treatment with the serotonin-selective reuptake inhibitor fluoxetine elicit significant changes in 5-HT2C pre-mRNA editing in C57BL/6 mice. In contrast, exposure of BALB/c mice to acute stress and chronic treatment of nonstressed BALB/c mice with fluoxetine elicit significant, site-specific increases in 5-HT2C pre-mRNA editing that increase the pool of mRNA encoding receptors with reduced function. These changes in 5-HT2C pre-mRNA editing resemble those detected previously in the prefrontal cortex of subjects with major depression. However, when chronic fluoxetine treatment is combined with stress exposure of BALB/c mice, these changes in 5-HT2C pre-mRNA editing are no longer detected. These findings illustrate that 5-HT2C pre-mRNA editing responses to stress and chronic fluoxetine are modulated by the genetic background, as well as the behavioral state of the animal. They suggest further that the changes in 5-HT2C pre-mRNA editing found in major depression reflect a previously unrecognized molecular response to stress that can be prevented by chronic antidepressant treatment.

title: Altered RNA editing of serotonin 2C receptor in a rat model of depression
abstract: Altered RNA editing of serotonin 2C receptor (HTR2C) has been suggested to be involved in the pathophysiology of major depression. Here we examined RNA editing status of HTR2C in the learned helplessness (LH) rats, one of well-established animal models of depression. LH rats showed the significantly increased RNA editing of site E, and tendency for increased RNA editing of other editing sites. Treatment with fluoxetine, a selective serotonin reuptake inhibitor, or imipramine, a tricyclic antidepressant, affected the RNA editing status of the LH rats. Although, these antidepressants differentially altered RNA editing status, they commonly reduced RNA editing efficiency of site E. We further revealed that altered RNA editing in the LH rats and by antidepressants was not explained by altered expression of RNA editing enzymes or their substrates (adenosine deaminases that act on RNA, HTR2C, and spliced form of HTR2C). These results suggest that alteration of RNA editing of HTR2C may play a role in the pathophysiology of depression and action of antidepressants.

title: A-to-I RNA Editing and Human Disease
abstract: The post-transcriptional modification of mammalian transcripts by A-to-I RNA editing has beenrecognized as an important mechanism for the generation of molecular diversity and alsoregulates protein function through recoding of genomic information. As the molecular players ofediting are characterized and an increasing number of genes become identified that are subject toA-to-I modification, the potential impact of editing on the etiology or progression of humandiseases is realized. Here we review the recent knowledge on where disturbances in A-to-I RNAediting have been correlated with human disease phenotypes.

title: RNA editing: a molecular mechanism for the fine modulation of neuronal transmission.
abstract: The term "RNA editing" is used to identify any mechanism responsible for producing mRNA molecules with sequence information not specifically encoded in the DNA. RNA editing is therefore an important event in gene modification, which takes place at a post-transcriptional level. The molecular mechanism of RNA editing involves site-selective deamination of adenosine to inosine in pre-mRNA, which leads to altering translation codons and splicing in nuclear transcripts, whereby functionally distinct proteins can be produced from a single gene. The mammalian editing enzymes ADARs (adenosine deaminases acting on RNA) are widely expressed in brain and other tissues: however, up until now their substrates have mainly been found in the Central Nervous System (CNS). Of particular relevance in the CNS is the editing occurring at the ionotropic glutamate receptors (GluRs) level. Three AMPA and two Kainate receptors are subject to RNA editing. The consequence of this process is the substitution of specific amino acids in functionally critical positions of the receptors. Depending on the GluR involved, the consequences of editing will involve: activation and/or inhibition of splicing sites; modulation of the trafficking of the receptor to the plasma membrane; the process of tetramerization of the receptor subunits; modification of the ions passage through the receptor channel; modulation of the desensitization and action potential recovery times. All these events are specific to the different GluRs and are genetically and developmentally controlled. RNA editing is therefore a crucial event involved in controlling transmission of the action potential at the postsynaptic level. This modulation involves the transmission of all sensory stimuli to the CNS and gives rise to the "Sensotype". The Sensotype therefore defines the "way" in which the information acquired from the environment by the sensory systems is transmitted to the brain. The signals and inputs deriving from the Sensotype are transmitted to the brain, which processes and stores these signals thus generating the "Brainotype". Brainotype and Sensotype are genetically and environmentally determined; they are individually unique and specific to every living organism with a nervous system. Their characteristics are, at least in part, dependent on the modulation of the "RNA editing" process since glutamate receptors represent the main neurotransmitter system in the CNS.

title: RNA editing in the Wilms' tumor susceptibility gene, WT1.
abstract: Rat kidney WT1 cDNAs contain either a thymidine or a cytosine residue at position 839. Genomic WT1 DNA contains only T839. To explain these results, we propose the WT1 transcript undergoes RNA editing in which U839 is converted to C, resulting in the replacement of leucine 280 in WT1 by proline. RNA editing at the same nucleotide was observed in WT1 cDNAs from human testis. In functional assays, the WT1-leucine280 polypeptide repressed the EGR-1 promoter in in vitro assays approximately 30% more efficiently than WT1-proline. Edited WT1-C839 mRNA was barely detectable in neonatal kidney, whereas adult rat kidneys contained both U839 and C839-WT1 mRNA, suggesting a role for the two protein isoforms in growth and differentiation.

title: The DNA methyltransferases of mammals.
abstract: The biological significance of 5-methylcytosine was in doubt for many years, but is no longer. Through targeted mutagenesis in mice it has been learnt that every protein shown by biochemical tests to be involved in the establishment, maintenance or interpretation of genomic methylation patterns is encoded by an essential gene. A human genetic disorder (ICF syndrome) has recently been shown to be caused by mutations in the DNA methyltransferase 3B (DNMT3B) gene. A second human disorder (Rett syndrome) has been found to result from mutations in the MECP2 gene, which encodes a protein that binds to methylated DNA. Global genome demethylation caused by targeted mutations in the DNA methyltransferase-1 (Dnmt1) gene has shown that cytosine methylation plays essential roles in X-inactivation, genomic imprinting and genome stabilization. The majority of genomic 5-methylcytosine is now known to enforce the transcriptional silence of the enormous burden of transposons and retroviruses that have accumulated in the mammalian genome. It has also become clear that programmed changes in methylation patterns are less important in the regulation of mammalian development than was previously believed. Although a number of outstanding questions have yet to be answered (one of these questions involves the nature of the cues that designate sites for methylation at particular stages of gametogenesis and early development), studies of DNA methyltransferases are likely to provide further insights into the biological functions of genomic methylation patterns.

title: Computational analyses show A-to-G mutations correlate with nascent mRNA hairpins at somatic hypermutation hotspots.
abstract: Activation-induced cytidine deaminase (AID) initiates Phase I somatic hypermutation (SHM) of antibody genes by deaminating deoxy-cytosine to deoxy-uracil (C-to-U). These lesions trigger Phase II, a poorly understood process of error-prone repair targeting A-T pairs by DNA polymerase eta (Pol eta). Since Pol eta is also a reverse transcriptase, Phase II could involve copying off RNA as well as DNA templates. We explore this idea further since in an RNA-based pathway it is conceivable that adenosine-to-inosine (A-to-I) RNA editing causes A-to-G transitions since I like G pairs with C. Adenosine deaminases (ADARs) are known to preferentially edit A nucleotides that are preceded by an A or U (W) in double-stranded RNA substrates. On this assumption and using a theoretical bioinformatics approach we show that a significant and specific correlation (P<0.002) exists between the frequency of WA-to-WG mutations and the number of mRNA hairpins that could potentially form at the mutation site. This implies roles for both RNA editing and reverse transcription during SHM in vivo and suggests definitive genetic experiments targeting the appropriate ADAR1 isoform (gammaINF-ADAR1) and/or Ig pre-mRNA templates.

title: Generation of G-to-A and C-to-U changes in HIV-1 transcripts by RNA editing.
abstract: RNA editing involves posttranscriptional alterations of messenger RNA (mRNA) sequences modifying the information content encoded by the genetic message. Here, it is shown that, in chronically infected H9 cells, human immunodeficiency virus-type 1 (HIV-1) mRNAs undergo guanine-to-adenine (G-to-A) and cytosine-to-uracil (C-to-U) changes. G-to-A modification in the untranslated region of exon 1 was present only in spliced HIV-1 mRNAs. The creation of stop codons in HIV-1 mRNAs may function to control the translation of viral proteins, such as viral protein R, that are involved in the regulation of HIV-1 expression and the survival of chronically infected cells.

title: Transmitter uptake and release in PC12 cells overexpressing plasma membrane monoamine transporters
abstract: Transmitter uptake and exocytosis of secretory vesicles are two essential aspects of neurotransmission. Here we show that transient overexpression of plasma membrane monoamine transporters in rat pheochromocytoma PC12 cells induced an approximate 20‐fold enhancement of cellular uptake of monoamines. Intravesicular amine concentration was greatly increased, as demonstrated directly by carbon fibre amperometry. However, the amount of stored monoamines diminished over a 5‐h period, unless monoamine oxidase was inhibited, indicating that monoamines leak out from secretory vesicles. This efflux of monoamines accounts for the reported dependence of vesicular monoamine content (the quantal size) on the kinetics of vesicular monoamine uptake. Measuring radiolabelled monoamines release from the cell population provided accurate determination of the secretory activity of the subpopulation (10–20%) of cells transfected with monoamine transporters, since they contained about 95% of the radiolabel. Accordingly, significant modification of the secretory responses was observed, at the cell population level, upon transient expression of the serotonin transporter and of proteins known to interfere with exocytosis, such as botulinum neurotoxin C1, GTPase‐deficient Rab3 proteins, truncated Rabphilin constructs or Rim. The co‐transfection assay described here, based on transient expression of monoamine transporters, should prove useful in functional studies of the secretory machinery.

title: Altered Editing of Serotonin 2C Receptor Pre-mRNA in the Prefrontal Cortex of Depressed Suicide Victims
abstract: Five adenosines within the coding sequence of the serotonin 2C receptor (5-HT2C) pre-mRNA are converted to inosines by RNA editing (named A, B, C' (E), C, and D sites). In human prefrontal cortex (PFC), the most abundant 5-HT2C mRNA sequences result from editing at the A site, or from the editing combinations AC'C, ABCD, and ABD. In suicide victims with a history of major depression, C' site editing is significantly increased, D site editing is significantly decreased, and the C site shows a trend toward increased editing. Treatment of mice with the antidepressant drug fluoxetine (Prozac) causes changes in C', C, and D site editing that are exactly opposite to those seen in suicide victims. Thus, one outcome of fluoxetine treatment may be to reverse the abnormalities in 5-HT2C pre-mRNA editing seen in depressed suicide victims.

title: RNA Editing of the Human Serotonin 5-HT2C Receptor: Alterations in Suicide and Implications for Serotonergic Pharmacotherapy
abstract: RNA encoding the human serotonin 5-HT2C receptor (5-HT2CR) 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-HT2CRs 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-HT2CR population differentially responsive to serotonergic drugs. An examination of the efficiencies of RNA editing of the 5-HT2CR 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-HT2CR. These findings suggest that alterations in RNA editing may contribute to or complicate therapy in certain psychiatric disorders.

title: RNA editing of serotonin 2C receptor in human postmortem brains of major mental disorders
abstract: The importance of serotonin 2C receptor (HTR2C) in mental disorders has been implicated by studies of HTR2C-deficient mice and linkage and association studies. Recent studies have revealed that RNA editing of HTR2C is involved in mental disorders. Here we examined RNA editing efficiencies of site A and D of HTR2C in the prefrontal cortex samples of patients with bipolar disorder, schizophrenia, and major depression as well as control subjects by using primer extension combined with denaturing high performance liquid chromatography. Postmortem samples were donated by the Stanley Foundation Brain Collection. We could not find significant alterations of RNA editing efficiencies of these sites in patients. However, we found trends for increased RNA editing efficiencies of site D in depressive patients (P=0.08) and site A in suicide victims (P=0.07). These findings are in accordance with the previous findings, and suggest that altered RNA editing of HTR2C may have some significance in major depression and suicide.

title: RNA editing of the 5-HT2C receptor is reduced in schizophrenia
abstract: 5-HT2C receptor (5HT2CR, serotonin-2C) RNA undergoes editing to produce several receptor variants, some with pharmacological differences. This investigation comprised two parts: the characterisation of 5-HT2CR RNA editing in a larger human control sample than previously examined, and a comparative study in subjects with schizophrenia. Secondary structure analysis of the putative edited region of the human 5-HT2CR gene predicted the existence of a double stranded (ds) RNA loop, essential for RNA editing in this receptor. RNA was then extracted from frontal cortex of five controls and five subjects with schizophrenia. RT-PCR products of the edited region were cloned and sequenced (n = 100). Reduced RNA editing, increased expression of the unedited 5-HT2C-INI isoform in schizophrenia (P = 0.001) and decreased expression of the 5-HT2C-VSV and 5-HT2C-VNV isoforms were detected in the schizophrenia group. In addition, two novel mRNA edited variants were identified: 5-HT2C-MNI and 5-HT2C-VDI. Screening of the 5-HT2CR gene did not reveal any mutations likely to disrupt the dsRNA loop, suggesting that the reduced RNA editing in schizophrenia may instead be caused by altered activity of the editing enzyme(s). Since the unedited 5-HT2C-INI is more efficiently coupled to G proteins than the other isoforms, its increased expression in schizophrenia may lead to enhanced 5-HT2CR-mediated effects. The results also illustrate that potentially important receptor alterations may occur in schizophrenia which are not detectable merely in terms of receptor abundance.

title: Modulation of Serotonin 2C Receptor Editing by Sustained Changes in Serotonergic Neurotransmission
abstract: Serotonin 2C (5-HT2C) receptor pre-mRNA is a substrate for RNA editing enzymes that convert five adenosines (named A, B, C′, C, and D editing sites) to inosines. Editing of two of these sites (C′ and C) is crucial for decreasing the efficiency of the receptor to activate G-protein. Nucleotide sequence analysis of mouse forebrain neocortical 5-HT2C mRNA isoforms revealed that editing at these two sites is regulated in a serotonin-dependent manner. In serotonin-depleted mice, C′- and C-site editing is significantly decreased. This results in an increased expression of 5-HT2C mRNA isoforms encoding receptors with higher sensitivity to serotonin. In contrast, a 4 d treatment with the 5-HT2A/2C agonist (±)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane significantly increases the editing frequency at the C′ site and leads to increased expression of 5-HT2C mRNA isoforms encoding receptors that activate G-protein least efficiently. None of the drug treatments led to alterations in cytoplasmic 5-HT2C mRNA levels. These data indicate that editing of 5-HT2C pre-mRNA is a mechanism that retains basic response properties of 5-HT2Creceptors in the face of changing synaptic input to keep receptor activation within an optimal range for information processing.

title: The snoRNA HBII-52 Regulates Alternative Splicing of the Serotonin Receptor 2C
abstract: The Prader-Willi syndrome is a congenital disease that is caused by the loss of paternal gene expression from a maternally imprinted region on chromosome 15. This region contains a small nucleolar RNA (snoRNA), HBII-52, that exhibits sequence complementarity to the alternatively spliced exon Vb of the serotonin receptor 5-HT2CR. We found that HBII-52 regulates alternative splicing of 5-HT2CR by binding to a silencing element in exon Vb. Prader-Willi syndrome patients do not express HBII-52. They have different 5-HT2CR messenger RNA (mRNA) isoforms than healthy individuals. Our results show that a snoRNA regulates the processing of an mRNA expressed from a gene located on a different chromosome, and the results indicate that a defect in pre-mRNA processing contributes to the Prader-Willi syndrome.

title: Neuronal Tryptophan Hydroxylase mRNA Expression in the Human Dorsal and Median Raphe Nuclei: Major Depression and Suicide
abstract: Major depressive disorder (MDD) and suicide are associated with deficient serotonergic neurotransmission. Tryptophan hydroxylase (TPH) is the rate-limiting biosynthetic enzyme for serotonin. Previously, we reported elevated levels of TPH protein in the dorsal raphe nucleus (DRN) of depressed suicides and now examine expression of neuronal TPH2 mRNA in a cohort of matched controls and depressed suicides (n=11 pairs). DRN TPH2 mRNA was measured by densitometric analysis of autoradiograms from in situ hybridization histochemistry experiments. TPH2 mRNA is confirmed as the raphe-specific isoform of TPH in human brain, and is expressed in neurons throughout the anteroposterior extent of the DRN and median raphe nucleus (MRN). TPH2 mRNA expression correlates with TPH protein distribution in the DRN, and has a negative correlation with age. In drug-free suicides, TPH2 expression is 33% higher in the DRN and 17% higher in the MRN as compared to matched nonpsychiatric controls. Higher levels of TPH2 mRNA were found throughout the entire extent of the rostrocaudal axis of the DRN, and were not specific to any single subnucleus. Higher TPH2 mRNA expression may explain more TPH protein observed in depressed suicides and reflect a homeostatic response to deficient brain serotonergic transmission.

title: Elevated expression of tryptophan hydroxylase-2 mRNA at the neuronal level in the dorsal and median raphe nuclei of depressed suicides
abstract: Deficient levels of serotonin are associated with suicide and depression. Paradoxically, in the dorsal raphe nucleus (DRN) there are more serotonin neurons and more neuronal tryptophan hydroxylase-2 (TPH2) expression postmortem in depressed suicides. In this study, we sought to determine whether greater TPH2 expression in depressed suicides was the result of more TPH2 expression per neuron. In situ hybridization and computer-assisted image analysis were performed on tissue sections throughout the extent of the raphe nuclei at the level of silver grains per neuron to systematically quantify TPH2 neuronal expression. Depressed suicides have 26.5% more TPH2 grain density per neuron in the DRN compared with matched controls (P=0.04). The difference in grain density is greater at mid- and caudal anatomical levels across the rostrocaudal axis of the DRN. Densitometric analysis of TPH2 expression in the DRN subnuclei showed that higher expression levels were observed at posterior anatomical levels of depressed suicides (121% of control in the caudal subnucleus). Higher TPH2 expression in depressed suicides may explain more TPH2 protein and reflect a homeostatic response to deficient serotonin levels in the brains of depressed suicides. Localized changes in TPH2 expression in specific subnuclei of the DRN suggest that the serotonergic compensatory mechanism in depression and suicide is specifically regulated within the DRN and has implications for regions innervated by this subnucleus.

title: Tryptophan hydroxylase 2 gene expression and promoter polymorphisms in bipolar disorder and schizophrenia
abstract: The tryptophan hydroxylase isoform-2 gene (Tph2) is located on chromosome 12 and is expressed primarily in brain tissue. Although the tryptophan hydroxylase isoform-1 gene (Tph1) has been reported to have a genetic association with bipolar disorder and schizophrenia, the Tph1 isoform is expressed at much lower levels than Tph2 (150-fold less in the mouse brain). We hypothesized that bipolar disorder and schizophrenia are associated with abnormal levels of TPH2 mRNA in the brain. TPH2 and β-actin mRNA levels in postmortem brain were quantified using real-time PCR. mRNA samples provided by the Stanley Foundation Array Collection were derived from the dorsolateral prefrontal cortex (Brodmann Area 46) of 35 bipolar, 35 schizophrenic, and 35 control subjects. There were significant differences in the mRNA levels among bipolar, schizophrenic, and normal subjects [F(2,102)=3.58; p=0.031]. A greater amount of TPH2 mRNA was found in the bipolar group in comparison with control subjects (Tukey's test: p=0.024). Further investigations of Tph2 are needed to clarify the potential role of this gene in the pathophysiology of bipolar disorder.

title: Genetic architecture of the human tryptophan hydroxylase 2 Gene: existence of neural isoforms and relevance for major depression
abstract: Impaired brain serotonin neurotransmission is a potential component of the diathesis of major depression. Tryptophan hydroxylase-2 (TPH2), is the rate limiting biosynthetic isoenzyme for serotonin that is preferentially expressed in the brain and a cause of impaired serotonin transmission. Here, we identify a novel TPH2 short isoform with truncated catalytic domain expressed in human brainstem, prefrontal cortex, hippocampus and amygdala. An exploratory study of 166 Caucasian subjects revealed association with major depression or suicide of a novel single nucleotide polymorphism (SNP) g.22879A>G located in exon 6 of this short isoform. This SNP and additional SNPs were discovered through a systematic characterization of the genetic architecture of the TPH2 gene for further genetic and functional investigations of its relationship to major depression and other psychopathology.

title: SNP and haplotype analysis of a novel tryptophan hydroxylase isoform (TPH2) gene provide evidence for association with major depression
abstract: Tryptophan hydroxylase (TPH), being the rate-limiting enzyme in the biosynthesis of serotonin plays a major role as candidate gene in several psychiatric disorders. Recently, a second TPH isoform (TPH2) was identified in mice, which was exclusively present in the brain. In a previous post-mortem study of our own group, we could demonstrate that TPH2 is also expressed in the human brain, but not in peripheral tissues. This is the first report of an association study between polymorphisms in the TPH2 gene and major depression (MD). We performed single-nucleotide polymorphism (SNP), haplotype and linkage disequlibrium studies on 300 depressed patients and 265 healthy controls with 10 SNPs in the TPH2 gene. Significant association was detected between one SNP (P=0.0012, global P=0.0051) and MD. Haplotype analysis produced additional support for association (P<0.0001, global P=0.0001). Our findings provide evidence for an involvement of genetic variants of the TPH2 gene in the pathogenesis of MD and might be a hint on the repeatedly discussed duality of the serotonergic system. These results may open up new research strategies for the analysis of the observed disturbances in the serotonergic system in patients suffering from several other psychiatric disorders.