Impact of RNA Editing on Functions of the Serotonin 2C Receptor in vivo

Transcripts encoding 5-HT2C receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV receptors. However, enhanced behavioral sensitivity to a 5-HT2C receptor agonist was also seen in mice expressing 5-HT2C-VGV receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV receptors had greater sensitivity to a 5-HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT2C receptor binding sites in the brains of mice solely expressing 5-HT2C-VGV receptors. We conclude that 5-HT2C-VGV receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5-HT2C receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C receptor binding sites in brain. We further caution that the pattern of 5-HT2C receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor

Alterations in Neurogenic Inflammatory Responses in Mice Lacking αCGRP

a-Calcitonin gene-related peptide (aCGRP) is a pleiotropic peptide neuromodulator that is widely expressed throughout the central and peripheral nervous systems. Although CGRP has been implicated in numerous physiological processes (including peripheral vasodilatation, acetylcholine receptor biosynthesis, nociception, and neurogenic inflammation), the precise physiological roles of CGRP remain to be elucidated. To provide a better understanding of the physiological role(s) mediated by this peptide neurotransmitter, we have generated aCGRP-null mice by targeted modification in embryonic stem cells

The Activity of the Serotonin Receptor 2C is Regulated by Alternative Splicing

The central nervous system-specific serotonin receptor 2C (5HT2C) controls key physiological functions, such as food intake, anxiety, and motoneuron activity. Its deregulation is involved in depression, suicidal behavior, and spasticity, making it the target for antipsychotic drugs, appetite controlling substances, and possibly anti-spasm agents. Through alternative pre-mRNA splicing and RNA editing, the 5HT2C gene generates at least 33 mRNA isoforms encoding 25 proteins. The 5HT2C is a G-protein coupled receptor that signals through phospholipase C, influencing the expression of immediate/early genes like c-fos. Most 5HT2C isoforms show constitutive activity, i.e., signal without ligand binding. The constitutive activity of 5HT2C is decreased by pre-mRNA editing as well as alternative pre-mRNA splicing, which generates a truncated isoform that switches off 5HT2C receptor activity through heterodimerization; showing that RNA processing regulates the constitutive activity of the 5HT2C system. RNA processing events influencing the constitutive activity target exon Vb that forms a stable double stranded RNA structure with its downstream intron. This structure can be targeted by small molecules and oligonucleotides that change exon Vb alternative splicing and influence 5HT2C signaling in mouse models, leading to a reduction in food intake. Thus, the 5HT2C system is a candidate for RNA therapy in multiple models of CNS disorders

Mutations underlying Episodic Ataxia type-1 antagonize Kv1.1 RNA editing

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 7 (2017): 41095, doi:10.1038/srep41095.Adenosine-to-inosine RNA editing in transcripts encoding the voltage-gated potassium channel Kv1.1 converts an isoleucine to valine codon for amino acid 400, speeding channel recovery from inactivation. Numerous Kv1.1 mutations have been associated with the human disorder Episodic Ataxia Type-1 (EA1), characterized by stress-induced ataxia, myokymia, and increased prevalence of seizures. Three EA1 mutations, V404I, I407M, and V408A, are located within the RNA duplex structure required for RNA editing. Each mutation decreased RNA editing both in vitro and using an in vivo mouse model bearing the V408A allele. Editing of transcripts encoding mutant channels affects numerous biophysical properties including channel opening, closing, and inactivation. Thus EA1 symptoms could be influenced not only by the direct effects of the mutations on channel properties, but also by their influence on RNA editing. These studies provide the first evidence that mutations associated with human genetic disorders can affect cis-regulatory elements to alter RNA editing.This work was supported by the Vanderbilt Molecular Endocrinology Training Program (T32DK007563; E.A.F.K.), a Ruth L. Kirschstein National Research Service Award (F31NS087911; E.A.F.K), a Vanderbilt Dissertation Enhancement Grant (E.A.F.K.), and the Vanderbilt Joel G. Hardman Chair in Pharmacology (R.B.E). Additional support for J.J.C.R. included NINDS (R0111223855, R01NS64259) and the Cystic Fibrosis Foundation Therapeutics (Rosent14XXO). Infrastructural support for J.J.C.R. was provided by NIGMS (P20GM103642), NIMH (G12-MD007600), and NSF (DBI 0115825, DBI 1337284)

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

Mice with altered serotonin 2C receptor RNA editing display characteristics of Prader–Willi syndrome

RNA transcripts encoding the 2C-subtype of serotonin (5HT2C) receptor undergo up to five adenosine-to-inosine editing events to encode twenty-four protein isoforms. To examine the effects of altered 5HT2C editing in vivo, we generated mutant mice solely expressing the fully-edited (VGV) isoform of the receptor. Mutant animals present phenotypic characteristics of Prader-Willi Syndrome (PWS) including a failure to thrive, decreased somatic growth, neonatal muscular hypotonia, and reduced food consumption followed by post-weaning hyperphagia. Though previous studies have identified alterations in both 5HT2C receptor expression and 5HT2C-mediated behaviors in both PWS patients and mouse models of this disorder, to our knowledge the 5HT2C gene is the first locus outside the PWS imprinted region in which mutations can phenocopy numerous aspects of this syndrome. These results not only strengthen the link between the molecular etiology of PWS and altered 5HT2C expression, but also demonstrate the importance of normal patterns of 5HT2C RNA editing in vivo

Altered RNA Editing in Mice Lacking ADAR2 Autoregulation

ADAR2 is a double-stranded-RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-selective conversion of adenosine to inosine. Previous studies from our laboratory have demonstrated that ADAR2 can modify its own pre-mRNA to create a proximal 3′ splice site containing a noncanonical adenosine-inosine dinucleotide. Alternative splicing to this proximal acceptor adds 47 nucleotides to the mature ADAR2 transcript, thereby resulting in the loss of functional ADAR2 protein expression due to premature translation termination in an alternate reading frame. To examine whether the editing of ADAR2 transcripts represents a negative autoregulatory strategy to modulate ADAR2 protein expression, we have generated genetically modified mice in which the ability of ADAR2 to edit its own pre-mRNA has been selectively ablated by deletion of a critical sequence (editing site complementary sequence [ECS]) required for adenosine-to-inosine conversion. Here we demonstrate that ADAR2 autoediting and subsequent alternative splicing are abolished in homozygous ΔECS mice and that ADAR2 protein expression is increased in numerous tissues compared to wild-type animals. The observed increases in ADAR2 protein expression correlate with the extent of ADAR2 autoediting observed with wild-type tissues and correspond to increases in the editing of ADAR2 substrates, indicating that ADAR2 autoediting is a key regulator of ADAR2 protein expression and activity in vivo