The serotonin2C (5-HT2C) receptor couples to both phospholipase C (PLC)-inositol phosphate (IP) and phospholipase A2 (PLA2)-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-HT2C 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 PLA2-AA pathways in CHO cells stably expressing two major, fully RNA-edited isoforms (5-HT2C-VSV, 5-HT2C-VGV) of the h5-HT2C receptor.
5-HT increased AA release and IP accumulation in both 5-HT2C-VSV and 5-HT2C-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-HT2C-INI) when receptors were expressed at similar levels.
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-HT2C agonists to traffic receptor stimulus is lost as a result of RNA editing.
These results suggest an important role for the second intracellular loop in transmitting agonist-specific information to signalling molecules.
British Journal of Pharmacology (2001) 134, 386–392; doi:10.1038/sj.bjp.0704255
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"The recoding of these DNA nucleotides by A-to-I RNA editing at these sites, generates up to 32 different mRNA variants that encode for up to 24 different protein isoforms, with varying biochemical properties. The fully edited receptor isoform (corresponding to the amino acids VGV) was shown to have a large reduction of agonist-stimulated G-protein coupling compared with the unedited receptor isoform (corresponding to the amino acids INI) (Berg et al. 2001). The RNA editing levels recorded for each of the four prominent sites A, B, C and D separately did not exhibit a statistically significant reduction in each of the three brain regions studied. "
[Show abstract][Hide abstract] ABSTRACT: Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer’s disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients’ brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing.
"Ribosomes decode inosine as guanosine, and editing events within open reading frames lead to production of edited isoforms of ion channel subunits and other proteins. Production of edited subunit isoforms affects the pharmacological properties of channels and receptors such as AMPA receptors, potassium Kv1.1 channels and serotonin 5HT2c receptors (3–6). Various neurological disorders including Motor Neuron Disease, depression and epilepsy have been linked to defects in RNA editing, particularly to ADAR2 and to the transcripts that it edits (7–11). "
[Show abstract][Hide abstract] ABSTRACT: RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of
proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar5G1 null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective
locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic
activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers;
Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar5G1 mutant phenotypes are ameliorated by feeding GABA modulators. We demonstrate that neuronal excitability is linked to dADAR
expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar5G1 null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic
conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability.
Full-text · Article · Oct 2013 · Nucleic Acids Research
"The E, C, and D editing sites on premRNA overlap with the targeted region. Posttranscriptional A to I editing has been reported to decrease the efficiency of G-protein coupling and therefore generates 5HT2c receptor variants with reduced activity (Berg et al. 2001; Vitali et al. 2005). Hence, perfect base complementarities displayed by the antisense element of Snord115 RNA to the alternatively spliced and posttranscriptionally edited exon of 5HT2c premRNA suggest a tempting model for regulation of serotonin receptor biogenesis by snoRNAs (Cavaille et al. 2000). "
[Show abstract][Hide abstract] ABSTRACT: The evolution of new genes can ensue through either gene duplication and the neofunctionalization of one of the copies or the formation of a de novo gene from hitherto nonfunctional, neutrally evolving intergenic or intronic genomic sequences. Only very rarely are entire genes created de novo. Mostly, nonfunctional sequences are coopted as novel parts of existing genes, such as in the process of exonization whereby introns become exons through changes in splicing. Here, we report a case in which a novel non-protein coding RNA evolved by intron-sequence recruitment into their structures. cDNAs derived from rat brain small RNAs, revealed a novel snoRNA originating from one of the Snord115 copies in the rat Prader-Willi-Syndrome locus. We suggest that a single point substitution in the Snord115 region led to the expression of a longer snoRNA variant, designated as L-Snord115. Cell culture and footprinting experiments confirmed that a single nucleotide substitution at Snord115 position 67 destabilized the kink-turn (K-turn) motif within the canonical snoRNA, while distal intronic sequences provided an alternate D-box region. The exapted sequence displays putative base pairing to 28S rRNA and mRNA targets.
Full-text · Article · Oct 2013 · Genome Biology and Evolution