The roles of phospholipase C activation and alternative ADAR1 and ADAR2 pre-mRNA splicing in modulating serotonin 2C-receptor editing in vivo

Abstract

The serotonin 2C receptor (5-HT2CR), a Gq-protein-coupled neurotransmitter receptor, exists in multiple isoforms that result from RNA editing of five exonic adenosines that are converted to inosines. In the adult brain, editing of 5-HT2C pre-mRNA exhibits remarkable plasticity in response to environmental and neurochemical stimuli. Here, we investigated two potential mechanisms underlying these plastic changes in adult 5-HT2CR editing phenotypes in vivo: activation of phospholipase C (PLC) and alternative splicing of pre-mRNA encoding the editing enzymes ADAR1 and ADAR2. Studies on two inbred strains of mice (C57Bl/6 and Balb/c) revealed that sustained stimulation of PLC-a downstream effector of activated Gaq protein-increased editing of forebrain neocortical 5-HT2C pre-mRNA at two sites known to be targeted by ADAR2. Moreover, changes in relative expression of the alternatively spliced ''a'' and ''b'' mRNA isoforms of ADAR1 and ADAR2 also correlate with changes in 5-HT2CR editing. The site-specific changes in 5-HT2CR editing detected in mice with different ''a'' over ''b'' ADAR mRNA isoform ratios only partially overlap with those evoked by sustained PLC activation and are best explained by the increased editing efficiency of ADAR1. Thus, activation of PLC and alternative splicing of ADAR pre-mRNA have both overlapping and specific roles in modulating 5-HT2CR editing phenotypes.

Evolutionarily conserved A-to-I editing increases protein stability of the alternative splicing factorNova1

“…In addition, many alternatively spliced genes that are not regulated by Nova are also RNA editing targets: even the function of both ADAR genes, which are responsible for RNA editing, is regulated through both auto-editing and AS. 30,31 This striking regulatory complexity illuminates the highly reticulate relationships underlying the complex transcriptomes of vertebrate brains.…”

Activity Regulation of Adenosine Deaminases Acting on RNA (ADARs)

“…While these two variants have a higher editing efficiency at the A-and C-sites, ADAR1a has higher activity at the B-site [69]. The relative expression of the alternatively spliced ADAR1a and ADAR1b mRNA isoforms correlates with the observed changes at the level of 5HT-2cR RNA editing [70].…”

“…In one study, negative feedback on RNA editing was shown to occur through the activation of phospholipase C (PLC) in response to serotonin stimulation [70]. PLC activation leads to the cleavage of phosphatidyl inositol 4,5-bisphosphate to form the second messengers diacylglycerol and inositol 1,4,5-triphosphate, whose successive phosphorylation ultimately leads to the formation of IP6.…”

“…The edited isoforms exhibit a reduction in G-protein coupling and a reduced affinity for serotonin [22,89,123,124]. Therefore, the serotonin-induced production of IP6 would cause increased production of active ADAR2, which, in turn, edits 5HT-2cR RNA to attenuate serotonin signaling [70]. An increase in IP6 levels may be relevant only for newly synthesized ADAR protein because it cannot move inside and outside of the protein [70].…”

“…Therefore, the serotonin-induced production of IP6 would cause increased production of active ADAR2, which, in turn, edits 5HT-2cR RNA to attenuate serotonin signaling [70]. An increase in IP6 levels may be relevant only for newly synthesized ADAR protein because it cannot move inside and outside of the protein [70]. Further investigation is needed to directly link PLC activation to the regulation of ADAR2 activity.…”