Abstract
The serotonin neurotransmitter system is widespread in the brain and implicated in modulation of neuronal responses to other neurotransmitters. Among 14 serotonin receptor subtypes, 5-HT2cR plays a pivotal role in controlling neuronal network excitability. Serotonergic activity conveyed through receptor 5-HT2cR is regulated post-transcriptionally via two mechanisms, alternative splicing and A-to-I RNA editing. Brain-specific small nucleolar RNA SNORD115 harbours a phylogenetically conserved 18-nucleotide antisense element with perfect complementarity to the region of 5ht2c primary transcript that undergoes post-transcriptional changes. Previous 5ht2c minigene studies have implicated SNORD115 in fine-tuning of both post-transcriptional events. We monitored post-transcriptional changes of endogenous 5ht2c transcripts during neuronal differentiation. Both SNORD115 and 5ht2c were upregulated upon neuronal commitment. We detected increased 5ht2c alternative exon Vb inclusion already at the stage of neuronal progenitors, and more extensive A-to-I editing of non-targeted sites A and B compared to adjacent adenosines at sites E, C and D throughout differentiation. As the extent of editing is known to positively correlate with exon Vb usage while it reduces receptor functionality, our data support the model where SNORD115 directly promotes alternative exon inclusion without the requirement for conversion of key adenosines to inosines, thereby favouring production of full-length receptor isoforms with higher potency.
Highlights
Serotonin receptor 2 C (5-HT2cR) is a member of the 7-transmembrane-spanning receptors that convey signals through coupling with intracellular guanine nucleotide-binding proteins (G proteins)
RNA-seq analysis (Fig. 2) built on available dataset[29] showed that neurons produced from P19 cell line express SNORD115 and 5ht2c genes at levels up to 2 orders of magnitude higher than the neuronal precursors with neuronal 5ht2c transcripts being 200-fold underrepresented compared to combined SNORD115 cluster members
Differentiating P19 cells were chosen as a model system to monitor 5ht2c post-transcriptional modifications in correlation with SNORD115 expression changes
Summary
Serotonin receptor 2 C (5-HT2cR) is a member of the 7-transmembrane-spanning receptors that convey signals through coupling with intracellular guanine nucleotide-binding proteins (G proteins). Whereas Vitali et al.[4] showed that SNORD115-guided ribose methylation inhibits A-to-I editing selectively at position C, Kishore and Stamm[24] demonstrated that SNORD115 affects alternative splicing in a methylation-independent manner, plausibly by transiently associating with a silencer element at the proximal splice site to promote exon Vb inclusion These studies were performed on neuroblasts and/ or fibroblasts transiently transfected with engineered 5ht2c minigenes that were either artificially targeted to nucleolus[4], the site of SNORD115 accumulation, or had their distal splice site (Fig. 1) mutated to mammalian consensus to activate splicing in immortalized cell line[24]. SNORD115 knock-down is complicated by redundancy as there are numerous homologous copies of the gene
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