Abstract
Ionotropic glutamate receptors (iGluRs) play key roles for signaling in the central nervous system. Alternative splicing and RNA editing are well-known mechanisms to increase iGluR diversity and to provide context-dependent regulation. Earlier work on isoform identification has focused on the analysis of cloned transcripts, mostly from rodents. We here set out to obtain a systematic overview of iGluR splicing and editing in human brain based on RNA-Seq data. Using data from two large-scale transcriptome studies, we established a workflow for the de novo identification and quantification of alternative splice and editing events. We detected all canonical iGluR splice junctions, assessed the abundance of alternative events described in the literature, and identified new splice events in AMPA, kainate, delta, and NMDA receptor subunits. Notable events include an abundant transcript encoding the GluA4 amino-terminal domain, GluA4-ATD, a novel C-terminal GluD1 (delta receptor 1) isoform, GluD1-b, and potentially new GluK4 and GluN2C isoforms. C-terminal GluN1 splicing may be controlled by inclusion of a cassette exon, which shows preference for one of the two acceptor sites in the last exon. Moreover, we identified alternative untranslated regions (UTRs) and species-specific differences in splicing. In contrast, editing in exonic iGluR regions appears to be mostly limited to ten previously described sites, two of which result in silent amino acid changes. Coupling of proximal editing/editing and editing/splice events occurs to variable degree. Overall, this analysis provides the first inventory of alternative splicing and editing in human brain iGluRs and provides the impetus for further transcriptome-based and functional investigations.
Highlights
The nervous system shows a remarkable degree of differentiation, despite being built from a limited set of molecular and cellular entities
The highest coverage was obtained for exons belonging to the NMDA receptor subunit GluN1 (GRIN1), which were covered on average with 30,080 reads per nucleotide position
We found that some previously reported isoforms occur at overall negligible amounts in human brain (e.g., GluK1-a and GluK5-b), whereas some other isoforms that have received little attention so far may occur at rather high abundance (e.g., GluA4-short/long and GluK1-1)
Summary
The nervous system shows a remarkable degree of differentiation, despite being built from a limited set of molecular and cellular entities. Splicing and editing are widespread in the central nervous system (CNS) [1], where they contribute to differentiation [2, 3], synaptic organization [4], and the tuning of voltage-gated channels and receptors [5,6,7]. 18 different iGluR subunits have been described [10, 11], which are grouped into four subfamilies: AMPA receptors (subunits GluA1-4) [12, 13], kainate receptors (subunits GluK1-5) [14, 15], delta receptors (GluD1 and GluD2) [16], and NMDA receptors [17, 18]. Heteromer formation appears to prevail within the AMPA and kainate receptor subfamilies, which allows for the integration of different functionalities within single receptor complexes [19,20,21]
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