iGluR transcripts in RNA-Seq data from human brain tissues
For our study, we chose 35 different publicly available RNA-Seq datasets from two unrelated large-scale transcriptome studies [66, 67]. In total, the datasets comprised 8.18∙109 reads of 50 or 100 nt length from different brain regions and individuals (693.5 Gbases; see Table S1 and Fig. S1). Alignment to the human reference genome hg38 showed reasonable overall data quality (see SI Methods, Fig. S2). Only a low percentage of reads mapped to intronic sequences, as expected for reads that mostly originate from processed transcripts. Subsequent analyses were performed with a user-defined reference genome, which only encompassed the iGluR genes and adjacent chromosomal regions (± 1 Mbp; see SI Methods).
To compare the transcript abundance of the different iGluRs, we determined the mean single-nucleotide coverages of their canonical exons across all datasets (Fig. 1 and Tables S2, S3). 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. The median iGluR average coverage was 1,848 reads per nucleotide position. GRIN2D and GRIN3A exons showed an average single-nucleotide coverage < 500; GRIN3B was hardly detectable (Fig. 1B).
As expected, transcript expression differed between different datasets and preparation methods (Fig. S3). Still, it should be noted that individual datasets cannot provide reliable information for comparing iGluR expression in different brain regions. Nevertheless, with the exception of GRIN3B, the aggregated data should allow for reliable detection and quantification of splice junctions as well as nucleotide mismatches caused by RNA editing.
Detection and analysis of splice junctions
Our aim was to identify and quantify iGluR splicing using direct experimental information. We thus focused our analysis on sequencing reads that mapped to splice junctions: These reads align at two distant gene regions, thereby revealing the corresponding splice donor and acceptor sites (Fig. 1A and Fig. S4).
From our alignment, we extracted 1,747,402 junction-spanning reads, which contained 1124 unique iGluR junctions (Table S4). A large fraction of these junctions were sampled rarely (55% in ≤ 10 reads; Fig. S5), but all shared the characteristics of major spliceosome U2 donor and acceptor splice sites (Fig. S6). We continued by classifying each junction as either (i) belonging to a canonical (reference) isoform, (ii) being an alternative junction, which has already been part of a human transcript reported in the Ensembl database (GENCODE; , or (iii) being a newly identified junction (Fig. 2A–C). Details on the workflow and de novo identification of splice junctions are reported in the Methods section.
For referencing purposes, we defined canonical iGluR isoforms, which typically represent the most frequent splice events (all are human Ensembl transcripts; Table S2). We detected all iGluR junctions belonging to the canonical isoforms (259/259), most of them with high coverage (Fig. S5, S7). The canonical junctions of GRIN1, which is the highest expressing iGluR gene, were covered with 17,541–78,603 reads. Even for low expressing iGluRs, such as GRIN2D and GRIN3A, we detected 60–967 reads per junction. Only the junctions of GRIN3B, which shows extremely low transcript levels (Fig. 1B), were covered poorly, i.e., with only a few reads. Within individual iGluR genes, the canonical junction abundance was rather uniform, as seen by a narrow distribution after normalization to the respective mean abundances (standard deviation 0.42; Fig. 2A and Fig. S7).
Besides the canonical junctions, we also detected 93 known alternative iGluR splice junctions out of 120 alternative junctions that were present in the human transcripts in the Ensembl database (Fig. 2B). To estimate the relative abundance of alternative junctions, we normalized their read counts to the corresponding canonical junctions. In cases, where this was not possible, we used the closest canonical junction for normalization (see SI Methods). Normalization shows that a large Fraction of the known alternative junctions occurs rather rarely, as 58% (54/93 junctions) had a relative abundance ≤ 0.05 (Fig. 2B). We thus limited our subsequent analysis to more abundant events: We classified junctions as likely relevant, if they were covered with ≥ 35 reads, and if they had either an overall (global) abundance of ≥ 0.05 or were clearly enriched in individual datasets (local abundance ≥ 0.15). These criteria were met by 46 of the 120 known alternative iGluR junctions (38%) (Fig. 2B), i.e., based on these criteria, more than half of the reported splice events may only play a minor role in adult human brain.
In addition to 352 known splice junctions, we detected 772 novel splice junctions (Fig. 2C). Of those, 728 encompass known donor and/or acceptor sites (primary novel junctions), and 44 contain both, a new donor and a new acceptor site (secondary novel junctions; Fig. S4A). However, most newly identified splice junctions had negligible abundance: Only 2.5% (19/772 junctions) met our relevance criteria and were analyzed further. The large number of low-abundance junctions likely reflects erroneous splice events, i.e., noise . In any case, it seems unlikely that we missed splicing events within our datasets that would have met our relevance criteria, since the number of unique canonical and relevant iGluR junctions saturated early (Fig. S8).
We next analyzed how the relevant alternative junctions differed from the canonical transcript junctions (Fig. 2D). In 37% (24/65 junctions), an alternative donor site (D) was present, in 34% (22/65) an alternative acceptor site (A), and in 20% (13/65) both an alternative donor and acceptor site (DA), which argues against particular detection biases. The remaining junctions, 9% (6/65), showed an alternative combination of canonical donor and acceptor sites (C), i.e., an exon skipping event. Applying our relevance criteria, we detected alternative splicing of all human iGluR genes, except for GRIK3, GRID2, GRIN2D, GRIN3A, and GRIN3B (Fig. 2D). Most relevant junctions were linked to either known or novel exons (Tables 1–4; Fig. S4B); only for 3/65 junctions we were not able to trace the junction to another exon, i.e., they appeared to recede in intronic regions. In the following sections, we summarize the relevant splicing events observed in AMPA, kainate, delta, and NMDA receptors and compare them to literature data.
Alternative splicing of AMPA receptor subunits
The relevant splice junctions belonging to the GRIA1-4 genes are shown in Table 1; for rare events, see Table S4. Information on the canonical reference isoforms is given in Table S2.
The AMPA receptor subunit GluA1 was the first cloned iGluR , which was followed by the identification of the subunits GluA2, GluA3, and GluA4 [10, 75]. At the same time, it was recognized that all four GluA subunits are expressed as flop and flip isoforms, due to the inclusion of mutually exclusive exons of 115 nt length . The corresponding flop and flip segments (38 aa) differ in 8–10 amino acid (aa) residues and are located at the end of the LBD S2 segment, where they contribute to the LBD dimer interface [26, 76] and the S2-TM4 linker region . The flop/flip isoform choice can have pronounced effects on desensitization [20, 23, 77], assembly and trafficking [78,79,80], as well as regulation by allosteric modulators, anions, and TARPs [26, 76, 81]. As expected, we identified junction-spanning reads for inclusion of the flop or flip exons in all four AMPA receptors (Table 1). The ratios of flop to flip junctions varied across datasets (Figs. S9, S10A), which is consistent with reported expression preferences in different brain regions and cell types [22, 23, 37, 40, 82, 83]. However, overall, flop and flip transcripts were detected at similar abundance (Table 1). Flop/flip splicing is thus one of the most frequent alternative splice events in iGluRs (Fig. S9), which underlines its physiological importance. The regulatory mechanisms of flop/flip splicing remain unknown, but activity-dependent changes were observed after neuronal silencing with TTX  and in a mouse model of Rett syndrome .
Next to the junctions that indicate proper splicing of the flop or flip cassettes, we also found junctions, which link the flop and flip exons (GRIA2 (d) 12%, GRIA3 (c) 11%, GRIA4 (g) 5%, Table 1; for GRIA1 0.7%, Table S4). Inclusion of both exons results in a frameshift introducing an early stop codon just before TM helix 4 (GluA1 806 aa; GluA2 813 aa; GluA3 824 aa; GluA4 814 aa) and points to erroneous and/or incomplete splicing. These transcripts should be degraded by nonsense-mediated decay (NMD), since the premature stop codon is followed by several downstream splice junctions > 50 bp away . In contrast, the numbers of junctions pointing to simultaneous removal of both the flop and flip exon were rather low for all subunits (global abundance < 1%; Table S4). These events again result in a frameshift, early truncation, and likely NMD. For GRIA2 and GRIA4, we detected additional junctions into and out of another cassette exon, which is located right before the flop exon (GRIA2 (f, g) and GRIA4 (e, f), Table 1). Also these transcripts code for truncated subunits and should be subject to NMD. Interestingly, these cassette exons are not conserved between GRIA2 and GRIA4, but across species (Fig. S10B).
For GRIA1, only one other splice junction met our relevance criteria, namely splicing from an alternative 5’-UTR exon to an alternative acceptor site in canonical exon 1 (GRIA1 (c), Table 1). However, this transcript would result in an N-terminally truncated subunit, as it lacks the original start codon, signal peptide, and a part of the ATD.
Also splicing in the AMPA receptor C-terminal regions is partly conserved between different subunits, which is exemplified by the GluA2-long isoform (901 aa) . This isoform results from an alternative splice donor site in the penultimate exon, which prolongs the reading frame to a stop codon in the last canonical exon. The C-terminus of GluA2-long lacks the C-terminal type II PDZ binding motif  and is homologous to the C-terminus of the canonical GluA1 and the GluA4-long isoform (Fig. S11). We detected the GluA2-long junction in 18/35 human datasets, but with low frequency compared to the corresponding canonical junction (global abundance 2%; Table 1). Also in rat, this isoform was reported to occur at < 10% abundance .
Several studies addressed variations in the 5′- and 3′-UTRs of GRIA2, which include a polymorphic GU-repeat domain in humans [84, 86] and different polyadenylation sites in the 3′-UTRs , which contain regulatory microRNA binding regions (see ). In addition, we found alternative splicing in the 5′-UTR (Table 1). However, two of these events, GRIA2 (c) and (e), would result in N-terminally truncated receptors without signal peptides; the third one occurs rather rarely (GRIA2 (i)).
GluA3 is subject to flip/flop splicing as described above (Table 1). A GluA3-long isoform does not exist, since the corresponding alternative 5’-donor site is missing in GRIA3 (Fig. S11; ). Apart from this, we detected junctions to two alternative exons, which, however, would result in a drastically shortened and altered ORF encoding 144 aa (GRIA3 (d, e)). Another GluA3 variant with a dominant-negative phenotype has been described to occur in the rat cochlea [89, 90]. In humans, this isoform would result from an alternative donor site in exon 13 (GRIA3 (f)), but in brain datasets, the corresponding junction was only present at low levels (< 1%; Table S4).
For GluA4, in addition to flip/flop splicing, alternative splicing is known to produce two C-terminal isoforms, GluA4-long (902 aa) and GluA4-short (884 aa; also named GluA4-c) [91, 92]. The GluA4-short C-terminus is homologous to the C-termini of GluA2-short (the canonical GluA2 isoform) and GluA3 (Fig. S11). In rodents, the GluA4-long isoform may prevail, since GluA4-short transcripts were mainly observed in the cerebellum . In humans, however, we detected the corresponding GluA4-short junction (GRIA4 (d), Table 1) frequently and almost ubiquitously in 31 of 35 datasets occurring with a global abundance of ~ 40% (Fig. S9), which is in line with previous RT-PCR data . Despite the abundance and loss of a PDZ binding motif in GluA4-long (Fig. S11C; [85, 93]), the physiological role of GluA4 C-terminal splicing has not been investigated so far.
Quite surprisingly, also no literature information can be found for an alternative GRIA4 splice junction that we detected at high abundance (194%) in all neuronal human datasets (GRIA4 (a), Table 1 and Fig. S9), but also in RNA-Seq data from other primate and murine species (Fig. S12). This junction connects canonical exon 10 to an alternative exon, which introduces an early stop codon after 433 aa followed by an alternative 3′-UTR and a polyadenylation signal (PAS) (Table 1 and Fig. S12). The resulting protein, which we termed GluA4-ATD (49 kD), would encompass the signal peptide and ATD of the full-length receptor followed by an additional 10 aa tail with a partly unique sequence (Fig. S12C). Using RT-PCR analysis with exon-specific primers, we independently confirmed the presence of the transcript in RNA from different human brain regions (Fig. S12D). Furthermore, mass spectrometric data suggest that this unusual isoform is expressed on the protein level, as we found a specific peptide matching the alternative GluA4-ATD (Fig. S12E) in a human brain proteome search [94, 95]. Future studies will have to address the expression of the GluA4-ATD isoform and possible physiological functions. In this context, it may be interesting to note, that the GluA4 ATD is involved in pentraxin interactions [96, 97]. Less frequent GRIA4 splicing events include the usage of an alternative 5′-UTR exon (GRIA4 (h)), and inclusion of a 39 nt cassette exon before the flop exon (GRIA4 (e, f)), see flip/flop splicing; Fig. S10B).
Alternative splicing of kainate receptor subunits
Several splicing events have been described for kainate receptors , most of them for the GluK1 subunit. When GluK1 was first cloned from rat , two isoforms were identified, GluK1-1 and GluK1-2. These isoforms differ by the inclusion of a cassette exon that codes for a 15 aa insertion close to the end of the ATD (Table 2; Fig. S13AB). Although this insertion is not seen in any other iGluR, it appears to prevail in GluK1, as we detected the junctions corresponding to GluK1-1 more frequently than the more typical GluK1-2 junction (GRIK1 (a)), which occurred at 53% abundance compared to GluK1-1. In ‘fetal brain’, however, the GluK1-2 isoform was threefold more abundant (Table S4). Queries of datasets from other species suggest similar overall abundances (Fig. S13C). Interestingly, the physiological and functional implications of GluK1-1/2 splicing remain unknown. The affected ATD/LBD-linker region has modulatory functions in other iGluRs (NMDA receptors; ), but experimental data are only available for the GluK1-2 isoform, with the exception of a study that reported a different sensitivity of GluK1-1 towards NS3763, a non-competitive inhibitor .
Moreover, four C-terminal GluK1 variants, GluK1-a–d, have been described [98, 101, 102]. In all analyzed human datasets (except ‘dura mater’), the GluK1-b isoform  junction was the most abundant junction. We thus defined GluK1-1b as canonical isoform [29, 103]. Also the two junctions reporting on the GluK1-c isoform occurred frequently in humans (24% and 30% abundance, respectively; GRIK1 (b,c)) Table 2) and other species (Fig. S13D). GluK1-c is known for poor trafficking in heterologous systems, but may play an important role in controlling presynaptic inhibition at immature synapses . The junction encoding the GluK1-d isoform (GRIK1 (d)) was only enriched in a single dataset (‘subiculum’), and we did not detect a single junction-spanning read for GluK1-a (GRIK1 (f)), which is the shortest reported GluK1 isoform . Specific queries showed that this isoform is also weakly expressed in mouse and rat brain (< 2% abundance; Table S6; Fig. S13D), which is in agreement with earlier studies that detected GluK1-a in the spinal cord and brain stem, but not the mouse forebrain [29, 104, 105]. Notably, the annotated GluK1-a 3′-UTR in human, mouse, and rat lack a polyadenylation signal. Nevertheless, most functional data reported in the literature were obtained with the GluK1-a isoform, as it shows favorable trafficking in heterologous expression systems. Besides known splicing variants we also detected skipping of canonical exon 2 (GRIK1 (e)), which preserves the signal peptide but deletes 56 aa in the N-terminal part of the ATD (864 aa), but overall this event occurs rather rarely.
For GluK2, two alternative isoforms, GluK2-b and GluK2-c, are known, which differ in their CTDs compared to the canonical GluK2-a isoform, due to the inclusion of different cassette exons before the last canonical exon [106,107,108]. In human datasets, the GluK2-a junction prevailed (Table 2), while we detected the two GluK2-b specific junctions (GRIK2 (b,c)) with ~ 8% abundance (see also Fig. S9). GluK2-a is known for particularly effective membrane trafficking , whereas heteromer formation with GluK2-b is thought to allow for additional intracellular interactions, e.g., with Ca2+ signaling associated proteins . We detected also detected reads belonging to the GluK2-c isoform junctions (GRIK2 (g,h); Table 2), but at even lower levels (Fig. S9). This is in line with previous reports that GluK2-c may be more common in human non-neuronal tissues . Similar abundances of these C-terminal isoforms are also seen in other species (Fig. S14C).
Apart from the known GluK2 isoforms, we identified a frequently occurring junction that points to an alternative human 5′-UTR (GRIK2 (a), maximal abundance 1.03; Fig. S14A). The corresponding splice event has been detected before , but the 5′-exon appears to be shorter than reported. Apart from splicing, a polymorphic TAA region is present in the 3′-UTR . Furthermore, we detected two splice events, which occur at low-to-moderate abundance, but encode truncated subunits. One is splicing to an early termination exon, which appears to contain an Alu element (AluSp) (GRIK2 (d), Table 2). In the other case, the inclusion of a cassette exon (GRIK2 (e,f)) introduces an early stop codon, which should mark the transcript for NMD.
For GluK3, one alternative splicing isoform has been described, GluK3-b. It results from a prolonged penultimate exon found in rat  and carries an alternative CTD that reduces surface trafficking . However, in human RNA-Seq data, we found no reads covering this alternative junction and inspection of genomic sequences shows that the corresponding splice donor site is absent in primates (Fig. S15). The human nucleotide coverage remains slightly increased beyond the canonical splice site, which may indicate a 369 nt elongation of canonical exon 15, which would then end with an alternative polyadenylation signal (GRIK3 (a)). However, the resulting C-terminal sequences appear to be poorly conserved between primates (Fig. S15), which suggests that this event does not constitute a major alternative GluK3 isoform. We also did not identify any other GRIK3 junctions with relevant global or local abundance (for rare events see Table S4).
To our knowledge, no splicing isoforms have been described for the ‘high-affinity’ kainate receptor subunit GluK4 (formerly KA1; ). We detected GRIK4 reads somewhat less frequently than reads of the other kainate receptor subunits, which is consistent with its limited expression in adult murine and human brain [114, 115]. However, we identified splice junctions that indicate that some of the transcripts may carry two alternative exons upstream of canonical exon 2, which would result in an alternative 5′-UTR (GRIK4 (a,b), Table 2). Another notable event is skipping of exon 9, which causes a 54 aa deletion in the GluK4 ATD (Table 2 and Fig. S16). The corresponding junction (GRIK4 (e)) occurs with moderate abundance of up to 20% in human datasets (6% global abundance), which was also confirmed by RT-PCR on human RNA samples (Fig. S16CD). Visual inspection shows that the structural integrity of the ATDs may be maintained by the sequence deletion in this potential GluK4-2 isoform (902 aa) (Fig. S16E). We detected the same exon skipping event also in other species, albeit at lower abundance (Fig. S16). Still, experimental work seems warranted to confirm the expression of this potential isoform and its functional consequences.
For GluK5, the second ‘high-affinity’ kainate receptor subunit (formerly KA2), an alternative GluK5-b isoform (981 aa), has been mentioned . However, no experimental data were reported for this isoform, which would result from splicing to an alternative last exon that encodes an alternative C-terminus and 3′-UTR (Fig. S17). Our analysis shows a very low coverage of the corresponding exon and splice junction (GRIK5 (c) junction abundance < 1%), with a slight enrichment in the ‘cerebellum’ dataset (junction abundance 5%; Fig. S9). The physiological significance of this event remains questionable, also because the GluK5-b exon is poorly conserved and rarely detected in chimpanzee and macaque (Fig. S17). In rat and mouse, a homologous exon is absent. Besides, we identified two novel junctions with low-to-moderate abundance (GRIK5 (a) and (b), Table 2), which do not yield full-length GluK5 subunits but may result in NMD; for rare events, see Table S4.
Alternative splicing of delta receptor subunits
The delta receptors GluD1 (GluRδ1) and GluD2 (GluRδ2) were cloned based on sequence homology to other iGluRs [116, 117]. They are expressed throughout the brain, and, although they do not appear to function as glutamate-gated ion channels per se, they play important roles in synapse maturation and plasticity [16, 118]. No delta receptor isoforms have been reported, so far. However, we identified two GRID1 junctions (GRID1 (a,b)), which indicate the insertion of a 91 nt cassette exon before the last canonical exon, exon 16 (Table 3 and Fig. 3A). Both, the single-nucleotide coverage and the relative junction abundances (Fig. 3B) suggest that this cassette exon may be included in ~ 10% of the transcripts. More, the splice event appears to be conserved across species, as we detected both alternative junctions at similar abundance in chimpanzee, macaque, rat, and mouse datasets (Fig. S18).
Importantly, this alternative splice event may lead to a functional but hitherto undescribed GluD1 isoform, which we termed GluD1-b. The cassette exon provides an alternative CTD sequence, which starts ~ 15 aa after TM helix 4 and ends with an early stop codon after 896 aa, in contrast to the canonical GluD1-a isoform with 1009 aa (Fig. 3C). The 3′-UTR and the polyadenylation signal are still provided by canonical exon 16.
To further validate the presence of GluD1 transcript isoforms, we performed reverse transcriptase (RT)-PCRs on RNA samples from human brain (Fig. 3D). Amplification with primers that bind to the alternative exon ‘ae’ and exon 16 (F2/R1) yielded a PCR product of the expected size, and sequencing confirmed the anticipated splice event (Fig. S18). A second primer pair, which binds to canonical exons 15 and 16 (F1/R1), yielded two PCR products, as expected for partial inclusion of the alternative GluD1-b exon. Semi-quantitative analysis showed inclusion in ~ 15% of the brain transcripts (Fig. 3E), which is in good agreement with our estimate based on RNA-Seq data. We also confirmed the presence of GluD1-b in RNA from human cerebral cortex, cerebellum, and testis (Fig. 3E and Fig. S18). We believe that this rather frequent and conserved isoform warrants further investigation, also because a recent study highlighted the role of the GluD1 C-terminus for trafficking in neurons . Apart from this, we detected one other GRID1 junction (GRID1 (c)), with low-to-moderate frequency, which reached from canonical exon 4 into the subsequent mega intron (222,579 bp). In this case, however, we were not able to identify exon-like features (Table 3).
For GRID2, we found lower transcript levels than for GRID1, apart from strong expression in the ‘cerebellum’ dataset (Fig. S3). This is in line with previous reports that showed some GluD2 expression throughout the mouse brain [118, 120] but prominent expression in cerebellar Purkinje cells [16, 117, 121]. We detected all canonical GRID2 junctions but no alternative junctions at clearly relevant levels (for rare events, see Table S4).
Alternative splicing of NMDA receptor subunits
Within the NMDA receptor subfamily [17, 122], alternative splicing has been foremost reported for the GluN1 subunit. GluN1 is abundantly and ubiquitously expressed in the CNS, and, being an obligatory subunit of all NMDA receptors, it is the only essential iGluR subunit.
Three different GluN1 splice events are known that combine to eight isoforms (e.g., [123,124,125]). The first event is the inclusion of a cassette exon between canonical exons 3 and 4 in humans (termed exon 5 in rodents), which encodes a 21 aa insertion in the ATD (denoted as GluN1-b isoforms; Table 4). Our analysis shows that this cassette exon is frequently used, but in most datasets, the canonical GluN1-a junction was somewhat more abundant (Fig. S9). Insertion of the GluN1-b segment, which is located at the ATD–ATD and ATD–LBD dimer interface, modulates glutamate affinity, Zn2+ and proton sensitivity, and deactivation kinetics depending on the partnering GluN2 subunit (e.g., [124, 126,127,128,129]). In the mouse cortex, GluN1-b isoforms may be primarily present in interneurons . Recent knockout studies directly show that this splice event is important for regulating synapse maturation as well as long-term potentiation in mice [130, 131]. Moreover, the GluN1-a/GluN1-b splice ratio has been shown to be affected by psychiatric diseases [51, 54].
The second event is the inclusion of a cassette exon between canonical exons 18 and 19, which adds a 37 aa segment in the CTD (GluN1-1 and GluN1-3 isoforms; Fig. 4A). The third event is splicing to an alternative acceptor site, which causes a 5′ extension of exon 19. This shifts the reading frame and results in different C-termini of GluN1-1 and GluN1-2 compared to GluN1-3 and GluN1-4, respectively. We detected junction-spanning reads for all four C-terminal splice combinations (GluN1-1 to GluN1-4) to significant extent (Table 4 and Fig. 4A). Notably, in some datasets, the GluN1-1 isoform (combination of cassette exon inclusion and splicing to the alternative acceptor site) is highly favored compared to the canonical GluN1-4 isoform (up to 31-fold; Fig. S9). The GluN1-2 isoform was the least abundant isoform and junction-specific queries showed that this isoform is also underrepresented in chimpanzee and macaque datasets (Fig. S19). In rat and mouse, GluN1-3 appears to be the least abundant isoform (Fig. S19; [125, 132]).
Given that the GluN1 CTD composition controls trafficking and protein interactions, for instance with PSD-95 and calmodulin (e.g., [8, 17, 28, 133,134,135,136]), we further analyzed the occurrence of different splice combinations within individual datasets. The C-terminal splice events are clearly correlated: Datasets with high levels of cassette exon inclusion (A+) also show high levels of alternative acceptor usage (B+) (Fig. 4B).
Since individual reads can provide direct information on how the two splice events are combined, we performed a more detailed correlation analysis by asking, whether the presence or absence of the cassette exon (A+ or A−) can be used to predict how much splicing to either acceptor site (B+ or B−) occurs, or, vice versa, whether the presence of either acceptor site may be predictive for cassette exon inclusion (Fig. 4C). We find that in the absence of the cassette exon (A−), the canonical acceptor site (B−) is strongly favored (86:14) over the alternative acceptor site. In the presence of the cassette exon (A+), the alternative acceptor (B+) is somewhat favored over the canonical site (63:37). Quite to the contrary, the type of acceptor site (B+ or B−) cannot be used to predict the inclusion of the alternative exon. Mechanistically, this could indicate that the absence or presence of the cassette exon controls the choice between the two splice acceptor sites. A strong correlation between cassette exon inclusion (A+) and alternative acceptor usage (B+) is also seen in other primate and rodent species (Fig. S19B). Further research seems warranted, also to address potential activity-dependent  and cell type-specific effects. Apart from these well-described isoforms, we only detected three other GluN1 splice events at low levels (Table 4). An early truncation (GRIN1 (g)) and a 3'-UTR exitron (intra-exonic splice junction GRIN1 (i); −448 nt) appear to encode for NMD transcripts, whereas the third junction (GRIN1 (h)) originates from an intronic region.
GluN2A was cloned from rat and mouse [137, 138], where no splicing isoforms are known. However, we detected an abundant exitron (GRIN2A (a)), which removes 343 nt from the last human canonical exon. The resulting frameshift alters and shortens the CTD to yield a GluN2A isoform with 1281 aa (Table 4 and Fig. S20). The same isoform, named GluN2A-short, was recently reported by Warming et al. to be expressed in human brain and to form functional receptors upon coexpression with GluN1 . Our junction analysis shows that the GluN2A-short isoform is abundantly expressed in human brain (global abundance 25%), in some datasets even on par with the canonical isoform (Fig. S9). We detected the GluN2A-short splice junction with similar frequencies in chimpanzee and macaque datasets (Fig. S20B), but the corresponding splice sites are absent in rat and mouse, which supports the suggestion that this is a primate-specific isoform . Shortening of the GluN2A CTD should have important functional consequences, as it results in a loss of several interaction motifs, including CaMKII and PSD-95 binding sites [27, 122]. We did not identify any other relevant splice junctions belonging to GRIN2A.
To our knowledge, no alternative splicing events have been reported for GluN2B other than variations in the mouse 5′-UTR . Similarly, we did not identify any significant junctions that would cause changes to the coding sequence of human GluN2B, but we detected two moderately abundant junctions, which define a chain of two additional 5′-UTR exons (GRIN2B (a,b), Table 4). Despite high coverage, we did not detect any new junctions in the long 3′-UTR (GRIN2B > 22 kb; GRIN2A 9.8 kb).
GluN2C is known for its high expression in the cerebellum (see [138, 141, 142]) and we observed the highest transcript levels in the ‘cerebellum’ dataset (Fig. S3). The most frequent splice events in our analysis point to the existence of alternative 5′-UTR exons, which can replace canonical exon 1 (GRIN2C (a) and (b), Table 4 and Fig. S21). The newly identified junction GRIN2C (a) was 3.22-times more abundant than the canonical 5′-exon junction and prevailed in all datasets with sufficient coverage (Fig. S9). This transcript may thus be considered the primary UTR isoform, which is also supported by the coverage track (Fig. S21A). The second alternative 5′-exon junction was somewhat less abundant than the canonical junction (GRIN2C (b), Table 4), and a 5′ elongation of exon 2 has been reported, as well (Fig. S21B). In contrast, the GRIN2C 5′-UTR variations described in mouse , and the two alternative translation starts reported in rat , have no correspondence in humans.
Another notable, newly identified GRIN2C event is the insertion of a 63 nt cassette exon between canonical exons 9 and 10, which would result in a 21 aa insertion in the LBD S2 segment (GRIN2C (f,g), Table 4 and Fig. S22). The junctions defining this potential GluN2C-b isoform (1254 aa) were present in several human datasets at low-to-moderate levels (up to 21% abundance). However, in chimpanzee and macaque, we found the corresponding junctions to minor extent (Fig. S22B), and in rat and mouse, no homologous sequences exist. Apart from these isoforms, several other junctions appear to result in non-functional variants (GRIN2C (c), (d), (e), (h), and (i)) which are mostly subject to NMD (Table 4). We did not detect reads pointing to an alternative donor site reported for rat GluN2C ; also previously reported splice events for human cerebellar GluN2C  were absent or not detected at significant levels (GluN2C-3 had a global abundance of 4%; Table S4). However, a large number of rare events were detected for GluN2C (Table S4).
The GluN2D subunit is known to be expressed at lower levels than the other GluN2 subunits [39, 141, 146]. The read numbers for GRIN2D were rather low (Fig. 1 and Fig. S7) and we did not detect any clearly relevant alternative splice events (for rare events, see Table S4). An exitron (−82 nt) has been reported in the last exon of rat , but no splice site consensus sequences are present in rodents or primates (Fig. S23A).
GluN3A and GluN3B subunits confer special signaling properties to NMDA receptors [17, 18]. For GRIN3A, we identified all canonical junctions at reasonable levels (Fig. S7; [147, 148]). However, we did not identify any reads that would correspond to the GluN3A-long isoform that has been found in rat and mouse but not in humans [149,150,151]. This isoform is characterized by 20 aa insertion in the CTD, which results from an alternative splice acceptor site that causes a 5′ extension of the last exon. A more detailed analysis showed that the corresponding splice acceptor site is missing in humans and that the corresponding region is not conserved between primates and rodents (Fig. S23B).
Despite very low expression in the CNS (Fig. 1B), we detected all canonical GRIN3B junctions (Fig. S7; [152,153,154,155]). However, given the low transcript coverage, we cannot comment on alternative splicing (cf. Table S4). Several alternative isoforms have been isolated from rat developing white matter  and a polymorphic poly(Q) stretch appears to be present in human exon 9 .
De novo identification of RNA editing events in iGluR transcripts
RNA-Seq data also contain direct information on single-nucleotide variations (Fig. 1A). Taking advantage of the high sequencing depth of the analyzed data and its origin from different human donors, we set up a strategy to identify potentially unknown editing sites (see Supplementary Methods; Figs. S1 and S24). For this, we considered frequent nucleotide mismatches that were present with ≥ 5% abundance in ≥ 30% of the datasets; still, 1220 mismatch positions were detected in exonic iGluR regions alone. We excluded poorly covered sites (see Supplementary Methods and Fig. S24) and obtained 67 mismatches, which could result from A-to-I or C-to-U editing in exonic regions (Fig. 5A and Table S7). Subsequent comparison to DNA-based data (dbSNP; NIH) revealed that 42 of these mismatches probably originate from widespread single-nucleotide polymorphisms (SNPs; Fig. S25). Some of the other mismatches showed different substitutions and/or were localized in non-coding regions (see Supplementary Methods). Eventually, our de novo identification resulted in ten mismatches in iGluR coding regions that could be unequivocally attributed to RNA editing (Fig. 5B), all of which had been described before.
We identified all eight major A-to-I iGluR editing events that are known to cause amino acid exchanges (Fig. 5B; Table S7; [8, 47]). Their editing frequencies are in good agreement with PCR-based quantifications (Fig. S26; [157, 158]). We found GluA2 Q/R editing to be the most abundant event (mean abundance 92.7%) followed by editing of the GluA3 R/G site (mean 86.3%). The least abundant, but still substantial editing events, result in silent amino acid exchanges: In GluA2, we found Q608Q editing, which is located + 4 nt of the Q607R site (mean 8.7%), and in GluK2, we found G615G editing −17 nt of the Q621R site (mean 23.5%). Due to their proximity to highly edited sites, these events may be considered secondary A-to-I editing events, which are controlled by the same editing site complementary sequence (ECS) [159, 160]. Other secondary events have been reported , but were detected with frequencies < 5%, such as GluA2-4 L/L editing −1 nt of the R/G site, as well as GluK1 G630G and GluK2 M620V editing (Table S7C). We also detected substantial editing of the intronic GluA2 editing hotspots + 60 and + 262 nt of the Q/R site , but, unlike in rodents no editing of adenosines at position + 263 and + 264.
In addition, we identified one exonic iGluR mismatch that could be caused by C-to-U editing, GluK1-d A870V (Fig. S25BC). However, this position is located in an alternative exon with poor coverage, the same substitution is encoded by a rare SNP, and the extent of C-to-U editing in the brain remains unclear [161, 162].
Correlations between RNA editing events and editing and splicing
We continued by investigating co-editing of sites that occur within read length. Using specific sequence queries (Table S8), we found that in GluA2, where Q608Q editing occurs less frequently (overall 15.4% frequency) than Q607R editing (overall 95.9%), the four combinations still show relative abundances, as they would be expected for independent editing of the two sites (Pearson’s chi-squared test of independence, p > 0.05; Fig. 5C). For GluK2 Y571C/I567V, a different behavior is seen, since reads only edited to I567V are strongly underrepresented (overall 1.2%) compared to the other three combinations, which occur with similar frequencies (25–44%), i.e., editing at these sites does not occur independently (p ≤ 0.0005). The same is true for GluK2 Q621R/G615G, where reads only edited at the G615G site are strongly underrepresented (overall < 1%) and reads edited at only the Q621R site are the largest class (67%).
Different mechanistic interpretations could explain this behavior. Apparently independent editing, like in GluA2, would be observed, if adenosine deaminase binding is not rate limiting, or, if different ADAR activities (ADAR1/ADAR2) are involved. In contrast, a clear correlation, as seen for GluK2, where G615G editing increases with Q621R editing, would be expected, if the presence or recruitment of deaminase is limiting, but deamination of the two sites proceeds with different efficiency. However, also more complex regulatory mechanisms may be at play, since also editing of more distant sites  and within single cells  has been reported.
We also analyzed the relation between editing and close-by splicing sites, such as editing of the R/G sites in GluA2, GluA3, and GluA4, which are located −2 nt of the splice donor sites that mediate splicing to either the flop or flip exon . Using sequence-specific queries, we investigated the interdependency of R/G editing and flip/flop splicing or intron retention (Fig. 5D; Table S8). Different patterns are seen for the three AMPAR subtypes: In GluA2, all R/G-flop/flip combinations are present at similar levels (overall 16.8–33.6%; |r|≤ 2.4). In GluA3, transcripts are mostly edited to G, but the R-flop combination remains particularly underrepresented (|r|≤ 4.7). In GluA4, which shows the strongest interdependence of editing and splicing, R-flop is again underrepresented, and G-flop is clearly overrepresented (|r|≤ 6.2). Similar observations have been reported in other human studies [157, 158]. However, it remains unclear, whether there is a mechanistic link between R/G editing and flip/flop splicing [50, 158, 163, 164], or whether these effects arise from cell type-specific effects, e.g., enrichment of certain splicing factors and deaminases . From a functional perspective, it is interesting to note that in the case of GluA2, the effects of R/G editing on the desensitization kinetics are more pronounced in the flop isoform than in the flip isoform [43, 80, 165].
In GluK1 and GluK2, the Q/R editing sites are close to the 3′ exon boundary (−6 nt), which allowed us to test, whether editing is coupled to splicing or intron retention (Fig. 5D; Table S8). In GluK1, Q/R editing (overall 64.6%) and intron retention (overall 12.4%) appear to be rather independent, as confirmed by Pearson’s chi-squared test (p ≥ 0.05). In GluK2, Q/R editing (overall 90.2%) and intron retention (overall 37.8%) are more frequent, but unedited reads in combination with intron retention are clearly underrepresented (1.0%; |r|≤ 2.7) compared to the other combinations (8.7–53.5%). The higher fraction of edited transcripts still containing the subsequent intron could be explained by the extended presence of the intronic ECS, which may result in more complete editing. This effect has recently been also found in a systematic analysis of mouse brain pre-mRNAs, including GRIA2, GRIA3, GRIK1, and GRIK2 . In the case of GluA2 Q/R editing, an even stronger control mechanism seems to exist. There, splicing can only proceed after editing, which may be controlled by the involvement of intronic editing hotspots [46, 164, 167, 168]. This ensures the low Ca2+ permeability of most adult AMPA receptors, which is critical for proper nervous system function [45, 47, 48, 169].