Modular Splicing Is Linked to Evolution in the Synapse-Specificity Molecule Kirrel3

Long-read transcript sequencing. Schematic of the long-read sequencing workflow starting with total mRNA from P14 hippocampal tissue. Pink indicates mRNA, blue cDNA, green DNA-barcodes, red raw Kirrel3 sequences, gray sequencing adapters, and black consensus Kirrel3 sequences. Extended Data Table 1-1 reports the sequence of each bar code used.

Mouse Kirrel3 gene, transcript isoforms, and proteins. A, Genomic organization of the mouse Kirrel3 gene, with exons (boxes) and introns (lines), including four independently spliced protein-coding exons (yellow, red, green, and blue). White boxes mark exons or exon parts with a stop-codon. Exons producing predicted secreted Kirrel3 isoforms are additionally marked with an asterisk. B, Alternative splicing of Kirrel3 exons is predicted to produce 13 different transmembrane isoforms. Isoforms are given letters, following the example of previously identified Isoforms A–E. Isoform E, the only transcript including exon 5, was not identified in the hippocampus. Exons 8, 9, 11, 13, 17, 19, and 20 are present in transcripts as short (part a) or extended (parts a + b) versions. Percent indicate the relative contribution of a particular Kirrel3 isoform to the total number of complete Kirrel3-transcripts featuring exon 2 and poly A-tail. Exons encode protein segments that are either extracellular (“extra”), intracellular (“intra”), or spanning the membrane (gray vertical bar). C, Six predicted secreted Kirrel3 isoforms (O–T) only comprise extracellular domains. D, Schematic of Kirrel3 protein isoforms. Percent are estimates of how frequent protein domains are present in hippocampal Kirrel3 based on isoform frequencies. E, Western immunoblots from hippocampi from three-week-old wild-type and Kirrel3 knock-out mice using antibodies directed against Kirrel3 amino acids 46–524 (αpanK3), the peptide encoded by exon 20b (α−20b), or house-keeping gene glyceraldehyde 3-phosphate dehydrogenase (αGAPDH). IG: Ig-domain, TM: transmembrane domain. Extended Data Table 2-1 reports the exact sequences for each mouse and human exon as well as SRA files.

Homophilic binding of Kirrel3 isoforms. A, Example of the CHO-cell aggregation assay. CHO cells are expressing mCherry (mCh) or GFP as a control, GFP-2A-Kirrel3F (K3F), mCherry-2A-Kirrel3K (K3K) or both Kirrel3 isoforms. B, Quantification of CHO cell aggregation assays. N = 3 independent trials for each condition. Error bars = SEM p = 0.003 for a one-way ANOVA and ***p < 0.001 for each pairwise post-test comparison with the control condition. C, Diagram of the cell junction assay in adherent HEK293 cells. D, Example of the cell junction assay. Note that Kirrel3F and Kirrel3K are both highly enriched in the cell-cell junction, but the control proteins, membrane bound GFP (mGFP) and Neuroligin-1 (Nlg1), are not. E, F, Quantification of the adherent cell junction assay. E, Bar graphs and error bars indicate SEM. Each dot indicates a cell and each color denotes cells from an independent culture. One-way nested ANOVA indicates p < 0.001 and **p < 0.01 from pairwise post-tests. F, Same data but graphed as a mean estimation plot with error bars showing SD, indicating that Nlg1 and mGFP are significantly different from Kirrel3F and Kirrel3K. G, Example image showing that K3F and K3K cluster heterophilically in junctions when they are expressed in different cells.

In situ detection of exons 20b and 22. A, Schematic of a coronal section through the hippocampus. Red boxes mark areas imaged in B-H. DG; dentate gyrus, LPN; lateral posterior nucleus of thalamus. B–H, Magnified images of regions shown in A that were hybridized with mRNA in situ probes for GAD1 (red) to mark GABAergic neurons, Kirrel3 exon 22 or 20b (white) as indicated to mark specific Kirrel3 isoforms, and the nuclear stain Hoechst (blue). B, Kirrel3 knock-out tissue produces no Kirrel3 mRNA signal using the larger exon 22 probe. C, D, Exon 22 and 20b transcripts are detected in DG neurons of wild-type mice. E, F, Exon 22 and 20b transcripts are detected in GABAergic neurons (white circles) in area CA3 of wild-type mice. G, H, Exon 22 and 20b transcripts are detected in the LPN of wild-type mice. I, J, Simultaneous use of probes for exon 22 and 20b indicate that individual neurons often express both isoforms. Extended Data Table 4-1 reports the HCR probe sets used.

Human Kirrel3 gene, transcript isoforms, and proteins. A, Genomic organization of the human Kirrel3 gene, with exons (boxes) and introns (lines), including five independently spliced protein-coding exons (yellow, red, green, purple, and blue). White boxes mark exons or exon parts with a stop-codon. Exons producing predicted secreted Kirrel3 isoforms are additionally marked with an asterisk. B, Alternative splicing of Kirrel3 exons is predicted to produce eight different transmembrane isoforms. Isoforms are given numbers, following the example of previously identified isoforms 1–3. White boxes indicate exons or exon parts with stop-codon. Exons encode protein segments that are either extracellular (“extra”), intracellular (“intra”), or spanning the membrane (gray vertical bar). C, Three predicted secreted Kirrel3 isoforms (9–11) only comprise extracellular domains. D, Western blot showing that Kirrel3 protein containing human exon 21 is found in brain lysates prepared from male and female postmortem tissue (see methods). Mouse wildtype (WT) and knockout (KO) lysates are used as a positive and negative control. E, Schematic of Kirrel3 proteins with the position of protein domains relative to the membrane (horizontal gray bar) and mutations associated with autism. IG: Ig-domain, TM: transmembrane domain.