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. 2022 Jul 6;110(13):2094–2109.e10. doi: 10.1016/j.neuron.2022.04.017

Figure 1.

Figure 1

Expression and detection of NRXN3 AS5+ proteoforms in mice

(A) Sashimi plots illustrating read distribution and splice junctions arising from mouse Nrxn3 AS5 in ribosome-associated mRNAs isolated from SST interneurons in mouse hippocampus (P28). Exons are depicted as boxes, and introns as dashed lines. Alternative exons and alternative acceptor sites (a,b) are marked in orange and constitutive exons in gray.

(B) Amino acids of exon 24 protein coding sequence in Nrxn3-AS5HA knockin mice. The HA epitopes (green), ω-site (red), and hydrophobic stretch conferring GPI-anchoring are indicated.

(C) Schematic illustrating introduction of a translational stop codon in AS5+ (exon 24-containing mRNAs). This results in production of shortened, GPI-anchored NRXN3 proteoforms encoded by mRNAs with a long 3′UTR encoded by exons 25a, 25b, and 25c. AS5- mRNA isoforms encode canonical transmembrane NRXN3 proteins.

(D) Western blot of whole neocortex (Cx), cerebellum (Cb), and hippocampal (Hc) extract from P28 wild-type and Nrxn3-AS5HA/HA knockin mice probed with anti-HA, antineuroligin (NLGN), and anti-beta-actin (β-ACT) antibodies. Position of α- and β-Neurexin proteoforms is indicated.

(E) Western blot of hippocampal brain extracts across development (postnatal days 2–60) from Nrxn3-AS5HA/wt knockin mice probed with anti-HA, antiNeurexin (NRXN), antineuroligin (NLGN), and anti-beta-actin (β-ACT) antibodies.

(F) Quantification of protein levels for HA-tagged NRXN3-AS5+ and for PAN-NRXN across development (P2-60), and corresponding mRNA levels assessed by qPCR for exon 24 (AS5+, GPI-anchored proteoform) and exon 25 (AS5-, transmembrane proteoform) of Nrxn3, n = 3 animals per time point.

(G) Distribution of endogenous NRXN3-AS5HA protein in subcellular fractionation of hippocampal cytosolic, membrane, and high-salt (HS) washed membrane fractions (equal percentage of total sample loaded in all lanes).

(H) Overexpressed HA-tagged NRXN3-AS5+, NRXN3 AS5-, and placental alkaline phosphatase proteins immunoprecipitated from transfected HEK293 cells after radiolabeling with 3H-ethanolamine. Immunoprecipitates were analyzed by autoradiography (left panel) or probed by western blotting with anti-HA antibodies (right panel).

Mean and SEM, one-way ANOVA.