Fig. 2: Human specific amino acid substitution does not affect mouse development, gene expression and RNA binding characteristics in NOVA1.

a Body weight comparison measured from 2-12 weeks postnatal. Nova1^wt/wt, N = 19, Nova1^hu/wt, N = 12, Nova1^hu/hu, N = 14. b Brain-to-body weight ratio comparison in 3-week-old mice (N = 8 per group). a, b Data are presented as mean values +/- SEM. Wilcoxon rank sum test, two-sided. c Gene expression correlations between Nova1^wt/wt and Nova1^hu/hu in midbrain at E18.5 and P21. Scatterplots show gene expression in average TPMs (log₂). The red dot marks one differentially expressed gene (Gkn3; p < 0.05, FDR < 0.1), and the yellow dot indicates Nova1. E18.5 midbrain: N = 6 per group; P21 midbrain: N = 4 per group. Statistics are performed using edgeR. d Representative autoradiography image from three independent NOVA1-CLIP experiments of 3-week-old midbrain in Nova1^wt/wt and Nova1^hu/hu mice. The yellow line marks the NOVA1 protein size, and the red outline highlights RNA extraction by sectioning. e Distribution of NOVA1 CLIP peaks on the mouse genome. f The most enriched binding sequence from NOVA1 CLIP peaks (top) and frequency of the UCAU binding sequence around the CLIP binding sites (bottom). g Scatterplot of CLIP tag number per peak between Nova1^wt/wt and Nova1^hu/hu mice at P21 midbrain (log₂). N = 3 per group. h Gene annotation analysis by Metascape¹²⁷ of NOVA1 bound transcripts. Transcripts with the top 1% peak heights (read count) for each genotype were analyzed. Genes expressed in the midbrain of P21 mice served as the background set. The term ‘behavior’ is highlighted with a black arrowhead. i Gel shift assay using purified NOVA1 protein and ³²P-labeled UCAU RNA oligo probe. The representative image from five independent experiments. The bottom band represents the free probe, and the top-shifted band indicates protein-bound RNA. j Quantified bands from the gel shift assay showing binding per protein concentration. Dissociation constants (Kd values) for each purified NOVA1 protein are plotted. Data are presented as mean values +/- SE. Two-tailed Student’s t-test. N = 5 for each point. Source data for (a, b, d, j) are provided as a Source Data file.