Fig. 5

NOVA promotes Sept8 exon 10b usage in MNs. a, b Volcano plot of differential ALE usage in MNs versus WSC (a) and Nova2 wild type (WT) versus KO mouse brains (b). ALEs with higher inclusion rates in MNs and Nova2 WT (dI ≥ 0.2, FDR ≤ 0.1) are labeled in green and red, respectively. ALEs with lower inclusion rates in MNs and Nova2 WT (dI ≤ 0.2, FDR ≤ 0.1) are labeled in purple and blue, respectively. The blue horizontal dashed lines denote FDR value 0.1. c UCSC genome browser images illustrating the correlation between differential NOVA binding pattern and Sept8 ALE usage in MNs. Partial gene and transcript structures of Sept8 are shown on top. Alternative last exons 10a and 10b are utilized in the X5 and X1 isoforms, respectively. Blue stars mark the two predominant alternative 3′ splice sites used in the adult spinal cord. Red octagons mark polyadenylation sites. For WSC and MN RNA-seq and NOVA CLIP tracks, see Fig. 4c, d legend for reference. Arrowheads mark significantly over-represented NOVA binding sites in MNs. E18.5 WT and Nova2 knockout mouse brain RNA-seq are displayed in black. Inclusion of exon 10b is dependent on NOVA, as highlighted by the orange box. Light gray box marks the genomic region included in the Sept8 minigene in d. d The Sept8 minigene construct. The 3′ end of Sept8 exon 10a and its adjacent intronic sequence were inserted downstream of the SV40 promoter and upstream of the SV40 poly(A) signal. Double-arrowed segments represent qPCR amplicons used for measuring transcription readthrough. The region surrounding the poly(A) signal is enlarged, with YCAY motifs marked in navy and the poly(A) signal in red. Mutant 1 minigene lacks the two YCAY motifs proximal to the poly(A) signal. The mutant 2 minigene is devoid of YCAY in the 150-nt region surrounding the poly(A) signal. e Sept8 minigene assay. COS-1 cells expressing minigene variants and indicated proteins were harvested for RNA isolation and qRT-PCR analysis. Two amplicons illustrated in Fig. 5d were used to measure RNA levels up- and downstream of exon 10a poly(A) sites, respectively. Y-axis represents percentage of downstream relative to upstream transcript level. Error bars represent standard error of the mean (SEM) based on three independent replicates