Figure 5. NOVA regulates cryptic NMD exons and transcript levels.

(A) Analysis of alternative spliced isoforms in transcripts chosen solely on exon array data showing NOVA-dependent steady-state mRNA changes and robust HITS-CLIP clusters in introns. Transcripts were then screened for the presence of cryptic NMD exons by RT-PCR using primers in exons bounding the intronic HITS-CLIP clusters. Data is divided into those transcripts down-regulated or up-regulated in Nova DKO, as indicated. Sequence analysis of RT-PCR products showed the presence of cryptic exons harboring premature stop codons (Figure 3—source data 1; Supplementary file 2). A diagram of the loci of each NMD exon present in Figure 5A is shown in Figure 5—figure supplement 1. For example, most transcripts down-regulated in Nova DKO brains show a larger, PTC containing exon in DKO; one exception is Actl6b, in which in the absence of NOVA there is a PTC, and in WT brain, an upper alternate isoform (exon) is present that corrects that frame-shift; (B) Effect of emetine on putative NOVA-regulated cryptic NMD exons. The steady-state level of six transcripts identified in Figures 3B, 4C and 5A were assessed by qRT-PCR in six DIV WT vs Nova DKO primary mouse neurons incubated for 10 hr in the presence or absence of emetine. The results were plotted with the Y-axis as a measure of the degree of putative NOVA-dependent NMD regulation (the fold change of transcript levels in DKO neurons in the presence or absence of emetine, divided by that of WT, in log₂ scale). For example, for Dlg3 the log₂ value is about 1.0 indicating that emetine treatment increased the Dlg3 NMD-isoform in DKO neurons relative to WT neurons by a factor of two, while emetine decreased the NMD isoform of Scn9a by ∼1.4-fold, leading to decrease or increase in the respective proteins in Nova DKO neurons (Figures 2 and 3 or Figure 4, respectively). Three independent experiments were performed and error bars represent standard deviation (p<0.05).

DOI: http://dx.doi.org/10.7554/eLife.00178.018

Figure 5—figure supplement 1. Diagrams of each of the NMD exons shown in Figure 5A.

The position of the exon harboring a premature termination codon is labeled in red (PTC).

DOI: http://dx.doi.org/10.7554/eLife.00178.019

Figure 5—figure supplement 2. NOVA regulates the expression of Stx2 (Syntaxin 2) mRNA and protein.

(A) RT-PCR from WT and DKO brain showed NOVA-dependent alternative splicing of a previously unknown, higher molecular weight isoform of Stx2. A (−) RT negative control is shown. (B) Immunoblot analysis of STX2 protein in biologic triplicate samples of WT vs Nova DKO brain. γ-Tubulin was used as a normalization control. (C) Quantitation of relative protein intensity (WT/DKO) from (B), plotted as relative ratio of STX2 in WT/DKO; error bars represent standard deviation (p<0.05). (D) Six DIV WT primary mouse culture were treated with vehicle (−) or emetine (+) for 10 hr and RT-PCR was performed for Stx2 as in (A). Quantitation revealed that the NMD exon (labeled) was increased twofold after emetine treatment. (E) DY547 direct-labeled siRNA targeting Upf1 was transfected into WT mouse primary cells, and immunofluorescence microscopy used to detect STX2 (green) and siRNA to Upf1 (red). The thin arrow (top right corner) indicates a cell which was not transfected with siRNA, and which shows a baseline signal of STX2 protein. The two thicker arrows indicate cells which had a good red signal (siRNA to Upf1) and stronger green signals (indicating increased STX2 protein). Scale bar: 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.00178.020