Figure 6. Identification of a novel, brain-specific, PTC-introducing, developmentally-regulated exon in Ptbp1.

(A) Top: Splice graph representation of a complex target LSV containing a previously unannotated, PTC-introducing exon in Ptbp1 (exon 14, green). Stop signs indicate multiple conserved premature termination codons. Bottom: UCSC Genome Browser tracks of RNA-seq reads from adrenal (red) and cerebellum (blue), and conserved Rbfox binding sites ([U]GCAUG) found within the bounds of this LSV. (B) Top panel: RT-PCR validation of RNA from replicate cerebellar and adrenal tissues with isoforms illustrated on the left. Asterisk denotes a background band that migrates non-specifically. Bottom panel: E[Ψ] violin plots of MAJIQ quantification for the colored junctions in (A). Matching isoforms are indicated on the left. (C) Top: RNA-seq reads from mouse cortices (Yan et al., 2015). Developmental time points indicated on the right with exons colored as in (A). Bottom: Ψ violin plots for the PTC-introducing exon 14 across brain development. (D) Top panel: Top regulatory motifs predicted by AVISPA to influence the neuronal-specific splicing of exon 14. Stacked bars represent the normalized feature effect (NFE) for each motif. Colors indicate the contribution of the corresponding motif in the region indicated in the inset. (E) MAJIQ Ψ quantification of the LSV shown in (A), using RNA-seq from one month old wild type whole brain (left) and nestin-specific Rbfox1 KO littermates (right).

DOI: http://dx.doi.org/10.7554/eLife.11752.014

Figure 6—figure supplement 1. Novel exon and PTCs in Ptbp1 are conserved, independent from known PTC event, and regulated by Rbfox1 and 2.

(A) PTBP1 domain structure (top) and splice graph from cerebellum data (bottom) highlighting approximate locations of known alternatively spliced linker region (dark grey) encoded by exon 13 (exon 9 in the literature), the novel PTC introducing exon 14 (red stop sign in protein, green exon in splice graph), and the known PTC upon exclusion of exon 16 (exon 11 in the literature). (B) UCSC genome browser view with sequence alignment and placental mammalian conservation. Novel exon 14 is highlighted in blue and boxed regions correspond to conservation of 3’ and 5’ splice sites and the in frame PTCs. (C) Locations of additional primers with RT-PCR from replicate cerebellum RNA. (D) UCSC genome browser view showing conserved Rbfox binding sites ([U]GCAUG) and brain RNA-seq reads from wild type one month old mice (top) and Rbfox1 KO littermates (bottom) corresponding to experiments quantified in Figure 6E. Exon 14 location is highlighted in blue. (E) MAJIQ Ψ quantification of junctions as illustrated in (C) from one month old wild type mice (top) and Rbfox2 KO littermates (bottom).

DOI: http://dx.doi.org/10.7554/eLife.11752.015

Figure 6—figure supplement 2. RT-PCR validation of complex Ptbp1  LSV across 11 mouse tissues. .

(A) Representation of Ptbp1 target LSV analyzed with primers indicated by arrows. (B) RT-PCR from replicates across tissues indicated with isoforms indicated on the left. (C) Representative RT-PCR from tissues indicated with isoforms indicated on the left. [Bstm: brainstem; Hyp: hypothalamus; Cer: cerebellum; Adr: adrenal gland; Kid: kidney; Hrt: heart; Mus: muscle; Bfat: brown adipose; Wfat: white adipose; Liv: liver]

DOI: http://dx.doi.org/10.7554/eLife.11752.016