Figure 2. ADD1 splicing isoforms are differentially expressed in the developing neocortex.
A) Alternative splicing of ADD1 exon10 and exon15 during human and mouse brain development. Top, genome (exon-intron) structure of ADD1 with indicated positions of exons 10 and 15. Left, RNA sequencing results and Sashimi plots showing the alternative 5’ splice site for exon10 and the inclusion/exclusion of exon15 between the cortical plate (CP, mostly neurons) and ventricular zone (VZ, mostly neural progenitor cells or NPCs) of gestational week (GW) 13-16 human fetal brains 40. Top right, RT-PCR results showing that human ADD1 exon10 and exon15 are differentially spliced between microdissected VZ and CP; Bottom right, RT-PCR results showing that mouse Add1 exon10 and exon15 are differentially spliced during dorsal cortex development.
B) ADD1 splice isoforms between neurons (top, NM_176801) and NPCs (bottom, NM_001119). The neuronal isoform has a longer neck domain due to extended exon10, but lacks the MARCKS-related domain due to inclusion of exon15, which introduces an in-frame stop codon. Positions of variants reported in this study are indicated.
C) CLIP-Seq peaks (green) showing that Ptbp1 binds to Add1 intron 14.
D) RT-PCR results showing that Ptbp1 shRNAs promote inclusion of Add1 exon15 in Neuro2a cells.
E) Western blot analysis of Add1 isoforms in E12.5, E14.5, E16.5 and E18.5 mouse dorsal brains, Neuro2a cells, and primary hippocampal neurons (DIV11).
See also Figure S2.