Figure 2.

PHF5A is globally required by GSCs for proper recognition of an unusual class of exons. (A) In GSCs but not NSCs, PHF5A knockdown causes a dramatic increase in missplicing of constitutive junctions (top row) as well as retention of constitutive introns (bottom row). (B) Many of the splicing changes induced by PHF5A knockdown in GSCs introduce in-frame stop codons, suggesting that the resulting transcripts will be degraded by NMD. Gene expression values were computed with RSEM (Li and Dewey 2011) and normalized with the TMM method (Robinson and Oshlack 2010). Confidence intervals indicate the first and third quartiles of expression. (C) Constitutive junctions that are misspliced following PHF5A knockdown in GSCs (center) have slightly shorter polypyrimidine tracts than do unaffected constitutive junctions (top); in contrast, retained constitutive introns have unusually C-rich polypyrimidine tracts (bottom). (D) Retained constitutive introns are much shorter. Plot illustrates the median intron length, and error bars indicate the standard error estimated by bootstrapping. (E) Retained constitutive introns have branch points that are unusually proximal to the 3′ splice site. Box plots indicate the first and third quartiles of the first upstream AG, a proxy for the branch point location (Gooding et al. 2006). See also Supplemental Figure S2.