Fig. 1

USP39 deficiency causes RP-like phenotypes and CS. (A) Representative eyes from control zebrafish larvae and usp39 crispants at 104 hours postfertilization (hpf) [retina is indicated by dark blue (control) and light blue (usp39 crispants) arrows] (left) and relative quantification of the retina/eye ratio (right) (n = 8; two-tailed t test; scale bars are 100 ?m). (B) Confocal image of representative eyes from control larvae and usp39 crispants treated with acridine orange at 104 hpf (dead cells are indicated by blue arrows) (left) and relative quantification of dead cells in the eyes (right) (n = 8; two-tailed t test; scale bars are 50 ?m). (C) Quantification of outer nuclear layer (ONL) thickness in control larvae and usp39 crispants at 104 hpf (n = 5; two-tailed t test). (D) Heat map showing gene expression (z-score) of retinal and photoreceptor genes in usp39 crispants and control larvae (n = 4) at 104 hpf. (E) Quantification of regulated alternative splicing (AS) events (RNA-seq) in si-USP39 HeLa cells (n = 3). (F) Sashimi plot of a representative si-USP39?regulated A5?SS event (n = 3). nt, nucleotides. (G) Percentage of annotated 5?SSs used in si-USP39 and si-Ctrl HeLa cells (n = 3). (H) SS strength for canonical 5?SS and A5?SS of exons with regular 5?SSs (nonregulated) or USP39-regulated 5?SSs (Wilcoxon signed-rank test; n = 3). MaxEnt represents the maximum entropy scores. (I) Kinetic modeling of 5?SS choice in wild-type (WT) and USP39-deficient cells. (J) Kinetic model fit to regulated and non-regulated 5?SS populations in USP39-deficient cells. Shown is the difference in 5?SS use between USP39-deficient and control conditions (?PSI) as a function of the percent spliced in (PSI) control; each dot represents the experimental observation for one splicing decision. Data points are colored according to classification into groups reflecting four types of 5?SS regulation by USP39 (regulated or nonregulated for canonical and cryptic 5?SS). The solid lines represent the model fit to all data points of a given group, with a confidence band calculated by bootstrapping (n = 2000). (K) Boxplots showing relative efficiency of U1-U2 bridging interactions, calculated as the ratio of cryptic and canonical efficiency (see methods) inferred by the best-fit models in USP39-deficient HeLa cells (Wilcoxon rank-sum test). Uncertainty was estimated by bootstrapping (n = 2000). For box-and-whisker plots, the center line represents the median, box limits are upper and lower quartiles, and whiskers are minimum and maximum values.