Fig. 5: DDX59 is required for the proper splicing of U12 introns; its deficiency disrupts the RNA exosome, the NEXT complex, and genes crucial for Oral-Facial-Digital syndrome (OFD) and ciliary function.

a Schematics of generating knockout (KO), Main, and CTRL(-) populations for RNA-seq analysis, in which respective traces are colored in red, green, and blue. These knockouts are rigorously controlled for growth conditions, transduction efficiency, and antibiotic selection, as, throughout the entire experiment, the cell populations grow together on the same plate until sorted by FACS. The IGV tracks are shown only as an illustration of color-coding that is further used in Figs. 5b–m, 6a–e, S3, S4, and S6. The schematic cell culture plates were created in BioRender. Che, J. (2025) https://BioRender.com/ag9ov57. b–d RT-qPCR quantification of intron retention in mRNAs of the exosome components EXOSC1 and EXOSC2 as well as the scaffold component of the Nuclear Exosome Targeting (NEXT) complex, ZCCHC8 after DDX59 knockout as described in (a). e–j Integrated Genome Viewer (IGV) traces of RNA-seq reads for minor intron-containing genes, the exosome components EXOSC1 and EXOSC2, as well as the scaffold component of the Nuclear Exosome Targeting (NEXT) complex, ZCCHC8, and genes associated with ciliary function, DCTN3 and OCRL, as well as C2CD3, which is also associated with Oral-Facial-Digital (OFD) syndrome. Cell populations with DDX59, RBM7, ZCRB1, and EXOSC10 knockouts were obtained as shown in (a) and their respective IGV traces are colored accordingly. Major and minor introns are labeled as U2 and U12, respectively. k–m RT-qPCR quantification of intron retention in mRNAs of genes associated with ciliary function, DCTN3 and OCRL, as well as C2CD3, which is also associated with Oral-Facial-Digital (OFD) syndrome. For RT-qPCRs, RNA levels were normalized to 18S RNA; ΔΔCt was used to quantify the relative expression levels. Data are presented as means of two biological × four technical replicates; error bars represent standard deviation. Statistically significant differences between knockout and control samples were determined by one-way ANOVA. Posthoc comparisons using Dunnett’s test were conducted to determine the overall difference between groups, and p values are labeled. Positions of qPCR primers are indicated and their sequences are listed in Supplementary Data S2.