Figure 5: DNMT3A recruits spliceosome components to RNA polymerase II and mRNA.

(A) Schematic of the inducible in vivo biotinylation system for DNMT3A in murine ES cells. Western blot validation of biotinylated DNMT3A after induction with 2ug/mL doxycycline in mouse ES cells (left) and immunoprecipitation with streptavidin coated magnetic beads (right). (B) Western blot after exogenous expression and co-immunoprecipitation of MYC-tagged DNMT3A in human embryonic kidney cells (HEK 293T). 10% input and eluates were probed for the indicated splicing-associated factors. HDAC1 was the positive control and β-actin was the negative control. (C) BiFC analysis of DNMT3A-interacting spliceosome components after lentiviral expression of human DNMT3A variants (n = 4 biologically independent samples and 2 independent experiments, mean values +/− SD, unpaired t-test with Welch's correction). (D) Proximity ligation assay (PLA) of DNMT3A and splicing-associated factors in mESCs. (E) PLA of DNMT3A and SF3B1 after treatment with 100nM Pladienolide B for 6 hours, and washout of the drug for 4 hours. (F) PLA of DNMT3A and SF3B1 at 0, 6h, and 12h after ATRA-stimulation in mESCs. Image represents interacting foci 12h after ATRA-stimulation (n = 3 biologically independent samples and 2 independent experiments). (G) PLA of SF3B1 and SRSF2/SC-35 or active RNA polymerase in ATRA-WT and ATRA-KO cells (n = 3 biologically independent samples and 2 independent experiments). (H) top western blot probing for SF3B1 after mRNA-IP in ATRA-WT and ATRA-KO cells. bottom SF3B1 mRNA-IP qPCR in ATRA-WT and ATRA-KO cells. All values relative to IgG control (in grey) (n = 3 biologically independent samples and 2 independent experiments, mean values +/− SD). Panels F-H – unpaired t-test with Welch’s correction. Scale bars on all immunofluorescent images represent 10μm.