Figure 5.

PRPF8 recognizes ubH2B via the C-terminal UBM. (A) RPRF8 recognizes ubH2B each other by co-IP and reciprocal co-IP assay. HCT116 cells were harvested. The chromatin fraction was isolated and the genomic DNA digested with benzonase to release nucleosomal histones. The lysates were examined by IP and western blot with indicated antibodies. (B) The deletion of C-terminal UBM of PRPF8 fails to recognize ubH2B. SFB-tagged full-length and UBM deletion mutant of PRPF8 (PRPF8 FL and PRPF8△C) were expressed in HCT116 cells. The interactions between PRPF8 FL or PRPF8△C and ubH2B were examined by IP and western blot. (C) The C-terminal UBM of PRPF8 recognizes ubH2B. SFB-tagged C-terminal UBM of PRPF8 (PRPF8-C) was expressed in HCT116 cells. The interaction between PRPF8-C and ubH2B was examined by IP and western blot. (D) The key residues in the UBM of PRPF8 are required for the interaction with ubH2B. Two key residues I2105 and L2106 in the UBM were mutated into alanines. This mutant (mutPRPF8) and wild-type PRPF8 were expressed in the PRPF8 knockdown HCT116 cells. The IP and western blot were performed with indicated antibodies. (E) Recombinant UBM of PRPF8 specifically pulls down ubH2B. GST-PRPF8 UBM or the I2105A/I2106A mutant was incubated with the histone fraction, followed by pull down with glutathione agarose and western blot analysis. (F) Ub null mutant of H2B cannot be recognized by UBM of PRPF8. K120 of H2B was mutated to Arg. HA-tagged H2B or the K120R mutant was expressed in HCT116 cells. The histone fraction was incubated with GST-PRPF8 UBM. Pull down and western blot assays were performed.