Figure 3

The C-terminal region of SETD5 is the main part of OGT-catalyzed O-glycosylation, which is essential for the functional relationship between SETD5, OGT, and RNA Pol II. (A) SETD5 interacted with OGT and was glycosylated. The interaction of SETD5 with OGT and the O-GlcNAcylation of SETD5 were analyzed by immunoprecipitation (IP) assay. 3× F-SETD5 and MYC-OGT were co-transfected into 293 T cells, followed by treatment of 100 nM OGA inhibitor (Thiamet-G; OGAi) for 24 h. FLAG-tagged SETD5 was immunoprecipitated by FLAG-affinity gel, and the immunoprecipitates were subjected to western blot. Symbols used: 3× F, 3× FLAG-tagged empty vector; MYC, MYC-tagged empty vector; 3× F-SETD5, 3× FLAG-tagged-SETD5 plasmid; MYC-OGT, MYC-tagged OGT plasmid; αAlpha-tub, Alpha-tubulin antibody; αRL2, O-GlcNAc-specific antibody. (B) SETD5 is O-GlcNAcylated by OGT. The SETD5 O-GlcNAcylation was determined by in vivo glycosylation assay using sWGA beads-based IP. Following immunoprecipitation, SETD5 was detected using a FLAG antibody as a primary antibody. The OGAi of 100 nM was treated for maximizing O-GlcNAcylation of SETD5 prior to the immunoprecipitation of SETD5. Symbols used: sWGA, IP using succinylated wheat germ agglutinin beads for the enrichment of O-GlcNAcylated proteins. (C,D) The O-GlcNAcylation site(s) of SETD5 were identified by LC–MS/MS analysis. The FLAG-tagged SETD5 was purified from 293 T cells and subjected to LC–MS/MS analysis to identify the potential O-GlcNAcylation site(s) (C). The O-GlcNAc sites were mostly found in the C-terminal region of SETD5 (D). (E) The schematic diagram for full-length SETD5 (WT, wild-type) and its three truncated forms (N, N-terminal part; Mid, middle part; C, C-terminal part) (left panel). Immunoprecipitations revealed the interaction between SETD5, OGT, RNA Pol II, and CTR9 (right panel). The SETD5 deletion mutants were co-transfected with MYC-tagged OGT into 293 T cells, then immunoprecipitated by FLAG affinity gel, and following western blot analysis.