ASPSCR1 promotes methylation of VCP by METTL21D.

<p>(A) In vitro methylation assays of VCP-His, UBXN6-His, ASPSCR1-His and fragments of ASPSCR1 with GST-METTL21D. Various combinations of the UBX proteins were added to reactions containing VCP. Coomassie staining of the gel is shown. (B) Linear representation of ASPSCR1 showing domain architecture of the protein (UBL, UBiquitin-Like domain; SHP, SHP box; UBX, UBiquitin regulatory X domain; CC, Coiled-Coil domain) and localization of residue 280 which marks the boundary between the N- and C- terminal fragments used in these experiments. (C) In vitro GST pull-down assays of VCP-His, UBXN6-His, ASPSCR1-His and fragments of ASPSCR1 with GST-METTL21D. Combinations of full-length ASPSCR1 and its fragments were once again employed with VCP. (D) Linear representation of VCP showing domain architecture of the protein (including double Ψ barrel superfold and 4-stranded β barrel of the N-terminal domain, Walker A and B motifs, as well as 4 α helices bundle of ATPase domains D1 and D2 as well as linker regions L1 and L2 and C-terminal domain) and localization of the mutants used in this study as well as the site of methylation. (E) In vitro methylation assays of wild-type VCP-His and IBMPFD and ALS-causing mutations R155H, R159G and R191Q in presence or absence of ASPSCR1-His. (F) In vitro GST pull-down assays of the same combination of proteins as in (E).