Fig. 1.

FMRP is sumoylated in vivo in the rat and mouse brain and the SUMO system targets the conserved residues K88, 130, and 614 of FMRP. a Representative immunoblot anti-FMRP (Ab#056) of P7 post-nuclear rat brain extracts prepared or not in the presence of the cysteine protease inhibitor NEM to prevent desumoylation. b Immunoblot anti-SUMO1 of NEM-treated P7 post-nuclear rat brain extracts subjected to immunoprecipitation with FMRP (Ab#056) antibody or control IgG. c Converse immunoblot with anti-FMRP (Ab#056) antibody of NEM-treated P7 post-nuclear rat brain extracts subjected to immunoprecipitation with SUMO1 antibody or control IgG. d Immunoblot anti-SUMO1 of NEM-treated P1 post-nuclear mouse brain extracts subjected to immunoprecipitation with FMRP (Ab#056) antibody or control IgG. *Non-specific band. e Immunoblot of post-nuclear mouse brain extracts (input) subjected to immunoprecipitation with FMRP antibody or control IgG and probed with anti-Ubc9 antibody. f Co-localization assays performed on cultured mouse neurons (20 DIV) with antibodies directed against Ubc9, FMRP (Ab#4317), SUMO1. Bar, 2 μm. Degree of co-localization (Manders’ coefficient) between FMRP and Ubc9 or SUMO1. N = 3 independent primary cultures with 60 dendrites analyzed for each condition. g Sequence alignments showing the evolutionary conservation of the potential SUMO-targeted lysine residues (stars) within the consensus sumoylation sites of FMRP. h, i Bacterial sumoylation assay. Representative immunoblots of purified fractions of N- and C-terminal WT or mutated parts of His-FMRP in a recombinant bacterial system and probed with anti-FMRP (h, Ab#1C3) or (i, #17722) and anti-SUMO1 antibodies as indicated. j COS7 sumoylation assay. Immunoblots with anti-FMRP (Ab#056) antibody of full-length WT or lysine-mutated FMRP expressed in COS7 cells with mcherry-SUMO1 WT or mutated (ΔGG) to prevent its conjugation. k Original X-ray structures fitted of three human N-terminal FMRP (PDB: 4OVA in green, 4QVZ in light green, 4QW2 in dark green) shown in cartoon representation. K88 and K130 are shown in sphere representation in red and blue, respectively. l Original model of FMRP (PDB: 4OVA) and SUMO1 (PDB: 4WJQ) structural links in cartoon and surface representation (with transparency), respectively, in green and light blue. Lysine residues 88 and 130 of FMRP are shown in sphere representation in red and blue, respectively