Figure 3. Patient FMRP contains a novel nuclear localization signal in the C-terminus.

1. Transfected HEK293 cells expressing a GFP-tagged FMRP in which the C-terminus is truncated following the G-ins. mutation site (GFP-FMR1^ΔCt). GFP-FMR1^ΔCt protein is exclusively cytoplasmic, suggesting that the truncation of the C-terminus alone is not sufficient to explain the localization change observed for the patient FMRP.
2. Transfected HEK293 cells expressing a modified version of GFP-tagged patient FMRP (GFP-FMR1^{G-ins. [NLSmutated]}). The novel amino acid sequence in the C-terminus of patient FMRP [RKRTRRKRTWRRLQRKRRSLPNR] contains stretches of arginines and lysines predicted to function as a nuclear localization signal (based on Expasy protein motif scanner, ScanProsite). Mutating adjacent R and K residues into alanines [RARTRAARTWARLQRAARSLPNR] abolishes the nucleolar localization of the patient FMRP, strongly suggesting that the novel amino acid sequence present in the patient FMRP C-terminus is a functional nuclear localization signal (NLS).

Data information: Scale bar represents 10 μm.