EGFP-FMRP forms proto-stress granules: A poor surrogate for endogenous FMRP

Abstract

Natalia Dolzhanskaya1, Wen Xie1, George Merz2 and Robert B. Denman1*

Overexpressed autofluorescent-tagged versions of the Fragile mental retardation protein (FMRP) such as EGFP-FMRP have been used in protein-protein interaction studies and in studies of the composition, the formation and the localization of neuronal granules. However, the question of whether these molecules truly recapitulate the properties of the endogenous protein has not been addressed. Here we demonstrate that overexpressed EGFP-FMRP forms three distinct granule types based on colocalization with various marker proteins. The majority of EGFP-FMRP-containing granules are larger and more amorphous than known granule types. Consistent with this, there is only partial colocalization with stress granule or P-body markers. Nevertheless, agents such as sodium arsenite, which create endogenous stress granules and P-bodies and hippuristanol, which induces stress granule formation, drive EGFP-FMRP exclusively into stress granules. Additionally, whereas inhibiting methyl-protein formation alters the composition of endogenous FMRP-containing stress granules, we found that such treatment had little effect on the formation of EGFP-FMRP granules, or their composition. Altogether these data suggest that many overexpressed EGFP-FMRP granules represent proto-stress granules requiring external stimuli for their conversion. More importantly, the inherent heterogeneity of these granules suggests that caution should be used in extrapolating results obtained with autofluorescent-tagged surrogates of FMRP to endogenous FMRP granules.