Abstract
AbstractOverexpressed EGFP-FMRP has been used as a substitute for endogenous fragile X mental retardation protein (FMRP) in protein-protein interaction studies, and in studies concerning the composition, the formation and the localization of neuronal granules. However, the question of whether this tool truly recapitulates 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 and no colocalization with a putative Drosophila ortholog neuronal granule marker. Nevertheless, agents such as arsenite and hippurstanol, which create endogenous stress granules and P-bodies, drive EGFP-FMRP into such granules. Additionally, whereas inhibiting cellular 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 EGFP-tagged surrogates of FMRP to endogenous FMRP granules.
Highlights
IntroductionAutofluorescent protein tags (AFPs) have been widely used as tools to study a variety of biological processes including, nervous system development (Brand, 1999), developmental abnormalities (Detrich III, 2008), neural stem cell development (Encinas and Enikolpov, 2008), localization of proteins in specific organelles (Di Giorgi et al, 1999), protein-protein interactions (Kedersha et al, 2005), protein-RNA interactions (Rackham and Brown, 2004), protein (Pierce and Vale, 1999) and mRNA trafficking (Querido and Chartrand, 2008), and membrane dynamics (Lippincott-Schwartz et al, 1999)
We examined whether enhanced green fluorescence protein fragile X mental retardation protein (FMRP) (EGFP)-FMRP granules contained asymmetrically dimethylated proteins
In contrast to the heterologous YFP-dFmr1 variant that human SmD3 did not colocalize with endogenous FMRP granules, FMRP arsenite-induced stress granules, TIA1 arsenite-induced stress granules, or with EGFPFMRP granules, Fig. 15A-C, nor did they colocalize with another Sm subtype, SmB/B’, Fig. 16
Summary
Autofluorescent protein tags (AFPs) have been widely used as tools to study a variety of biological processes including, nervous system development (Brand, 1999), developmental abnormalities (Detrich III, 2008), neural stem cell development (Encinas and Enikolpov, 2008), localization of proteins in specific organelles (Di Giorgi et al, 1999), protein-protein interactions (Kedersha et al, 2005), protein-RNA interactions (Rackham and Brown, 2004), protein (Pierce and Vale, 1999) and mRNA trafficking (Querido and Chartrand, 2008), and membrane dynamics (Lippincott-Schwartz et al, 1999). Treating cells with sodium arsenite for 20 min following transfection produces stress granules and P-bodies (Kedersha et al, 2007) Under these conditions Dcp1a granules associate more closely with EGFP-FMRP granules, they generally do not overlap, Fig. 6B (panel f) and Table 1. The above data suggested that reduced methylation of endogenous proteins does not drastically alter the composition of EGFP-FMRP granules; it does not inform one as to whether asymmetric dimethylation affects the formation of EGFPFMRP granules To address this question, HeLa cells were pre-treated with AdOx for twenty-four hours prior to transfection. The expression of five different protein arginine methyltransferases (PRMTs) was unaffected by treatment with AdOx, Fig. 13 As these data unequivocally demonstrate sDMA is harbored in arsenite-treated EGFP-FMRP granules we endeavored to ascertain whether endogenous stress granules contained sDMA-modified proteins. We found that neither class II PRMT (PRMT5 nor PRMT7) colocalized with TIA1-containing stress granules
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