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First published online April 24, 2006
doi: 10.1242/10.1242/jcs.02882

Journal of Cell Science 119, 1933-1946 (2006)
Published by The Company of Biologists 2006
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Methylation regulates the intracellular protein-protein and protein-RNA interactions of FMRP

Natalia Dolzhanskaya1, George Merz2, John M. Aletta3 and Robert B. Denman1,*

1 Biochemical Molecular Neurobiology Laboratory, Department of Molecular Biology, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA
2 Tissue Culture Laboratory, Department of Pathological Neurobiology, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA
3 Department of Pharmacology and Toxicology, Center for Neuroscience, University at Buffalo School of Medicine State University of New York, 102 Farber Hall, Buffalo, NY 14214, USA


Figure 1
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  		Fig. 1. FMRP and FXR1P are co-translationally methylated by an endogenous MT in RRL. (Top panels) FMRP or (middle panels) FXR1P was produced in RRL in the presence of [3H]SAM and in the presence (lane 3) or absence (lane 2) of 6 µM AdOx, as indicated. Aliquots (20 µl) were resolved by SDS-PAGE and subjected to fluorography, while 5 µl aliquots were blotted and probed with anti-FMRP mAb or anti-FXR1P pAb. Lane 1 shows background methylation in the absence of the plasmids used in the lanes 2 and 3 translations. (Lower panels) Purified histone H3 (2 µg) from calf thymus was incubated with RRL in the presence of [3H]SAM and in the presence (lane 3) or absence (lane 2) of 6 µM AdOx. Aliquots (10 µl) were resolved by SDS-PAGE and subject to fluorography, while 10 µl aliquots were subjected to Coomassie Blue staining.

 

Figure 2
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  		Fig. 2. Methylation affects the protein-protein interactions of FMRP. (A) Methylated FXR1P but not non-methylated FXR1P heterodimerizes with FMRP in vitro. Methylated and non-methylated FXR1P was bound to equal amounts of biotinylated-FMRP. Heterodimers were isolated by affinity capture on SoftLink resin. The unbound (U) and bound (B) fraction for each reaction was resolved by SDS-PAGE, blotted and probed with anti-FXR1P pAb. As a control, FXR1P binding to SoftLink resin in the presence of a mock RRL reaction without FMRP plasmid (Mock) is shown. The far right-hand panel shows that the anti-FXR1P pAb does not detect biotinylated-FMRP under these conditions. (B) The percent binding of 35S-FXR1P to biotinylated-FMRP produced in the presence of various concentrations of AdOx is plotted. Binding to FMRP produced in the presence of 3 µM AdOx was significantly less than in its absence (P<0.01, ANOVA). Binding to FMRP produced in the presence of 6 µM AdOx was also significantly less than in its absence (P<0.006, ANOVA). The number of determinations for each concentration is shown in the bar. (C) Methylated FMRP but not non-methylated FMRP heterodimerizes with FXR1P in vitro. Biotinylated-methylated and biotinylated-non-methylated FMRP produced in RRL was bound to equal amounts of methylated-FXR1P. Heterodimers were isolated and detected as described in A. For controls, FXR1P binding to SoftLink resin in the presence of a mock RRL reactions without FMRP plasmid and with or without AdOx (Mock and Mock+AdOx) is shown. (D) The percent binding of 35S-FMRP to biotinylated-FMRP produced in the absence or presence 6 µM AdOx is plotted. The number of determinations for each concentration is shown in the bar.

 

Figure 3
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  		Fig. 3. Methylation enhances the distribution of FMRP in dense granules. (A) Western blot of HeLa cell proteins that pellet through 50% sucrose (lanes 1, 2) or pellets from HeLa cells that were treated for 24 hours with 20 µM AdOx (lanes 4, 5). Proteins in lanes 2 and 4 were from pellets treated with 25 mM EDTA prior to sedimentation. Lanes 3 and 6 are total HeLa cell extracts (20% v/v of the pellet). Blots were probed with anti-FMRP mAb, anti-PABP pAb, anti-Hsp70cp mAb, and anti-EF-1A and anti-PRMT1 mAbs. Boxed lanes are for presentation purposes. The lower panel shows Coomassie staining of the proteins in each fraction. (B) Densitometric analysis of the ratio of FMRP in the pellet fraction of EDTA-treated lysates (E) to FMRP in the pellet fraction of non-treated lysates (P) in the presence or absence of 20 µM AdOx. The results of three sets of cell lysates are presented. (C) Western blot analysis of cell lysates prepared as in A, but treated or not treated with RNAse A prior to sedimentation. Lanes 1 and 4 represent total cell proteins (20% v/v of the pellet), lanes 2 and 5 are pellet proteins treated with RNAse A, lanes 3 and 6 are untreated pellet proteins. Boxed lanes are for presentation purposes. (D) Densitometric analysis of three sets of cell lysates prepared in C.

 

Figure 4
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  		Fig. 4. FMRP is often found in small cytoplasmic granules. (A) Confocal z-series images of FMRP granules in a single HeLa cell. Arrows indicate a single granule in panels 2-4. (B) FMRP small cytoplasmic granules normally do not colocalize with PABP or TIA-1, constituents of stress granules. HeLa cells grown in the absence or presence of 20 µM AdOx for 24 hours were immunostained with anti-FMRP mAb (red) and anti-PABP pAb (green) left panel, or anti-FMRP mAb (red) and anti-TIA-1 pAb (green) right panel. Quantitative analysis of the number of FMRP-granule containing cells for each treatment is presented in the graph below. The number of cells analyzed for each treatment is shown in each bar.

 

Figure 5
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  		Fig. 5. Arsenite induces the accumulation of FMRP in stress granules. (A) HeLa cells grown in the absence of treatment (naive), the presence of 20 µM AdOx for 24 hours, the presence of 0.5 mM sodium arsenite for 30 minutes, or pre-treated with 20 µM AdOx for 24 hours and then treated with 0.5 mM sodium arsenite for 30 minutes were immunostained with anti-PABP pAb (green) and anti-FMRP mAb (red) as in Fig. 3. (B) FMRP small cytoplasmic granules lack asymmetric dimethyl-arginine. HeLa cells treated as in A were immunostained with anti-mRG pAb (green) and anti-FMRP mAb (red).

 

Figure 6
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  		Fig. 6. (A) Stress granules do not have uniform composition. HeLa cells grown in the absence of treatment (naive), the presence of 20 µM AdOx for 24 hours, or the presence of 0.5 mM sodium arsenite for 30 minutes, or pre-treated with 20 µM AdOx for 24 hours and then treated with 0.5 mM sodium arsenite for 30 minutes were immunostained with anti-FXR1P pAb (green) and anti-FMRP mAb (red) as in Fig. 3. Arrows show granules that do not contain FXR1P. (B) AdOx alters the distribution of FXR1P-containing granules. HeLa cells grown in the presence of 0.5 mM sodium arsenite for 30 minutes, or pre-treated with 20 µM AdOx for 24 hours and then treated with 0.5 mM sodium arsenite for 30 minutes were immunostained with anti-FXR1P pAb (red) and anti-PABP (green). The area outlined in red shows a cluster of granules lacking PABP, while the areas outlined in green show clusters of granules lacking FXR1P. The grayscale images of FXR1P and PABP immunostaining alone highlight the effect of AdOx. (C) AdOx treatment results in a loss of FMRP from FXR1P immunoprecipitates. HeLa cells were treated with 20 µM AdOx for 24 hours and then immunoprecipitated with anti-FXR1P pAb as described. The resulting supernatant (S) or immunoprecipitate (P) fractions were resolved by SDS-PAGE and then probed with anti-FMRP mAb. As controls, untreated HeLa cells were subject to immunoprecipitation with anti-FXR1P pAb and AdOx-treated cells were run through the immunoprecipitation protocol without anti-FXR1P pAb.

 

Figure 7
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  		Fig. 7. Methylation affects the ability of FMRP to bind specific mRNAs and regulate their translation. (A) Semi-quantitative RT-PCR of mRNAs associated with HeLa cell FMRP immunoprecipitates (IP) in the absence of AdOx, and following treatment of the cells with 20 µM AdOx for 24 hours prior to immunoprecipitation. Primer pairs for FMR1 mRNA, EF-1A mRNA, Tip60a mRNA, ßAPP mRNA and dynamin A1 were used to amplify each message from total HeLa cell RNA (T) and the FMRP IP (P) for each treatment. The five panels (left) show representative results for each message. The specific amplicons are labeled; non-specific amplicons and primer-dimers are marked by an asterisk. The graph (right) shows the percentage of the mRNA associated with the FMRP immunoprecipitate for AdOx-treated and non-treated cells. Values for three independent immunoprecipitations are shown. Asterisks indicate significant decreases in AdOx-treated cells (P<0.004 by ANOVA). (B) Comparison of the percent change in mRNAs associated with FMRP in AdOx-treated versus non-treated cells with the differences in in vitro binding of the mRNAs in the presence and absence of AdOx [results taken from Denman (Denman, 2002Go)]. (C) Western blot analysis of HeLa cell proteins from cells treated with 0 or 20 µM AdOx for 24 hours. Duplicate blots (left) were probed simultaneously with anti-FMRP mAb and anti-dynamin mAb, or anti-EF-1A mAb and anti-Hsp70cp mAb. The graph (right) shows the ratio of AdOx-treated to non-treated protein expression normalized to Hsp70cP. The number of determinations for each protein is shown in the bar. Double asterisk indicates that the AdOx-treated expression level is significantly different from the control (P<0.004, ANOVA); the single asterisk indicates that the AdOx-treated expression level is significantly different from the control (P<0.06, ANOVA).

 

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© The Company of Biologists Ltd 2006