Arginines of the RGG box regulate FMRP association with polyribosomes and mRNA

Abstract

Fragile X syndrome is caused by the loss of expression of the fragile X mental retardation protein, FMRP. FMRP is an RNA-binding protein that is highly expressed in neurons and undergoes multiple post-translational modifications including methylation on arginine. FMRP is methylated on the high-affinity RNA-binding motif, the RGG box, at positions 533, 538, 543 and 545 of murine FMRP. To identify the arginines important for FMRP function, we examined their role in polyribosome and mRNA association. We found that arginines 533 and 538 were required for normal FMRP polyribosome association whereas all four arginines played a role in RNA binding, depending on the identity of the RNA. The model G-quadruplex RNA sc1 required arginines 533 and 538 for normal association with FMRP, whereas AATYK mRNA did not. In vitro methylation of FMRP-bearing arginine substitutions inhibited sc1 binding but not AATYK binding. In addition, we found that PRMT1 co-immunoprecipitated with FMRP isolated from cells and that siRNAs directed against PRMT1 led to reduced FMRP methylation. Thus, two lines of experimentation demonstrate that PRMT1 acts on FMRP in cells. In summary, we provide evidence for the important role of the RGG box in polyribosome association. We also demonstrate for the first time that the different arginines of the RGG box are important for the binding of different RNAs. Finally, we show that PRMT1 methylates FMRP in cells, suggesting a model where methylation of the RGG box modulates either the quantity or the identity of the RNAs bound by FMRP.

Figure 1.

Arginines 533 and 538 of the FMRP RGG box are required for normal polyribosome association. Extracts from FMR1 knock-out STEK cells transfected with FMRP (WT); 543,545m; ΔRGG; 533,538,543,545m or 533,538m were overlaid on a 15–45% sucrose density gradient that was centrifuged and fractionated into 650 μl fractions by bottom displacement using a gradient fractionator with the ribosomal profile monitored at 254 nm. (A) Representative polyribosomal profile of transfected STEK. (B) Immunoblots of the transgene-encoded FMRP indicated on the left. (C) Bar graphs indicate the results of the distribution of FMRP through the first eight fractions in three independent experiments and were calculated using NIH Image J and Microsoft Excel. The histograms in (C) represent averaged values; *P-value <0.05 and **P-value <0.01 compared with WT using the unpaired Student's t-test (n = 3). Error bars represent standard deviation. (D) Schematic of the RGG box in murine FMRP (nm_008031). The methylated arginines are underlined and italicized.

Figure 2.

Arginines 533 and 538 of the FMRP RGG box are critical for sc1 RNA association. (A) In vitro synthesized, ³⁵S-methionine-radiolabeled FMR proteins as indicated above the blot were used in an RNA capture assay and detected by autoradiography. One of three independent assays is shown (A); biotinylated sc1 was able to capture, on average, 15% of 533,538,543,545m; 23% of 533,538m and 44% of 543,545m compared with WT FMRP. The amount of ΔRGG captured was too small to accurately detect using densitometry and is therefore reported as 0% compared with WT FMRP. (B) Similar amounts of transgene-encoded FMRP as indicated were used for each capture assay. (C) The histogram summarizes the results of three independent experiments showing the decrease in capture observed for each FMR protein. WT was normalized to a value of 1, the differences between each construct and WT were compared using the one sample Student's t-test (n = 3) and comparisons of 533,538,543,545m versus 533,538m and 533,538,543,545m versus 543,545m were performed using the unpaired Student's t-test (n = 3). *P-value <0.05. Error bars represent standard deviation.

Figure 3.

Arginines 533 and 538 do not contribute to binding AATYK mRNA. (A) In vitro synthesized, ³⁵S-methionine-radiolabeled FMR proteins as indicated above the blot were used in an RNA capture assay and detected by autoradiography. One of three independent assays is shown; biotinylated AATYK mRNA was able to capture, on average, 21% of ΔRGG; 67% of 533,538,543,545m; 108% of 533,538m and 54% of 543,545m compared with WT FMRP from three independent experiments. (B) Similar amounts of transgene-encoded FMRP as indicated were used for each capture assay and correspond to the constructs shown in (A). (C) The histogram summarizes the results of three independent experiments showing the decrease in capture observed for each FMR protein. WT was normalized to a value of 1 and the values for ΔRGG; 533,538,543,545m and 543,545m are significantly different from WT using the one sample Student's t-test (n = 3). **P-value <0.01. The differences between WT and 533,538m (P = 0.61) and 533,538,543,545m and 543,545m (P = 0.10) are not statistically significant (n.s.) using the one-sample and unpaired Student's t-test, respectively (n = 3). Error bars represent standard deviation.

Figure 4.

FMRP co-immunoprecipitates with PRMT1. (A) Untransfected COS-7 cells (mock) and COS-7 cells expressing WT FMRP, ΔRGG or one of the arginine substitutions indicated were immunoprecipitated with the 7G1-1 antibody to capture transgene-encoded FMRP that was then incubated with PRMT1 or BSA (as a negative control) and S-adenosyl-l methyl-³H-methionine ([³H]-SAM) as described in Menon et al. (20). Top panel, immunoblot for FMRP; bottom panel, autoradiogram. (B) Untransfected L-M(TK−) cell lysate (VC) or Flag-FMRP stably expressing L-M(TK−) cell lysate (WT) was immunoprecipitated using anti-Flag M2 agarose beads (Flag-IP), resolved on a 12% SDS–PAGE and immunoblotted for PRMT1. Lower panel, immunoblot for FMRP showing that FMRP was only immunoprecipitated from the WT-expressing cell line (the KI antibody does not detect FMRP from cell lysate because Flag-FMRP in L-M(TK−) cells is not overexpressed (54)).

Figure 5.

Methylation of arginines 533 and 538 or 543 and 545 inhibits sc1 RNA association. WT, ΔRGG, 543,545m or 533,538m was synthesized in an in vitro transcription/translation reaction and then treated with recombinant PRMT1 (+) or an equivalent amount of BSA (−) in the presence of [³H]-SAM. The FMRP was then incubated with 5′ end biotinylated sc1 and captured with streptavidin-coated Dynal beads. The samples were split and subjected to FMRP immunoblot analysis (top) or autoradiography (bottom). Input protein is shown in the middle by immunoblot for FMRP.

Figure 6.

Methylation of arginines 533 and 538 or 543 and 545 does not inhibit AATYK association. (A) In vitro synthesized ³⁵S-methionine-radiolabled FMR protein was generated, a fraction of the reaction was retained to measure input protein and the remaining reaction was equally split and treated with either recombinant PRMT1 (+) or an equivalent amount of BSA (−) in the presence of [³H]-SAM. The FMRP was then incubated with biotinylated AATYK mRNA, captured with streptavidin-coated Dynal beads and examined by autoradiography (top panel). The bottom panel shows input protein using autoradiography. The signal generated by ³⁵S-methionine is 10-fold stronger than that of ³H, so the ³H signal is unlikely to contribute substantially in the top panel. (B) The histogram summarizes the results of three independent experiments showing the decrease in capture observed for each FMR protein when compared with 533,538m treated with BSA. 533,538m treated with BSA was normalized to a value of 1 and the values of 533,538m with PRMT1 (119%) and 543,545m with PRMT1 (103%) are not statistically significant (n.s.) using the one-sample Student's t-test (P = 0.4642 and P = 0.8753, respectively, n = 4). *P-value <0.05. The difference between 543,545m treated with PRMT1 and 543,545m treated with BSA (67%) is not statistically significant using the unpaired Student's t-test (P = 0.1166, n = 4). Error bars represent standard deviation.

Figure 7.

PRMT1 methylates FMRP in cells. COS-7 (A) or HeLa (B) cells were transfected with Flag-FMR1 and treated with either an irrelevant siRNA or an siRNA specific for PRMT1. siRNA treatment resulted in an 80% reduction in PRMT1 expression in COS-7 cells and a 30% reduction in HeLa cells, as detected by immunoblot; eIF5 was used as a loading control. Immunoprecipitated FMRP is indicated by immunoblot and methylated FMRP is visualized by autoradiography (³H-methyl-methionine). PRMT1 siRNA treatment led to a 34% decrease in FMRP methylation in COS-7 cells and a 48% decrease in HeLa cells. Percent reduction values were determined using NIH Image J.