Pur alpha binds to rCGG repeats and modulates repeat-mediated neurodegeneration in a Drosophila model of fragile X tremor/ataxia syndrome

Abstract

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a recently recognized neurodegenerative disorder in fragile X premutation carriers with FMR1 alleles containing 55-200 CGG repeats. Previously, we developed a Drosophila model of FXTAS and demonstrated that transcribed premutation repeats alone are sufficient to cause neurodegeneration, suggesting that rCGG-repeat-binding proteins (RBPs) may be sequestered from their normal function by rCGG binding. Here, we identify Pur alpha and hnRNP A2/B1 as RBPs. We show that Pur alpha and rCGG repeats interact in a sequence-specific fashion that is conserved between mammals and Drosophila. Overexpression of Pur alpha in Drosophila could suppress rCGG-mediated neurodegeneration in a dose-dependent manner. Furthermore, Pur alpha is also present in the inclusions of FXTAS patient brains. These findings support the disease mechanism of FXTAS of rCGG repeat sequestration of specific RBPs, leading to neuronal cell death, and implicate that Pur alpha plays an important role in the pathogenesis of FXTAS.

Figure 1. Identification of rCGG repeat-binding proteins (RBPs)

A. Gel shift assay with mouse brain lysates. Lane 1, rCGG probe only; Lane 2, rCGG probe with mouse brain lysates; Lanes 3-7, rCGG probe and mouse brain lysates with the indicated increasing amounts of unlabeled rCGG repeats (molar ratio). B. Gel shift competition assay with fly brain lysates. Lane 1, rCGG probe only; Lane 2, rCGG probe with fly brain lysates; Lanes 3-5, rCGG probe and fly brain lysates in the presence of 100-fold unlabeled triplet repeat RNA, as indicated (molar ratio). C. Identification of RBPs. Coomassie Blue staining gel with RBPs is shown, and distinct bands were cut for protein identification. The identities of those proteins are indicated on the right.

Figure 2. Pur α displays a sequence-specific interaction with rCGG repeats

A. Mouse Pur α protein binds to rCGG repeats. The biotinylated r(CGG)₁₀₅ repeats were incubated with mouse cerebellar cytoplasmic lysates and captured by DynaBeads. Western blot analysis using antibody against Pur α/β is shown. B. An antibody specifically recognizes Drosophila Pur α protein. Western blot analysis with recombinant proteins (GST-dPur α and GST alone), and wildtype fly brain lysates was shown to test the specificity of this antibody. C. Drosophila Pur α protein also binds to rCGG repeats. The biotinylated rCGG repeats were incubated with fly brain lysates. The captured proteins were eluted and used for Western blot analysis with anti-dPur α antibody. D. Drosophila Pur α protein directly binds to rCGG repeats. The biotinylated rCGG repeats were used for binding reactions with in vitro-translated dPur α or dFmrp. The inputs (10%) and bound fractions are shown. E. Drosophila Pur α protein binds to rCGG repeats specifically. Shown is dPur α protein bound to rCGG repeats in the presence of 100-fold excess (molar ratio) different triplet repeat RNAs. F. Drosophila Pur α protein is associated with rCGG repeat-containing mRNA in vivo. dPur α protein was immunoprecipitated from fly larve expressing either (CGG)₉₀-EGFP or EGFP alone, and the co-immunoprecipitate RNA was isolated. The input (10%) and immunoprecipitated RNAs were used for RT-PCR using the primers specific for EGFP.

Figure 3. Pur α is present in the rCGG-induced inclusions

A. Western blot of protein extracted from fly heads, detected with either anti-dPur α antibody or anti-Hsp70 antibody. Arrows indicate migration position of SDS-soluble dPur α and Hsp70 proteins. The aggregated SDS-insoluble protein complex remained in the stacking gel. The genotypes of flies used are w¹¹¹⁸; gmr-GAL4 in trans to w¹¹¹⁸ (control) and UAS-(CGG)₉₀-EGFP. B. Drosophila Pur α protein is part of rCGG-induced inclusions. Confocal images are shown of the brain transverse sections from 7-day-old flies of either EGFP alone (control) or (CGG)₉₀-EGFP in trans to gmr-GAL4, stained with antibodies against Hsp70 (green) and dPur α protein (red). The nuclei were stained with DAPI (blue).

Figure 4. Overexpression of Pur α suppresses rCGG-mediated neurodegeneration in the fly

A. Schematic representation of pUAST-FLAG-dPur α construct. The full-length cDNA of the Drosophila Pur α gene with FLAG inserted into the downstream of the ATG translational start site was cloned into pUAST plasmid. B. Column 1, flies expressing (CGG)₉₀-EGFP only; Column 2, flies expressing both (CGG)₉₀-EGFP and moderate levels of fly dPur α; Column 3, flies expressing both (CGG)₉₀-EGFP and high levels of fly dPur α. Shown are SEM eye images.

Figure 5. Pur α is present in the inclusions of FXTAS patient brain

Confocal images are shown of the sup-mid temporal cortex section from a FXTAS patient, stained with antibodies against Ubiquitin (red) and Pur α protein (green). The nuclei were stained with DAPI (blue). Arrow indicates the inclusion containing both ubiquitin and Pur α protein.