RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppress fragile X CGG premutation repeat-induced neurodegeneration in a Drosophila model of FXTAS

Abstract

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a recently described neurodegenerative disorder of older adult carriers of premutation alleles (60-200 CGG repeats) in the fragile X mental retardation gene (FMR1). It has been proposed that FXTAS is an RNA-mediated neurodegenerative disease caused by the titration of RNA-binding proteins by the CGG repeats. To test this hypothesis, we utilize a transgenic Drosophila model of FXTAS that expresses a premutation-length repeat (90 CGG repeats) from the 5' UTR of the human FMR1 gene and displays neuronal degeneration. Here, we show that overexpression of RNA-binding proteins hnRNP A2/B1 and CUGBP1 suppresses the phenotype of the CGG transgenic fly. Furthermore, we show that hnRNP A2/B1 directly interacts with riboCGG repeats and that the CUGBP1 protein interacts with the riboCGG repeats via hnRNP A2/B1.

Figure 1. CUGBP1 modulates rCGG-mediated neurodegenerative eye phenotype

Over-expression of CUGBP1 suppresses rCGG-mediated eye toxicity. From left to right; fly expressing (CGG)₉₀-EGFP (GMR-GAL4 UAS: CGG90/UAS: EGFP) has disorganized fused ommatidia; middle, fly expressing (CGG)₉₀-EGFP and CUGBP1 (GMR-GAL4 UAS: CGG90/+; UAS: CUGBP/+) has restored organized ommatidia, fly expressing CUGBP1 (GMR-GAL4/+; UAS: CUGBP/+) has organized ommatidia. The crosses were performed at 22°C and Gmr-GAL4 was used.

Figure 2. CUGBP1 interacts with rCGG repeats through hnRNP A2/B1

Immunoprecipitation (IP) was performed on mouse brain lysates using anti-CUGBP1 (A) or anti-hnRNP A2/B1 (B) antibody with IgG as a negative control. The immunoprecipitates were analyzed by immunoblotting with antibodies specific to hnRNP A2/B1, hnRNP A3, FMRP or CUGBP1. FMRP and hnRNP A3 westerns were performed using different gels. 10% of the input used for IP was run in parallel. C. Immunoprecipitation (IP) was performed on mouse brain lysates with RNase using anti-CUGBP1 and analyzed by immunoblotting to hnRNP A2/B1 D. In vitro translated protein were incubated with biotinylated rCGG repeats, western blot data shows that CUGBP1 interacts with rCGG repeats in the presence of hnRNP A2/B1 (lanes 3 and 4)

Figure 3. Over-expression of Drosophila hnRNP A2/B1 homologues suppress the CUGBP1 rough eye phenotype

From left to right: fly expressing CUGBP1 (GMR-GAL4/+; UAS: CUGBP/+) has disorganized ommatidia, fly expressing CUGBP1 and Hrb87F (GMR-GAL4/+; UAS: CUGBP/Hrb87F) or fly expressing CUGBP1 and Hrb98DE (GMR-GAL4/+; UAS: CUGBP/Hrb98DE) have more well organized ommatidia. The crosses were performed at 25°C.

Figure 4. Interaction between rCGG repeats and hnRNP A2/B1 modulates rCGG-mediated neurodegeneration

A. Gel super shift assay shows the binding of hnRNP A2/B1 to the rCGG repeats. Lane 1, 100-bp RNA ladder; Lane 2, rCGG probe only; Lanes 3, rCGG probe with mouse cerebellar lysates; Lane 4-6, rCGG probe and mouse cerebellar lysates with increasing amounts of anti-hnRNP A2/B1 antibody. B. Mammalian hnRNP A2/B1 binds to rCGG repeats. Western using an antibody against hnRNP A2/B1 is shown. The biotinylated rCGG repeats were incubated with mouse cerebellar lysates and captured by Dynal beads. FT represents flow through while E indicates the eluted proteins that bind to rCGG repeats. Both nuclear and cytoplasmic lysates were used and indicated. 30, 105 rCGG repeats or beads alone were used in the binding reactions. The last two lanes are nuclear protein lysates and cytoplasmic lysates as inputs. C. hnRNP A2/B1 directly interacts with rCGG repeats and shows increased binding to 105 rCGG repeats. Equal amount of in vitro translated hnRNP A2/B1 was incubated with identical molar quantities of either biotinylated 30 rCGG repeats or 105 rCGG repeats; beads alone as control. Input and bound fractions are shown. D. Over-expression of hnRNP A2/B1 and its Drosophila orthologs suppress the rough-eye phenotype caused by the expression of CGG repeats, and this suppression is maintained when co-expressed with CUGBP1 (supplemental figure 2). From left to right, GMR-GAL4 UAS: CGG90/UAS: EGFP, GMR-GAL4 UAS: CGG90/+; UAS: Hrb98DE/+, GMRGAL4 UAS: CGG90/+; UAS: Hrb87F/+, GMR-GAL4 UAS: CGG90/UAS: EGFP, GMR-GAL4 UAS: CGG90/UAS: hnRNP A2/B1. Flies shown in the first three panels are grown at 25°C and the last two panels at 22°C.