MicroRNA-277 And Retrotransposon Gypsy Contribute To Fragile X Premutation RCGG-mediated Neurodegeneration

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder identified among fragile X syndrome premutation carriers. The most common clinical feature of FXTAS is a progressive action tremor with ataxia. The major neuropathological hallmark for FXTAS is eosinophilic, ubiquitin-positive intranuclear inclusions located in broad distribution throughout the brain in neurons, astrocytes, and in the spinal column. Previous studies have proposed that FXTAS is an RNA-mediated neurodegenerative disease. Several lines of evidence suggest that rCGG premutation repeats may sequester specific RNA-binding proteins, Pur a, hnRNP A2/B1, and CUGBP1, and reduce their ability to perform their normal cellular functions, thereby contributing significantly to the pathology of this disorder.Recent genome-wide studies have shown that less than 2% of the mammalian genomic text consists of protein-coding regions. The vast majority produces many thousands of regulatory non-coding elements, including simple and tandem repeats, transposable elements, pseudogenes, functional non-coding RNAs, regulatory elements et al. A range of evidence suggests that these non-coding elements act through complex networks to fulfil important and diverse roles as transcriptional and post-transcriptional regulators and as guides of chromatin-modifying complexes. Multiple lines of evidence increasingly link mutations and dysregulations of noncoding elements to diverse human diseases. Particularly, noncoding RNAs have been recently implicated in the molecular pathogenesis of some neurodegenerative disorders. Here we utilized our biochemical and genetic study on the proposed mechanisms that noncoding elements may give rise to the molecular pathogenesis of FXTAS, especially the mechanisms including misregulation of selective miRNAs, including miR-277 and activation of a specific retrotransposon, gypsy in modulating the neurodegeneration caused by fragile X premutation rCGG repeats.1. MicroRNA-277 contributes to the neurodegeneration caused by fragile X premutation rCGG repeats1.1 Fragile X premutation rCGG repeats alter the expression of selective miRNAsTo determine whether fragile X premutation rCGG repeats could influence the expression of miRNAs, we profiled the expression of 72 known miRNAs using rCGG repeat transgenic flies. We identified a subset of miRNAs that consistently displayed altered expression in rCGG repeat flies relative to control group. Seven miRNAs with a≥2-fold increase and two miRNAs with expression decreased by≥1.5-fold have been found in rCGG repeat flies. These results suggest that fragile X premutation rCGG repeats could lead to the dysregulation of a subset of specific miRNAs.1.2 MiR-277 modulates the neurodegeneration caused by fragile X rCGG repeatsTo assess the potential involvement of the identified miRNAs with altered expression in FXTAS fly brain, we examined the genetic interaction between specific miRNAs and rCGG-mediated neuronal toxicity. We found that overexpression of miR-277 enhances rCGG repeats-induced neuronal toxicity. To further explore the potential regulatory effect of miR-277 on rCGG-mediated neurodegeneration, we generated transgenic miR-277 sponge (miR-277SP) line, which could block the activity of miR-277, to test blocking effect on rCGG-induced neurodegenerative eye phenotype. We crossed miR-277SP transgenic flies with the flies expressing 90 CGG repeats, and found that the expression of miR-277 sponge could suppress rCGG-mediated neuronal toxicity. This result suggests that blocking the activity of miR-277 could mitigate the neurodegeneration caused by fragile X premutation rCGG repeats.1.3 MiR-277 differentially regulates the expression of Drep-2 and Vimar mRNAsTo seek the mechanisms by which miR-277 modulates rCGG-mediated neurodegeneration, we referenced TargetScanFly 5.1 to identify potential miR-277 targets. We then carried out a genetic screen on the rCGG90 neurodegenerative eye phenotype to identify potential miR-277 targets that could modulate the rCGG-mediated neuronal toxicity. We crossed gmr-GAL4, UAS-(CGG)9o-EGFP transgenic flies with fly mutants in genes coding for the top candidates of miR-277 target genes. Through this screen, we identified two modifiers of rCGG-mediated neurodegeneration, Drep-2 and Vimar. Partial loss of Drep-2 could enhance the rCGG90 eye phenotype by increasing ommatidial disorganization. Overexpression of Drep-2 could suppress the rCGG-induced eye phenotype. A heterozygous loss-of-function mutant of Vimar was also found to aggravate the rCGG90 eye phenotype. These data together indicate that Drep-2 and Vimar could modulate rCGG-mediated neurodegeneration. By introducing 3'-UTR dual luciferase assays, we further tested that miR-277 could indeed target to Drep-2 or Vimar. Next we went on to examine the steady-state levels of Drep-2 and Vimar mRNA in rCGG repeat flies, in which the expression of miR-277 is increased. We saw a significant reduction of endogenous Drep-2 mRNA in rCGG repeat flies relative to control flies. While the Vimar mRNA expression in rCGG repeat flies remains similar to those in control flies. These observations suggest that miR-277 could regulate Drep-2 and Vimar mRNAs differentially. While miR-277 regulates the expression of Drep-2 mainly at the mRNA level, the regulation of Vimar by miR-277 is achieved through the translational suppression instead.1.4 rCGG-repeat-binding protein, hnRNP A2/B1, regulates the expression of miR-277Two rCGG-repeat-binding proteins, Pur a and hnRNP A2/B1, were previously identified to bind rCGG repeats directly and modulate rCGG-mediated neuronal toxicity. Intriguingly, recent studies have shown that multiple heterogeneous nuclear ribonucleoproteins (hnRNPs) could interact with heterochromatin protein 1 (HP1) to bind to genomic DNA and modulate heterochromatin formation. So we tested whether hnRNP A2/B1 could interact directly with genomic regions proximal to miR-277. We performed hnRNP A2/B1-specific chromatin immunoprecipitation (ChIP) followed by real-time quantitative PCR across a 6-kb region surrounding miR-277. Immunoprecipitation of chromatin chemically crosslinked to DNA with a hnRNP A2/B1-specific antibody demonstrated that a region 1.5 kb upstream of miR-277 was enriched～7-fold relative to IgG control as well as adjacent regions. This result suggests that hnRNP A2/B1 could directly bind to the upstream region of miR-277 and regulate its expression.We provide both biochemical and genetic evidence to support a novel miRNA-mediated gene regulation in the pathogenesis of neurodegenerative disorders. Identification of these miRNAs and their targets could reveal potential new targets to develop therapeutic interventions for FXTAS as well as other neurodegenerative disorders.2. Retrotransposon gypsy activation contributes to fragile X premutation rCGG-mediated neurodegeneration2.1 Fragile X rCGG repeats cause the activation of specific retrotransposons in the brainWe observed a consistent upregulation of several retrotransposons when we conducted gene expression profiling using rCGG-repeat transgenic flies. Based on this finding, we systematically examined the expression of retrotransposons in our FXTAS Drosophila model by RT-PCR. Compared with the control flies, we saw a significant increase in three retrotransposons (I-element, gypsy and copia) and one repetitive sequence (Mst40) in rCGG-repeat transgenic flies. We further examined the expression of these transposon elements in transgenic flies expressing reduced rCGG repeats. We observed a reduced increase in gypsy and copia expression, but detected no elevation in the expression of I-element and Mst40 transcripts in these flies. These results together suggest increased expression of specific retrotransposons in rCGG-repeat transgenic flies is rCGG-repeat dosage-dependent.2.2 HnRNP A2/B1 interacts with HP1 to modulate the activity of gypsy and copiaTo determine the role of retrotransposon activation in rCGG-mediated neurodegeneration, we further examined the genetic interaction between specific transposons and rCGG-mediated neuronal toxicity. We crossed gmr-GAL4, UAS-(CGG)90-EGFP transgenic flies with two flamenco mutant fly lines that are permissive for gypsy expression. We observed that derepressing gypsy expression could enhance the neurodegenerative eye phenotype of rCGG-repeat transgenic flies, suggesting that the activation of gypsy could directly modulate rCGG-mediated neurodegeneration. HP1, a conserved critical component of heterochromatin, is known to be required for retrotransposon silencing. Intriguingly, HP1 is found to interact with multiple hnRNPs to modulate heterochromatin formation. Given that one of the rCGG-repeat-binding proteins that we identified previously is hnRNP A2/B1, we examined the potential association between HP1 and hnRNP A2/B1 proteins. We performed immunoprecipitation and found that hnRNP A2/B1 is associated with HP1, and potentially heterochromatin. Previous chromatin immunoprecipitation (ChIP) assays indicated that HP1 could associate with the majority of the flamenco locus to control retroelement activity. Given that fragile X rCGG repeats could cause the activation of selective retrotransposons and hnRNPA2/B1 is one of the rCGG-binding proteins, we determined the interaction between hnRNP A2/B1 and retrotransposons induced by fragile X rCGG repeats using a ChIP assay. We found that hnRNP A2/B1 is significantly associated with the genomic regions containing retrotransposons with increased expression in the presence of fragile X rCGG repeats, including gypsy, copia and Mst40. The retrotransposons without altered expression in the presence of fragile X rCGG repeats are not associated with hnRNP A2/B1. Furthermore, overexpression of hnRNPA2/B 1 in control fly brain has no effect on gypsy expression, whereas overexpression of hnRNPA2/B1 in rCGG-repeat transgenic flies could suppress upregulated gypsy expression; similar suppression was also seen with copia expression. Our results from co-immunoprecipitation experiments, ChIP assays and genetic studies suggest that hnRNP A2/B1 could bind to selective retrotransposons and recruit HP1 for transposon silencing. In the presence of fragile X rCGG repeats, hnRNP A2/B1 will be sequestered by excess rCGG repeats. The depletion of hnRNP A2/B1 potentially leads to less HP1 being recruited to the genomic regions containing those retrotransposons, and subsequent activation of retrotransposons.2.3 Reduction of gypsy expression suppresses rCGG-mediated neurodegenerationTo further investigate the physiological relevance of retrotransposon activation in fragile X premutation rCGG-mediated neurodegeneration, we generated fly UAS lines that could express dsRNAs against either gypsy or copia in the presence of a GAL4 driver. We then crossed these transgenic lines with the rCGG-repeat transgenic lines that exhibit eye neurodegeneration. To our surprise, we found that expression of dsRNAs against gypsy, but not copia, could suppress rCGG-mediated neurodegeneration. Expression of these dsRNAs has no effect on wild-type fly eyes. This observation strongly suggests that the dysregulation of retrotransposon activation plays significant role in the molecular pathogenesis of rCGG-mediated neuronal toxicity.Here we provide both biochemical and genetic evidence to demonstrate a surprisingly active role for retrotransposition in fragile X premutation rCGG repeats-mediated neurodegeneration. It would be interesting to explore further whether the activation of transposable elements could be a common mechanism underlying neurodegeneration in general.