Proposed model of miR-277 modulating fragile X premutation rCGG repeat-mediated neurodegeneration.

<p>HnRNP A2/B1, an rCGG repeat-binding protein, could be sequestered by fragile X premutation rCGG repeats from their normal cellular functions. Sequestration of hnRNP A2/B1 could de-repress the expression of miR-277. MiR-277 could recognize and bind to the 3′-UTR of its mRNA targets, such as Drep-2 and Vimar, to induce their mRNA degradation or translational suppression. Misregulation of Drep-2 or Vimar, which are involved in cell apoptosis or mitochondrial regulation, could lead to the neuronal cell death associated with FXTAS.</p

MiR-277 regulates the expression of Drep-2 and Vimar post-transcriptionally.

<p>A. The two miR-277 target sites in the Drep-2 3′-UTR and three miR-277 target sites in the Vimar 3′-UTR were predicted by TargetScan. We created the mutant Drep-2 3′-UTR (Drep-2 3′-UTR^ΔmiR-277) with two miR-277 binding sites deleted and the mutant Vimar 3′-UTR (Vimar 3′ UTR^ΔmiR-277) with three miR-277 binding sites deleted, as shown. B. The expression of a luciferase reporter gene containing the Drep-2 3′-UTR was suppressed by miR-277 in HEK293FT cells. The suppression is specific to the miR-277 seed region within the Drep-2 3′-UTR, as deletion of the miR-277 target sites in the Drep-2 3′-UTR alleviated repression by miR-277. (*-P<0.05; ns-P>0.05). C. Similar miR-277-mediated suppression was observed with Vimar 3′-UTR constructs. D. Endogenous Drep-2 mRNA expression was significantly reduced in rCGG repeat flies relative to control flies (elav-GAL4), whereas no obvious change of Vimar mRNA expression was observed in the rCGG repeat flies compare to control flies. E. Endogenous Drep-2 mRNA expression was significantly altered in the flies expressing either miR-277 or miR-277 SP, *-P<0.05.</p

Mutant alleles corresponding to the mRNA targets of miR-277 used for genetic screen.

<p>Mutant alleles corresponding to the mRNA targets of miR-277 used for genetic screen.</p

Modification of rCGG-mediated neurodegenerative eye phenotype by Drep-2 and Vimar.

<p>(A–C) SEM eye images from control flies expressing rCGG₉₀, rCGG₉₀ and a heterozygous mutation in Drep-2 (Drep-2^LOF), rCGG₉₀ and overexpression of Drep-2 (Drep-2^OE), and rCGG₉₀ and a heterozygous mutation in Vimar (Vimar^LOF). rCGG₉₀ flies manifest ommatidial disorganization and fusion (A). This phenotype is enhanced in flies carrying a heterozygous loss-of-function mutation in Drep-2 (B), while the eye phenotype could be suppressed by overexpression of Drep-2 (C). Mutation of Vimar enhances the rCGG₉₀-mediated eye phenotype (F). All the mutants alone do not cause any abnormal eye phenotype (D, E, G). Genotypes are A-<i>gmr-GAL4,UAS-CGG₉₀-EGFP/+</i>; B -<i>gmr-GAL4, UAS-CGG₉₀-EGFP/Drep-2^KG02396</i>; C-<i>gmr-GAL4, UAS-CGG₉₀-EGFP/Drep-2^d00223</i>; D-<i>gmr-GAL4/Drep-2^KG02396</i>; E-<i>gmr-GAL4/ Drep-2^d00223</i>; F -<i>gmr-GAL4, UAS-CGG₉₀-EGFP/ Vimar^K16722</i>; G-<i>gmr-GAL4/ Vimar^K16722</i>.</p

Expression of miR-277 is regulated by hnRNPA2/B1.

<p>A. Schematic of the six kb proximal to miR-277 on chromosome 3R assayed in ChIP experiments. B. HnRNP A2/B1-specific ChIP assay indicates the enrichment of DNA 1.5 kb upstream of the miR-277 genomic locus. Relative enrichment is calculated relative to IgG-only nonspecific control and normalized to the empty vector. Error bars indicate mean ± SEM. C. Ectopic expression of hnRNP A2/B1 in fly brain could suppress the expression of miR-277, *-P<0.05.</p

Identification of the miRNAs with altered expression in the brains of an FXTAS <i>Drosophila</i> model.

<p>Relative quantity of miRNA with ≥two-fold change in expression shown for rCGG₆₀ flies, calibrated to control (elav-GAL4) flies. Control relative quantity = 1.</p

Overexpression of miR-277 enhances rCGG-mediated neurodegeneration.

<p>Shown are scanning electron microscope (SEM) eye images from seven-day-old flies. WT fly eyes show the normal organization of ommatidia (A). Expression of rCGG90 causes disorganized, fused ommatidia (B). Flies overexpressing miR-277 alone present with a mild rough eye phenotype (C). The rCGG90 eye phenotype is enhanced by miR-277 overexpression with aggravated disorganized, fused ommatidia (D). Alteration of bantam, let-7, or miR-1 does not modify the rCGG-induced eye phenotype (F, H, J, L, N). Alteration of bantam, let-7 or miR-1 alone does not cause an abnormal eye phenotype (E, G, I, K, M). Genotypes are B<i>-gmr-GAL4,UAS-CGG₉₀-EGFP/+</i>; C<i>-gmr-GAL4/UAS-miR-277</i>; D<i>-gmr-GAL4, UAS-CGG₉₀-EGFP/UAS-miR-277</i>; E-<i>gmr-GAL4/+;UAS-bantam/+</i>; F-<i>gmr-GAL4,UAS-CGG₉₀-EGFP/+;UAS-bantam/+</i>; G-<i>gmr-GAL4/+;UAS-Let-7/+</i>; H-<i>gmr-GAL4,UAS-CGG₉₀-EGFP/+;UAS-Let-7/+</i>; I-<i>gmr-GAL4/UAS-GFP;UAS-miR-1/+</i>; J-<i>gmr-GAL4,UAS-CGG₉₀-EGFP/UAS-GFP;UAS-miR-1/+</i>; K-<i>gmr-GAL4/+;ban¹²/+</i>; L-<i>gmr-GAL4,UAS-CGG₉₀-EGFP/+;ban¹²/+</i>; M-<i>gmr-GAL4/+;ban²⁰/+</i>; N-<i>gmr-GAL4,UAS-CGG₉₀-EGFP/+;ban²⁰/+</i>.</p

Blocking the activity of miR-277 suppresses rCGG-mediated neurodegeneration.

<p>A. Shown is the diagram depicting the strategy for generation of transgenic microRNA sponge lines. The sponge construct contains 10 repetitive sequences complementary to a microRNA downstream of EGFP in a UAS-containing P-element vector. When these UAS lines are crossed to GAL4 driver lines, the F1 progeny generate tissue- or cell-specific expression of microRNA sponge to block the activity of specific miRNA in <i>Drosophila</i>. B. The miR-sponge contains bulged sites that are mismatched opposite microRNA at positions 9–12. The bulge is designed to protect Ago2-mediated endonucleolytic cleavage. C. Scanning electron microscope (SEM) eye images from seven-day-old flies. Block of miR-277 activity by miR-277SP could rescue the rCGG-mediated neurodegenerative eye phenotype. Knockdown of miR-277 alone does not cause an abnormal eye phenotype. Genotypes are (left)<i>-gmr-GAL4,UAS-CGG₉₀-EGFP/+</i>; (middle)<i>-gmr-GAL4,UAS-CGG₉₀-EGFP/UAS-miR-277SP</i>; (right)-<i>gmr-GAL4/UAS-miR-277SP</i>.</p

Relative fertility of different mutant flies.

<p>Individual female was crossed with wildtype (<i>w¹¹¹⁸</i>) male flies and the number of fertile eggs was quantified.</p>*<p><i>P<0.01</i> when the trans-heterozygotes were compared with either WT or single heterozygotes.</p