Activating frataxin expression by repeat-targeted nucleic acids - PubMed (original) (raw)

Activating frataxin expression by repeat-targeted nucleic acids

Liande Li et al. Nat Commun. 2016.

Abstract

Friedreich's ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression.

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Conflict of interest statement

D.R.C. and L.L. have filed a patent application related to this study. M.M. declares no competing financial interest.

Figures

Figure 1

Figure 1. RNA-mediated activation of FXN expression.

(a) Schematic of repeat expansion within intronic FXN mRNA and binding of AGO:RNA complexes. The longer mutant repeat is predicted to bind more AGO:RNA complexes than the shorter wild-type repeat. (b) Schematic of R-loop formation at FXN locus and potential to influence histone modification and gene expression (Adapted from Groh _et al._19). (c) Complementarity of guide strand RNAs (25 nM) to FXN RNA. (d,e) Effect of anti-GAA duplex RNAs on d, FXN mRNA (_n_=3) and e, protein expression. (f) A dose-response profile of upregulation of FXN protein expression by siGAA. FRDA patient-derived fibroblast cells (GM03816) were used. siExon2 is a duplex siRNA that targets FXN exon and is expected to decrease FXN expression. CM is a negative control RNA that is not complementary to FXN RNA. RNA scramble is a duplex RNA in which the sequences of siGAA and siAAG are mixed to preserve nucleotide composition by alter their order. si5mmAAG is similar to siAAG but has five mismatches relative to the repeat region within FXN mRNA target. Cells were collected at day 3 for RNA extraction and day 4 for protein extraction. Error bars:±STDEV. **P<0.01, by Student _t_-test.

Figure 2

Figure 2. Comparison of RNA-mediated activation of FXN expression in patient cells to FXN expression in normal cells and FXN activation by a histone deacetylase inhibitor.

(a) Activation of FXN protein expression in FRDA cells (GM03816) and wild-type fibroblast cells (GM02153). (b,c) Effect of HDAC inhibitor BML210 (5 μM) treatment on expression of b FXN mRNA (_n_=3) and (c) protein (inset, western analysis, _n_=4) expression in FRDA patient fibroblast cells (GM03816). All data are presented as mean±STDEV.

Figure 3

Figure 3. Mechanism of FXN activation by repeat-targeted duplex RNAs.

(a) RIP examining the association of Ago2 with FXN pre-mRNA after treatment with 50 nM duplex RNA and analysis by real-time PCR. An arrow marks the PCR product of FXN pre-mRNA, which was confirmed by sequencing (Supplementary Fig. 7) (b) Anti-GAA duplex RNA with central mismatches (siGAA 9,10 mm with mismatches on both strands; 25 nM) activates FXN expression at a level similar to the analogous fully complementary duplex RNA (_n_=3). siExon3 is a positive control for transfection efficiency targeting exon 3 of FXN. (c) ChIP for RNAP2 using four different primer sets (_n_=4). (d) ChIP for transcription-associated histone modification markers H3K4me3, H3K9me2, H3K9me3, H3K9Ac, H3K27me3 and H4Ac (_n_=4–8). (e) FXN mRNA stability assay. Cells were transfected with duplex RNAs siGAA or CM at 25 nM (_n_=3). Actinomycin D (5 μg ml−1) was added with fresh media 3 days after transfection and cells were collected at the indicated time points. HPRT expression was measured for normalization. All experiments were performed in GM03816 patient-derived cells. All data are presented as mean±STDEV. *P<0.05, **P<0.01, by Student _t_-test.

Figure 4

Figure 4. LNA-mediated activation of FXN expression.

(a) Structure of LNA. (b) Western analysis of the effect of LNAs with phosphodiester (PO) backbone on FXN protein expression. (c) Quantitation of western analysis (_n_=2). (d) Western analysis of the effect of LNAs with phosphorothioate (PS) backbone on FXN protein expression. (e) Quantitation of quadruplicate western analysis. (f,g) qPCR showing effect on FXN mRNA expression of (f) PO LNAs (_n_=5) or (g) PS LNAs (_n_=3). PO-control-LNA5 is a negative control LNA with PO backbone that is not complementary to FXN RNA. PO-control-LNA6 has five mismatches relative to the repeat region within FXN RNA target. PS-control-LNA7 is a negative control LNA similar to PO-control-LNA5 but with PS backbone. FRDA patient fibroblast cells (GM03816) were treated with 12.5 nM duplex LNAs. Cells were collected at day 3 for RNA extraction and day 4 for protein extraction. All data are presented as mean±STDEV. NT, no treatment; **P<0.01, by Student _t_-test.

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