Reversal of RNA Dominance by Displacement of Protein Sequestered on Triplet Repeat RNA

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Science  17 Jul 2009:
Vol. 325, Issue 5938, pp. 336-339
DOI: 10.1126/science.1173110


Genomic expansions of simple tandem repeats can give rise to toxic RNAs that contain expanded repeats. In myotonic dystrophy, the expression of expanded CUG repeats (CUGexp) causes abnormal regulation of alternative splicing and neuromuscular dysfunction. We used a transgenic mouse model to show that derangements of myotonic dystrophy are reversed by a morpholino antisense oligonucleotide, CAG25, that binds to CUGexp RNA and blocks its interaction with muscleblind-like 1 (MBNL1), a CUGexp-binding protein. CAG25 disperses nuclear foci of CUGexp RNA and reduces the overall burden of this toxic RNA. As MBNL1 is released from sequestration, the defect of alternative splicing regulation is corrected, thereby restoring ion channel function. These findings suggest an alternative use of antisense methods, to inhibit deleterious interactions of proteins with pathogenic RNAs.

Myotonic dystrophy type 1 (DM1) is representative of a group of dominantly inherited disorders in which expression of a toxic RNA leads to neuromuscular degeneration (15). A feature common to these pathogenic RNAs is the presence of an expanded repeat. In DM1, the disease-inducing transcript is the DM protein kinase (DMPK) mRNA containing an expanded CUG repeat in its 3′ untranslated region (3′UTR) (6). These CUG repeats bind to muscleblind-like 1 (MBNL1), a splicing regulator, with high affinity (7, 8). Because each mutant transcript typically contains thousands of CUG repeats, the capacity for protein binding is large, causing MBNL1 to become sequestered in ribonucleoprotein complexes. These complexes are observed in DM1 cells as foci of CUGexp-MBNL1 aggregates in the nucleus (7, 911).

If sequestration of MBNL1 contributes to symptoms of DM1, inhibitors of MBNL1-CUGexp binding may reverse these effects. To test this possibility, we used CAG25, an antisense 25-nucleotide morpholino oligonucleotide composed of CAG repeats. Antisense morpholinos do not trigger cleavage of their target RNAs (12), which suggests that they could be used to bind CUGexp RNA and release sequestered proteins, without risk of degrading other transcripts that contain CUG repeats. A potential obstacle for this approach, however, is that CUGexp RNAs form hairpins with high thermal stability (13, 14) and extensive MBNL1 binding (8), which potentially could limit their access to antisense oligonucleotides. In vitro CAG25 was able to invade CUGexp hairpins and form a stable RNA-morpholino heteroduplex (Fig. 1A and figs. S1 and S2). CAG25 was also able to block the formation of CUGexp-MBNL1 complexes and disrupt complexes that had already formed (Fig. 1, B to D).

Fig. 1

CAG25 inhibits formation of CUGexp-MBNL1 complexes. (A) Gel shift assay demonstrates that addition of CAG25 (indicated concentration) to labeled (CUG)109 (2 nM) results in slower migration of (CUG)109-CAG25 heteroduplex. (B) CAG25 prevents CUGexp-MBNL1 complex formation (top) and displaces MBNL1 protein from preformed complex (bottom). Lane C shows migration of labeled (CUG)109 hairpin. Addition of excess MBNL1 produces complexes of variable size that migrate as a broad smear (lane 0.0). Addition of CAG25 at increasing concentration reconstitutes (CUG)109-CAG25 heteroduplex as the dominant band (27). (C) Microtiter plate/gel assay confirms that CAG25 prevents the formation of (CUG)109-MBNL1 complex (top) and displaces MBNL1 from preformed complexes (bottom). Bands indicate the amount of recombinant MBNL1 protein that remains in ribonucleoprotein complex at the indicated concentration of CAG25 (27). (D) The percent of MBNL1 bound to CUGexp is expressed as the mean ± SD of protein retained on plate. Median inhibitory concentration (IC50) for “prevention” is 462 ± 31 nM and for “displacement” is 1032 ± 117 nM. (E) Fluorescence in situ hybridization of single flexor digitorum brevis (FDB) muscle fibers from HSALR mice. Probe (red) binds to HSALR transcripts upstream from CUG repeat; nuclei are blue. CAG25, but not control morpholino, causes dispersal of RNA foci. (F) MBNL1 (immunofluorescence, green) shifts from punctate to diffuse nuclear distribution after injection of FDB with CAG25. Scale bars, 5 μm.

To examine whether CAG25 can influence CUGexp interactions in vivo, we tested its effects in a transgenic mouse model of DM1. HSALR transgenic mice express human skeletal actin transcripts that have (CUG)250 inserted in the 3′UTR. These mice accumulate CUGexp RNA and MBNL1 protein in nuclear foci in skeletal muscle (1), a process that is thought to depend on CUGexp-MBNL1 interaction (15). We therefore examined the effect of CAG25 on foci in muscle cells. We loaded CAG25 into muscle fibers by intramuscular injection followed by in vivo electroporation. Muscle tissue was examined 1 to 3 weeks later by means of fluorescence in situ hybridization, using probes that hybridize to the CUG repeat or to sequences flanking the repeat. Injection of CAG25, but not a control morpholino of unrelated sequence, caused a marked reduction of nuclear foci and a redistribution of MBNL1 protein (Fig. 1, E and F, and fig. S3).

To determine whether CAG25 can reverse the biochemical consequences of MBNL1 sequestration, we examined its effects on alternative splicing. HSALR transgenic mice show alternative splicing changes similar to those observed in human DM1 (10). The splicing misregulation is improved when MBNL1 levels are increased (16), aggravated when MBNL1 levels are reduced, and reproduced by ablation of Mbnl1 (17), suggesting that splicing defects in this model are primarily caused by MBNL1 sequestration. For each DM1-affected exon that we examined, the alternative splicing was normalized or nearly corrected at 3 weeks after injection of CAG25 (Fig. 2). Effects of CAG25 on alternative splicing persisted at 14 weeks (fig. S4, A and B) but not at 8 months after a single injection. In contrast, CAG25 did not correct the misregulated splicing of these same exons in Mbnl1 knockout mice or alter their splicing patterns in wild-type (WT) mice (fig. S5), indicating that its effects are mediated through CUGexp RNA rather than acting directly on the respective precursor mRNAs (pre-mRNAs). The Capzb and Itgb1 transcripts also show developmentally regulated alternative splicing in skeletal muscle, but they do not depend on MBNL1, they are not misregulated in HSALR mice (10), and CAG25 had no effect on splicing of either transcript.

Fig. 2

Reversal of misregulated alternative splicing by CAG25. (A) Reverse transcription polymerase chain reaction (RT-PCR) analysis of alternative splicing for ClC-1, Serca-1, m-Titin, and Zasp, 3 weeks after a single injection of CAG25 in tibialis anterior (TA) muscle of HSALR transgenic mice. The contralateral (con) TA was injected with vehicle (saline, mice 1 and 2) or morpholino with inverted sequence (GAC25, mice 3 and 4). Splice products from untreated HSALR transgenic and WT TA muscle (n = 3 mice) are shown. Int 6, intron 6 retention. (B) Quantification of results in (A), expressed as the percentage of splice products that include or exclude the indicated exon. *P = 0.003 and **P < 0.001 for CAG25 versus contralateral (Student’s t test). untr, untreated HSALR.

To determine whether CAG25 can rescue the physiological deficits of DM1, we examined the expression and function of chloride channel 1 (ClC-1), the muscle-specific chloride channel. Delayed muscle relaxation and repetitive action potentials (myotonia) are cardinal features of DM1, resulting from loss of ClC-1 channels (18, 19). Affected individuals and mouse models show inclusion of an additional exon in the ClC-1 mRNA, resulting in frameshift and loss of channel activity (20). CAG25 corrected the defect of ClC-1 alternative splicing (Fig. 2 and fig. S4, A and B) and restored the expression of ClC-1 protein to the surface membrane (Fig. 3A and fig. S4C). Furthermore, transmembrane chloride ion conductance was normalized (Fig. 3B), and myotonia was markedly reduced (Fig. 3C and figs. S4D and S7).

Fig. 3

ClC-1 protein expression and function are rescued by CAG25. (A) Immunofluorescence for ClC-1 protein expression in sections from HSALR TA muscle. Scale bar, 20 μm. (B) ClC-1 current density in FDB fibers isolated from 15-day-old HSALR mice injected with CAG25 or control morpholino. Fibers from WT mice injected with CAG25 or control morpholino serve as controls (fig. S6). (C) Electromyographic myotonia analysis 3 weeks after injection of CAG25 into TA muscle. The contralateral side was injected with vehicle (saline) alone or control morpholino (27). n = 11 mice examined; *P < 0.0001 for CAG25 versus saline (Student’s t test).

The effects of MBNL1 sequestration on gene expression are not limited to alternative splicing. For example, transcription of Eda2r, Uchl1, and Sarcolipin is highly upregulated in Mbnl1 knockout mice, and similar changes are induced by the expression of CUGexp (21). Although the mechanism for this effect has not been determined, it was partially reversed by CAG25 (fig. S6). These data indicate that CAG25 ameliorates both transcriptional and posttranscriptional effects of toxic RNA.

We next determined whether CAG25 can overcome the nuclear retention of CUG-expanded transcripts (22). Despite reducing the overall level of CUGexp RNA in muscle (see below), CAG25 increased the amount of this transcript in the cytoplasm (Fig. 4A and fig. S8). Moreover, because reduced translation of DMPK mRNA may contribute to cardiac symptoms of DM1 (23), we also tested whether CAG25 could enhance the translation of CUGexp-containing transcripts. We derived transgenic mice that express luciferase mRNA containing (CUG)270 in the 3′UTR. The luciferase transcript is retained in nuclear foci, and basal levels of luciferase activity are accordingly reduced. In vivo bioluminescence imaging showed that CAG25 induced a focal increase of luciferase activity in the injected hindlimb (Fig. 4B), which is consistent with increased translation of the CUG-expanded mRNA.

Fig. 4

CAG25 increases the cytoplasmic level and translation of CUGexp-containing mRNA despite reducing the overall level of CUGexp RNA. (A) RT-PCR assay of transgene mRNA in the cytoplasm. Human (transgene) and mouse (endogenous) skeletal actin transcripts were coamplified by the same primers; the species origin was revealed with AluI cleavage (27). h, human muscle. (Bottom) Depletion of nuclear RNA from cytoplasmic fraction (c) relative to nuclear pellet (n), analyzed by means of RT-PCR for the 5′ external transcribed spacer (5′ETS) (nuclear-retained) of ribosomal RNA (rRNA). Numbers refer to different mice treated with CAG25. (B) CAG25 increases luciferase activity in LLC9/Rosa-CreER bitransgenic mice. (Top) LLC9 transgene for conditional expression of CUGexp RNA. (Bottom) In vivo bioluminescence imaging (BLI) of different bitransgenic mice (27). For quantification, luciferase activity (indicated in yellow and orange) in CAG25-injected muscle was normalized to the contralateral side that was injected with saline or control morpholino (n = 7 mice). (C) Northern blot of total cellular RNA shows decreased HSALR mRNA in muscle injected with CAG25 as compared with contralateral muscle injected with vehicle (saline). Mouse actin is loading control. (D) CUGexp-CAG25 heteroduplex is not cleaved by RNase H. (CUG)109 RNA was incubated with CAG25 morpholino or DNA oligonucleotide of identical sequence (CAG25-DNA). The heteroduplex was incubated with the indicated concentration of RNase H then separated on polyacrylamide gels. Lane c is the control with (CUG)109 alone. (E) (CUG)109 RNA was incubated with CAG25 morpholino or CAG25-DNA to form heteroduplex as in (D), to which HeLa cell extract was added for the indicated time.

CAG25 caused a ~50% reduction in the overall burden of CUGexp RNA (Fig. 4C). There was no parallel reduction of transgene pre-mRNA (fig. S9), suggesting that downregulation of this transcript results from accelerated decay. However, previous work has shown that antisense morpholinos do not support RNA cleavage by ribonuclease H (RNase H) (12). Consistent with these observations, CAG25 did not induce cleavage of CUGexp RNA by recombinant RNase H or by HeLa cell extracts in vitro, whereas an equivalent DNA oligonucleotide caused extensive CUGexp degradation (Fig. 4, D and E). Moreover, CAG25 did not reduce levels of endogenous CUG-repeat-containing transcripts (fig. S10), nor did it reduce transgene mRNA in mice that express an equivalent HSA transcript containing a nonexpanded CUG repeat (fig. S11). Taken together, these results are consistent with an indirect effect of CAG25 on the accumulation of CUGexp RNA. However, therapeutic effects of CAG25 do not derive solely from downregulation of the repetitive RNA because the residual levels of CUGexp were not below the threshold for inducing RNA disease (fig. S12).

DM1 presents a complex phenotype that results from trans-dominant effects of mutant RNA on many different transcripts. To intervene at an upstream site and accomplish a general correction of DM1, we used an antisense methodology to inhibit the protein interactions of expanded RNA repeats. This strategy has several effects that are potentially beneficial, including the release of MBNL1 protein from ribonucleoprotein foci, enhanced transport of CUGexp transcripts to the cytoplasm, and a reduced burden of CUGexp RNA. The mechanism for the latter effect remains to be determined, but we postulate that it may result from accelerated decay of CUGexp RNA once it has been transported to the cytoplasm. Considering that the extent of MBNL1 sequestration in DM1 is variable in different nuclei from the same individual (10), and that ~50% of normal MBNL1 levels are sufficient to ensure normal splicing regulation in mice (10, 17), even partial release of sequestered MBNL1 probably improves splicing abnormalities in DM1. The mass of CUGexp RNA in muscle from HSALR mice is two- to eightfold higher than in muscle tissue from patients with DM1 (fig. S13), suggesting that similar therapeutic effects can be achieved in humans if the antisense can be effectively delivered. Indeed, treatment with CAG25 of DM1 cells in tissue culture led to a reduction of intranuclear RNA foci (fig. S14). Among several CUGexp-protein interactions that may contribute to DM1 pathogenesis, we have focused on MBNL1 because it has the highest CUGexp-binding affinity (8, 24) and most complete sequestration (11) of any factor so far identified. However, it seems likely that interactions of CUGexp with other RNA-binding proteins, such as CUG-binding protein 1 (25), will also be inhibited by this approach, which may favorably affect signaling abnormalities in DM1 (26). Taken together, these data supply proof of concept that agents inhibiting deleterious RNA-protein interactions have therapeutic potential in RNA-dominant disorders.

Supporting Online Material

Materials and Methods

Figs. S1 to S14

Table S1


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. This work comes from the University of Rochester Wellstone Muscular Dystrophy Cooperative Research Center (U54NS48843) and Center for RNA Biology, with support from NIH (AR046806, AR/NS48143, and NIDCR-T32DE07202 to J.D.L.), the Muscular Dystrophy Association, and a postdoctoral fellowship to K.S. from the Foundation for Polish Science, Run America Foundation, and the Saunders Family fund. We appreciate help from L. Richardson and S. Leistman. The University of Rochester has applied for a patent based partly on the work described here.

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