Reversal of RNA dominance by displacement of protein sequestered on triplet repeat RNA

Abstract

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.

Fig. 1

CAG25 inhibits formation of CUG^exp-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 CUG^exp-MBNL1 complex formation (upper panel) and displaces MBNL1 protein from pre-formed complex (lower panel). 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 (upper panel) and displaces MBNL1 from pre-formed complexes (lower panel). Bands indicate the amount of recombinant MBNL1 protein that remains in ribonucleoprotein complex at the indicated concentration of CAG25 (27). (D) The % MBNL1 bound to CUG^exp is expressed as the mean ± SD of protein retained on plate. 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 HSA^LR mice. Probe (red) binds to HSA^LR 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.

Fig. 2

Reversal of misregulated alternative splicing by CAG25. (A) RT-PCR analysis of alternative splicing for chloride channel-1 (ClC-1), Serca-1, m-Titin, and Zasp, 3 weeks after a single injection of CAG25 in tibialis anterior (TA) muscle of HSA^LR 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 HSA^LR transgenic and wild-type 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 vs contralateral (t-test). “untr” = untreated HSA^LR.

Fig. 3

Chloride channel 1 protein expression and function are rescued by CAG25. (A) Immunofluorescence for ClC-1 protein expression in sections from HSA^LR TA muscle, bar = 20 μm. (B) ClC-1 current density in FDB fibers isolated from 15-day-old HSA^LR mice injected with CAG25 or control morpholino. Fibers from wild-type mice injected with CAG25 or control morpholino serve as controls (see also 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 vs. saline (t-test).

Fig. 4

CAG25 increases the cytoplasmic level and translation of CUG^exp-containing mRNA, despite reducing the overall level of CUG^exp RNA. (A) RT-PCR assay of transgene mRNA in the cytoplasm. Human (transgene) and mouse (endogenous) skeletal actin transcripts were co-amplified by the same primers, species origin was revealed by AluI cleavage (27). “h”, human muscle; “wt”, wild-type mouse. Lower panel shows depletion of nuclear RNA from cytoplasmic fraction (“c”) relative to nuclear pellet (“n”), analyzed by RT-PCR for the 5′ external transcribed spacer (5′ ETS, nuclear-retained) of ribosomal RNA. Numbers refer to different mice treated with CAG25. (B) CAG25 increases luciferase activity in LLC9/Rosa-CreER bitransgenic mice. Upper panel shows LLC9 transgene for conditional expression of CUG^exp RNA. Lower panels show in vivo bioluminescence imaging (BLI) of different bitransgenic mice (27). For quantification, luciferase activity (indicated in yellow-orange) in CAG25-injected muscle was normalized to the contralateral side injected with saline or control morpholino (n = 7 mice). (C) Northern blot of total cellular RNA shows decreased HSA^LR mRNA in muscle injected with CAG25, compared to contralateral muscle injected with vehicle (saline). Mouse actin is loading control. (D) CUG^exp-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”, 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.