A fly model for the CCUG-repeat expansion of myotonic dystrophy type 2 reveals a novel interaction with MBNL1

Abstract

Expanded non-coding RNA repeats of CUG and CCUG are the underlying genetic causes for myotonic dystrophy type 1 (DM1) and type 2 (DM2), respectively. A gain-of-function of these pathogenic repeat expansions is mediated at least in part by their abnormal interactions with RNA-binding proteins such as MBNL1 and resultant loss of activity of these proteins. To study pathogenic mechanisms of CCUG-repeat expansions in an animal model, we created a fly model of DM2 that expresses pure, uninterrupted CCUG-repeat expansions ranging from 16 to 720 repeats in length. We show that this fly model for DM2 recapitulates key features of human DM2 including RNA repeat-induced toxicity, ribonuclear foci formation and changes in alternative splicing. Interestingly, expression of two isoforms of MBNL1, MBNL135 and MBNL140, leads to cleavage and concurrent upregulation of the levels of the RNA-repeat transcripts, with MBNL140 having more significant effects than MBNL135. This property is shared with a fly CUG-repeat expansion model. Our results suggest a novel mechanism for interaction between the pathogenic RNA repeat expansions of myotonic dystrophy and MBNL1.

Figure 1.

Characterization of transgenic flies expressing various-length CCUG RNA repeats. (A) Design of transgenic constructs. CCTG non-coding repeat expansions were expressed under the control of the GAL4/UAS system. The control contained 16 CCTG-repeat units whereas the expanded repeat ranged from 200 to 720 units. (B) DNA Southern blot to determine the CCTG-repeat length in six representative lines. From left to right, repeat lengths are 16, 200, 475, 520, 700 and 720, respectively. (C) Northern blots to determine RNA transcript levels in UAS-(CCTG) flies with various repeat lengths. Non-coding CCUG-repeat RNA was expressed with hs-gal4. The SV40 3′ UTR probe was used as for detection. These lines express similar levels of the CCUG-repeat transgene.

Figure 2.

Repeat length-dependent CCUG toxicity in the fly eye. CCTG-repeat expansions of various lengths were expressed in the eye using the gmr-gal4 driver. 1d animals. (A) External eye and (B) internal retinal structure were examined. (A) Repeat RNA expression caused length-dependent toxicity as reflected by loss of pigmentation, necrotic patches and a degenerate eye surface indicative of disruption to the ommatidial organization (arrows). (B) Internally, the expanded repeat impacts depth and structure of the retina at repeat lengths of 475 and longer. Genotypes: gmr-gal4 in trans to UAS-(CCTG)16, UAS-(CCTG)200, UAS-(CCTG)475, UAS-(CCTG)525, UAS-(CCTG)700, UAS-(CCTG)720.

Figure 3.

Expanded CCUG-repeat RNA accumulates in nuclear foci and impacts alternative splicing. (A) Confocal images of CCUG-repeat foci detected with (CAGG)5-FITC probe, nuclei stained with DAPI and merged images between ribonuclear foci and nuclei in body-wall muscles of third instar larvae. Genotypes: w¹¹¹⁸, 24B-gal4 in trans to UAS-(CCTG)200, UAS-(CCTG)475, UAS-(CCTG)475/MBNL1₃₅. (B) Mean number of foci in each nucleus in flies obtained in three independent experiments. Genotypes: 24B-gal4 in trans to UAS-(CCTG)200, UAS-(CCTG)475, UAS-(CCTG)475/MBNL1₃₅ (**P < 0.05, ANOVA test). (C) Mean volume of foci obtained in three independent experiments. Genotypes: 24B-gal4 in trans to UAS-(CCTG)200, UAS-(CCTG)475, UAS-(CCTG)475/MBNL1₃₅ (**P < 0.05, ANOVA test). (D) Expression of expanded repeat CCUG in photoreceptor neurons promoted exclusion of the second exon of sTNI as determined by radioactive PCR. Shown in the top panel is a representative gel image for determining the densitometry ratio between 140-bp and 110-bp PCR products, corresponding to the splicing isoform with (140 bp) or without (110 bp) the second exon of sTNI minigene reporter, respectively (38). Shown in bottom panel is quantification of three independent experiments. With expression of expanded CCUG repeats, the ratio decreased, such that the second exon was preferentially excluded. Genotypes: rh1-gal4 UAS-hcTNT in trans to UAS-(CCTG)16, UAS-(CCTG)200, UAS-(CCTG)475, UAS-(CCTG)520, UAS-(CCTG)700. *P < 0.05, **P < 0.01 comparing to CCTG16 control (ANOVA followed by Dunnett's post-test).

Figure 4.

MBNL1₃₅ and MBNL1₄₀ mildly suppress CCUG-repeat toxicity and mediate upregulation and cleavage of non-coding CUG/CCUG-repeat expansions. (A) Mild suppression of the external (arrows) and internal retinal toxicity of the CCUG repeat occurs upon expression of MBNL1₄₀ or MBNL1₃₅. Genotypes, from left to right, gmr-gal4 UAS-(CCTG)700 in trans to w¹¹¹⁸, UAS-MBNL1₃₅, UAS-MBNL1₄₀. (B) Expression of MBNL1₃₅ and MBNL1₄₀ mediates upregulation and cleavage of expanded CCUG-repeat RNA. Genotype of flies (from the left lane to right lane): gmr-gal4, UAS-(CCTG)700 in trans to: w¹¹¹⁸, UAS-MBNL1₃₅, UAS-MBNL1₄₀ and w¹¹¹⁸ alone as the negative control. (C) Expression of MBNL1₃₅ and MBNL1₄₀ also mediate cleavage and upregulation of expanded CUG-repeat RNA. Genotype of flies (from the left lane to right lane): gmr-gal4, UAS-DsRed-(CTG)250 in trans to: w¹¹¹⁸, UAS-MBNL1₃₅, UAS-MBNL1₄₀ and w¹¹¹⁸ alone as the negative control. (D) In contrast to findings with MBNL1, expression of DNAJC17 does not lead to upregulation and cleavage of expanded CCUG-repeat RNA. Genotype of flies (from the left lane to right lane): gmr-gal4, UAS-(CCTG)700 in trans to: w¹¹¹⁸, UAS-MBNL1₃₅, UAS-MBNL1₄₀ and dDNAJC17. (E) Integrity and level of coding CAG repeats are not dramatically affected by MBNL1. Genotype of flies (from the left lane to right lane): w¹¹¹⁸ alone as the negative control and gmr-gal4 UAS-SCA3trQ78 in trans to: w¹¹¹⁸, UAS-MBNL1₃₅ and UAS-MBNL1₄₀.