Triplet repeat-derived siRNAs enhance RNA-mediated toxicity in a Drosophila model for myotonic dystrophy

Abstract

More than 20 human neurological and neurodegenerative diseases are caused by simple DNA repeat expansions; among these, non-coding CTG repeat expansions are the basis of myotonic dystrophy (DM1). Recent work, however, has also revealed that many human genes have anti-sense transcripts, raising the possibility that human trinucleotide expansion diseases may be comprised of pathogenic activities due both to a sense expanded-repeat transcript and to an anti-sense expanded-repeat transcript. We established a Drosophila model for DM1 and tested the role of interactions between expanded CTG transcripts and expanded CAG repeat transcripts. These studies revealed dramatically enhanced toxicity in flies co-expressing CTG with CAG expanded repeats. Expression of the two transcripts led to novel pathogenesis with the generation of dcr-2 and ago2-dependent 21-nt triplet repeat-derived siRNAs. These small RNAs targeted the expression of CAG-containing genes, such as Ataxin-2 and TATA binding protein (TBP), which bear long CAG repeats in both fly and man. These findings indicate that the generation of triplet repeat-derived siRNAs may dramatically enhance toxicity in human repeat expansion diseases in which anti-sense transcription occurs.

Figure 1. CTG repeat transcripts cause repeat-length dependent toxicity.

A. DNA constructs for DM1 fly model. A pure, uninterrupted CTG repeat was placed in the 3′UTR of a control protein DsRed. B. Southern blot, probed with DsRed sequence, was used to determine CTG repeat length in transgenic lines. w¹¹¹⁸ was the negative control. C. Northern blot to determine RNA expression levels. 3′UTR sequence was used as the probe. D. Western blot to compare DsRed protein level. Heat shock, with a hs-gal4 driver, was used for expression in B–D. E. External eye and internal retinal structure of flies expressing distinct length CTG repeat transcripts at 1d (top panels) and 14d (bottom panels). Genotypes of flies from left to right: Gmr-gal4 in trans to, UAS-DsRed-(CTG)19, UAS-DsRed-(CTG)130, UAS-DsRed-(CTG)200, UAS-DsRed-(CTG)230, UAS-DsRed-(CTG)270. The effect of UAS-DsRed-(CTG)270 was variable (see also Figure S1); shown here are examples of mild (m) and severe (s) effects. Arrows highlight necrotic patches on external eyes and loss of retinal tissue internally.

Figure 2. Interaction between expanded CAG and CTG repeat transcripts causes biogenesis of small RNAs.

A. External eye (top) and internal retinal sections (bottom). Left, co-expression of transgenes with short repeats shows no deleterious effect. Right, co-expression of expanded (CAG)250 with expanded (CTG)200 repeat transcripts leads to a disrupted eye externally, with severe loss of retinal integrity internally. Genotypes: left, gmr-gal4 in trans to UAS-DsRed-(CTG)19 UAS-DsRed-(CAG)34 and right, gmr-gal4, UAS-DsRed-(CTG)200/+; UAS-DsRed-(CAG)250/+. Age of flies: 1d. B. Northern blot. The expression level of the (CTG)250 transcript is reduced when co-expressed with the (CAG)250 transcript. *: a non-specific band overlapping with Dsred-(CTG)19. C. Small repeat RNAs were generated when expanded CAG and CTG repeat transcripts were co-expressed. Genotype of flies in B and C: hs-gal4 in trans to UAS-DsRed-(CTG)19 UAS-DsRed-(CAG)34, w¹¹¹⁸, UAS-DsRed, UAS-DsRed-(CAG)250, UAS-DsRed-(CTG)250 and UAS-DsRed-(CAG)250 UAS-DsRed-(CTG)250.

Figure 3. Toxicity and small RNA biogenesis of co-expressed CTG and CAG transcripts are dependent on dcr2 and ago2.

A. Loss of dcr2 rescues the toxicity caused by co-expression of (CAG)250 and (CTG)250. With normal dcr2 gene function (wildtype), (CAG)250(CTG)250 caused lethality at the pre-adult pupal stage, with dissected animals showing severely disrupted eyes externally and internally. In the dcr2 null background, these flies were now viable and displayed a dramatically improved retinal structure. Genotypes: gmr-gal4 in trans to UAS-DsRed-(CTG)250 UAS-DsRed-(CAG)250 in normal or homozygous dcr2 null background. Age of flies: 1d. B. Mutation of dcr2 also rescued lethality of flies co-expressing expanded repeat transcripts. Survival of adult flies was scored 50 hr after 30 min heatshock induction of transgene expression with hs-gal4. *: p<0.01 when compared to dcr2 null background. ANOVA and Newman-Keuls post-test. Genotypes: hs-gal4 in trans to UAS-DsRed-(CAG)34 UAS-DsRed-(CTG)19, 2xUAS-DsRed-(CAG)250, 2xUAS-DsRed-(CTG)250, and UAS-DsRed-(CAG)250 UAS-DsRed-(CTG)250 in normal or homozygous dcr2 background. C. Co-expression of (CAG)100 and (CTG)130 in muscle with 24B-gal4 leads to developmental lethality, which is rescued by dcr2 mutation. Genotype of parental flies: 24B-gal4 ∶ UAS-DsRed-(CAG)34 UAS-DsRed-(CTG)19/TM6B, Tb. 24B-gal4 ∶ 2xUAS-DsRed-(CTG)130/TM6B, Tb. 24B-gal4/TM6B, Tb ∶ 2xUAS-DsRed-(CAG)100. 24B-gal4 ∶ 2xUAS-DsRed-(CAG)100 UAS-DsRed-(CTG)130/ TM6B, Tb. dcr2^L811fsX; 24B-gal4/ TM6B, Tb ∶ dcr2^L811fsX; UAS-DsRed-(CAG)100 UAS-DsRed-(CTG)130/TM6B, Tb. *: p<0.05 when compared to flies in wildtype background. ANOVA and Newman-Keuls post-test. D. Homozygous loss ago2 suppressed the toxicity caused by co-expression of (CAG)250 and (CTG)200, with flies showing improved external eye. Flies were raised at 29 °C. Age of flies: 1d. Genotypes: gmr-gal4 in trans to UAS-DsRed-(CAG)250 UAS-DsRed-(CTG)200 in normal or homozygous ago2 null background. E. Loss of dcr2 restored levels of full-length repeat transcripts. Head RNA was subject to Northern blot. 18S rRNA, loading control. F. Biogenesis of triplet repeat-derived small RNAs is dcr2-dependent. Small RNA isolated from fly heads was analyzed by Northern blot. 2S rRNA, loading control. Genotypes E and F: hs-gal4 in trans to UAS-DsRed-(CAG)250 UAS-DsRed-(CTG)250 in wildtype or dcr2 null background. G. Triplet repeat-derived small RNAs were methylated at the 3′ end by Hen1 shown by oxidation and ß-elimination reactions. Small RNA from heads was analyzed by Northern blot and probed with (CAG)5. Note that triplet repeat-derived small RNAs from hen1 null mutants run as a range of faster-migrating species after ß-elimination. 2S rRNA blot served as the control. Genotype: hs-gal4 in trans to UAS-DsRed-(CAG)250 UAS-DsRed-(CTG)270.

Figure 4. Expression of expanded (CAG) and (CTG) repeat transcripts disrupts expression of genes containing short triplet repeat stretches.

A. Biogenesis of the endogenous siRNAs, hp4068B and esiRNA-sl-1, and microRNAs, miR-277 and miR-8, is not affected in flies co-expressing CAG with CTG repeat transcripts. Genotypes: Hs-gal4 in trans to UAS-DsRed-(CAG)34 UAS-DsRed-(CTG)19, w¹¹¹⁸, UAS-DsRed, UAS-DsRed-(CAG)250, UAS-DsRed-(CTG)250 and UAS-DsRed-(CAG)250 UAS-DsRed-(CAG)250. B. Levels of transcripts containing short (CAG) stretches are reduced in flies co-expressing (CAG)250(CTG)250. Two transcripts containing at least five consecutive (CAG) repeats, atx2 and tbp, were chosen for analysis. Retrotransposon 412, ß-tubulin and appl were included as controls. Realtime PCR on fly head RNA, 9 hr after heat shock. Genotypes: Hs-gal4 in trans to UAS-DsRed, UAS-DsRed-(CAG)250, UAS-DsRed-(CTG)250, UAS-DsRed-(CAG)250 UAS-DsRed-(CAG)250. (*: p<0.01, **: p<0.001, compared to DsRed. ANOVA and Bonferroni's post test, n = 4). C. Dicer-2 dependent cleavage of transcripts of tbp and atx2 in flies co-expressing expanded CAG/CTG transcripts as determined by RLM-RACE assay. Genotypes: hs-gal4 in trans to: w¹¹¹⁸, UAS-DsRed-(CAG)34 UAS-DsRed-(CTG)19 and UAS-DsRED-(CAG)250 UAS-DsRED-(CTG)250 in either wild type or dcr2 null background. Nested PCR products were analyzed on agarose gels; bands labeled with asterisk were reproducible among four repeat experiments. They were sequenced and confirmed to be derived from cleaved transcripts of atx2 and tbp. D. Cleavage sites on transcripts of atx2 and tbp mapped by sequence analysis of PCR products of the RLM-RACE assay. Frequencies of cleavage at certain sites are shown in the linear map of atx2 and tbp transcripts. Black boxes represent CAG rich regions. Atx2 has multiple splicing isoforms and isoform B is the form abundantly expressed in fly heads. E. A model for repeat toxicity in CTG diseases that includes possibility of anti-sense CAG transcripts. Sense transcripts containing the CUG repeat RNA expansion exert toxicity through misregulation of RNA binding proteins such as Muscleblind and CUG-BP1, resulting in aberrant alternative splicing , , , , . Antisense transcripts containing CAG repeat expansions could be translated into toxic polyglutamine proteins (as in SCA8 [14]) and they may also be toxic on their own . Our data here suggest that CTG and CAG transcripts may also interact, leading to the generation of ∼21 nt triplet repeat derived siRNAs, which may target other transcripts that contain CAG repeat stretches through the RNA interference pathway.