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Title: Triplet Repeat–Derived siRNAs Enhance RNA–Mediated Toxicity in a Drosophila Model for Myotonic Dystrophy}
Article:
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.
Introduction:
Trinucleotide repeat expansions within non-coding regions of RNA cause pathogenesis in a number of human diseases, including myotonic dystrophy type 1( DM1), fragile X-associate tremor and ataxia syndrome( FXTAS), spinocerebellar ataxia type 8( SCA8), and Huntington's disease-like 2( HDL2)[1]-[3]. The causative mutations of DM1, SCA8 and HDL2 are CTG repeat expansions. In DM1, the CTG expansion is located within the 3′ untranslated region( 3′UTR) of the dystrophia myotonica-protein kinase( DMPK) gene[4],[5]. The expanded CUG repeat RNA forms ribonuclear foci, and mislocalizes and misregulates RNA binding proteins such as CUG-BP1 and MBNL1 that influence alternative splicing[6]-[8]. Similarly, expanded CUG RNA also contributes to pathophysiology of SCA8 and HDL2[9],[10]. These findings indicate that CTG-based RNA expansion diseases may have the accumulation of RNA foci, sequestration of MBNL1, and disruption of alternative splicing as common components. It is now recognized, however, that more than 70% of genomic loci show evidence of transcription from both sense and anti-sense strands in the mammalian genome[11], thus many of the trinucleotide repeat disease loci may display bidirectional transcription. Indeed, anti-sense transcripts have been detected for most trinucleotide repeat disease loci, including DM1 and SCA8[12]-[16]. In DM1 cells, the sense and antisense transcripts can cause regional chromatin modification[15]. In SCA8, an antisense CAG transcript can be translated into a polyglutamine-encoding protein[14]. Given that anti-sense transcription occurs widely in the human genome[11],[17],[18], defining the range of potential roles and impact of anti-sense repeat transcripts on trinucleotide repeat diseases may provide novel insight into disease pathogenesis. Drosophila has proven a powerful system to reveal insight into neurological and neurodegenerative disease with relevance to the human situation[19]-[22]. Thus, to gain insight into CUG RNA toxicity in the DM1 disease situation, we established transgenic flies that express pure, uninterrupted CTG repeat expansions in the 3′UTR of a control protein DsRed. These flies recapitulate major features of human CUG RNA expansion diseases. Given the finding that ∼70% of genes show anti-sense transcription, we then tested the effect of co-expressing CTG and CAG disease transcripts. These data revealed dramatically enhanced toxicity upon co-expression of these transcripts; studies indicate this is due to the generation of triplet repeat-derived siRNAs which can target other repeat containing transcripts. These findings suggest that sense and anti-sense expanded repeat transcripts may interact in vivo to generate small RNAs that may dramatically enhance pathology in disease situations.
Discussion:
Like many genes within the mammalian genome[11], the DM1 gene displays bi-directional transcription, generating an anti-sense CAG repeat transcript in addition to the disease-associated CTG transcript[15]. These transcripts have been shown to interact in human cells to generate small RNAs, with one effect being local gene silencing[15]; however additional ways in which this may contribute to pathogenicity in disease is largely unknown. In order to provide new insight into DM1, we generated a Drosophila model by expressing pure, uninterrupted CTG repeat expansions; fly models for various disorders have revealed critical insight into a number of human disease situations( Clark et al., 2006; Fernandez-Funez et al., 2000; Jin et al., 2003; Meulener et al., 2005; Warrick et al., 1998). Interestingly, targeted expression of the long CTG repeats in the fly eye caused a variable toxic effect( see Figure S1). This was also observed in a fly model of SCA8, which carries an uninterrupted CTG repeat expansion[30]. In contrast, fly models generated using interrupted CTG repeats were not reported to show variable phenotype[24],[25]. It is thus possible that phenotypic variability may be a feature of pure repeat sequences, which is in line with the fact that DM1 is among the most variable human disorders. To define potential effects of bi-directional transcription, we then co-expressed expanded CAG repeat transcripts with the DM1 CTG repeats. This resulted in dramatically enhanced toxicity concomitant with the generation of triplet repeat-derived siRNAs. Our results are in striking contrast with previous findings that co-expression of CGG and CCG expansions in flies leads to mitigated toxicity in a ago2-dependent manner[31], suggesting that toxicity derived from interactions between sense and anti-sense repeat transcripts may be specific to CTG/CAG situations. Both CAG and CUG strands can be processed into ∼21 nt small RNAs when coexpressed and small RNAs derived from both strands are methylated in a Hen1-dependent manner( see Figure 2C, Figure 3G, Figure S4B and S4C). These results suggest that both CAG and CUG small RNAs can be loaded into mature, holo-RISCs presumably due to the symmetrical thermodynamic properties of the repeat small RNA duplex[28],[32]-[34]. In our studies, we detected direct cleavage of CAG containing transcripts, and we were unable to detect cleavage of CUG containing transcripts mediated by CAG small RNAs( see Figure 4B–4D). Although underlying reasons for this differential effect remain unclear, CUG and CAG transcripts may have differential expression levels or translation efficiencies, and/or CUG-containing and CAG-containing transcripts may be associated with different RNA binding proteins of various affinities, making CUG-transcripts less accessible to the RISC complex than CAG-containing transcripts[35]. A number of CUG-binding proteins have been defined, such as MBNL1, CUGBP1 and PKR[36]-[38]. Interestingly, in-vitro gel retardation analysis indicated that MBNL1 has a much lower affinity for CAG repeat RNA than CUG repeat RNA[36]. Moreover, expanded CAG transcripts, although co-localizing with MBNL1 in ribonuclear foci similarly to expanded CUG transcripts, do not appear to cause mis-regulation of alternative splicing in cells[39], further highlighting differential properties of these repeats in interacting with RNA binding proteins. The toxicity caused by co-expression of expanded CAG and CTG was associated with deleterious effects on transcripts of other CAG containing genes within the genome; additional mechanisms that contribute to toxicity may also exist. A large number of genes contain CAG stretches in fly and human genomes( Table S1 and Table S2). The enhanced toxicity we observed in flies expressing expanded CAG and CTG may therefore be reflecting an additive effect of knockdown of multiple CAG-containing genes, with each individual gene contributing only partially to the overall outcome. Although further reducing atx2 dosage did not enhance toxicity of co-expressed CTG/CAG expansions( ZY and NB, unpublished observations), the compromised activities of many target genes may be involved and further compromising any single one has minimal impact. The toxic effects seen of the CAG/CTG situation may also be complicated by the later-onset and progressive nature of the toxicity. Further study will clarify the contribution of this mechanism, and key targets among all possible transcripts, to the overall phenotype of the disease. Moreover, the deleterious effects caused by triplet repeat derived small RNAs may be further exacerbated by the wide prevalence of CAG stretches in the human transcriptome( Table S2) and the relative low specificity of RNA interference when siRNAs and/or RNA targets contain simple repeats like CAG[40],[41]. Such interactions may represent a novel activity of endo-siRNAs that characterize disease situations where bi-directional transcription spanning the repeat region occurs( Figure 4E). We confirmed that two of CAG containing genes, atx2 and tbp, are targets of the triplet repeat-derived siRNAs. Interestingly, CAG repeat expansions in ATXN2( the human Ataxin-2 gene) and TBP define two of the CAG-repeat expansion diseases( SCA2 and SCA17, respectively). In such diseases, the expanded polyglutamine domain is thought to confer toxicity[1],[2]; however, increasing evidence suggests that the loss-of-function of gene activity, and not just dominant activities of the protein with an expanded polyglutamine region, occur in disease[42],[43]. Our findings raise the possibility that bi-directional transcription of the repeat region in diseases like DM1 may confer additional components of pathogenicity due to deleterious interactions between the two overlapping repeat-containing transcripts through the generation and activity of triplet repeat-derived siRNAs. Studies indicate that bi-directionally transcribed RNAs, and presumably resultant endogenous double-stranded RNAs, are processed into ∼21–23 nt small RNAs in human cells[44],[45]. This is despite the fact that in most mammalian cells, long exogenous double-stranded RNAs can elicit the interferon response[46],[47]. That response presumably occurs in a threshold-dependent manner; cells may also respond differentially to long exogenous double-stranded RNAs versus endogenous double-stranded RNAs. Thus, these findings suggest that the biogenesis pathway of small RNAs from endogenous double-stranded RNAs is conserved in mammalian cells. Many loci are bi-directionally transcribed throughout the mammalian genome[11],[17],[18], and among these are a number of human trinucleotide disease genes, including SCA8 and DM1[12],[16]. In SCA8, an anti-sense transcript is proposed to encode a polyglutamine protein, which itself may have deleterious actions[14]. In DM1, the two transcripts interact to produce small RNAs that can have local effects on gene silencing[15]. Our findings raise another possibility, that processing of co-expressed transcripts containing CUG/CAG expansions into triplet repeat-derived siRNAs in vivo, may contribute to toxicity with widespread deleterious effects. These effects may include downregulating the expression of other genes containing CAG repeats. Among the genes that could be targets are the polyglutamine disease genes themselves, one of which is TBP. Expansion of the TBP polyglutamine repeat underlies SCA17[48]; intriguingly, general transcriptional compromise has been shown to be a component of repeat expansion diseases[49],[50]. Our studies raise the possibility that perhaps another reason why these diseases share transcriptional compromise may be that they share bi-directional transcript interactions that compromise common elements like TBP. This possibility underscores the idea of shared therapeutic targets and mechanisms in repeat expansion diseases. It has been proposed that siCAG and siCUG may be used for therapy of triplet repeat expansion diseases based on findings in cell culture that these siRNAs seem to specifically target mutant transcripts with expanded repeats[51]. Our data suggest caution in designing such siRNA-based therapy, as in the intact organismal situation, pathogenic activities may be noted. Although previous findings suggest that expanded CUG alone can be processed into small RNAs,[51], our data suggest that both expanded CAG and CTG are required for triplet repeat-derived siRNA generation and toxicity in vivo( see Figure 2C and Figure S5). Thus, co-expressed CAG and CTG expansions may contribute to DM1 pathogenesis through a fundamentally different mechanism from that of CTG expansions alone. Although our studies were conducted in fly models, the findings may apply to human trinucleotide expansion diseases. Targeting these diseases at the transcriptional level may therefore be a promising therapeutic approach that would minimize not only the effects of single expanded repeat transcripts, but deleterious interactions between sense and anti-sense repeat transcript domains.
Over 20 diseases are caused by the expansion of simple repeat sequences in the human genome . Among these are the polyglutamine protein diseases and other primarily RNA–based diseases like myotonic dystrophy ( DM1 ) and spinocerebellar ataxia type 8 ( SCA8 ) . Recently , it has also become clear many genes are transcribed into RNAs from both strands . These include genes causing DM1 and SCA8 . Small interfering RNAs ( siRNAs ) are short RNAs that are cleaved from double-stranded RNAs . These siRNAs can direct degradation of other complementary RNA sequences to reduce their expression . To study consequences of bi-directional transcription of pathogenic repeat sequences , we introduced repeat RNAs containing both CUG and CAG in a Drosophila model of DM1 . We found that these repeat RNAs can be cleaved into repeat-derived siRNAs that are highly toxic to the animal . The mechanisms include targeting RNAs of other genes containing simple repeat sequence for degradation . These findings indicate that repeat-derived siRNAs generated from bidirectional transcription may , in a disease situation , contribute novel pathogenic components .