Abstract
Myotonic dystrophy disorders are caused by expanded CUG repeats in non-coding regions. To reveal mechanisms of CUG repeat pathogenesis we used C. elegans expressing CUG repeats to identify gene inactivations that modulate CUG repeat toxicity. We identified 15 conserved genes that function as suppressors or enhancers of CUG repeat-induced toxicity and modulate formation of nuclear RNA foci by CUG repeats. These genes regulated CUG repeat-induced toxicity through distinct mechanisms including RNA export and RNA clearance, suggesting that CUG repeat toxicity is mediated by multiple pathways. A subset is shared with other degenerative disorders. The nonsense-mediated mRNA decay (NMD) pathway plays a conserved role regulating CUG repeat RNA transcript levels and toxicity, and NMD recognition of toxic RNAs depends on 3′UTR GC nucleotide content. Our studies suggest a broader surveillance role for NMD where variations in this pathway influence multiple degenerative diseases.
Keywords: CUG repeats, myotonic dystrophy, DM1, RNA toxicity, repeat disorders, nonsense-mediated decay, Caenorhabditis elegans
Introduction
Expansions in nucleotide repeat sequences cause many neuromuscular degenerative disorders1 and can occur in noncoding as well as coding regions of genes. Expansions of CTG repeats in the 3′ untranslated region (3′UTR) of the DMPK protein kinase gene causes myotonic dystrophy 1 (DM1), an autosomal dominant degenerative disease2. DM1 CTG expansions range up to >2,000 repeats; normal CTG lengths range from 5–36 repeats. RNA toxicity is the cause of DM1 pathology, where transcripts containing expanded CUG repeats accumulate in the nucleus as discrete RNA foci3. The length of repeat expansion correlates with DM1 disease onset and severity4,5. Expanded CUG repeat RNA transcripts disrupt alternative RNA splicing mediated by muscleblind-like (MBNL)6 and the CUG binding protein 1 (CUG-BP1)7 RNA binding protein families, causing toxicity. However, disruption of these splicing factors, in particular of MBNL1, does not explain the many phenotypes observed in DM disorders. The identification of new factors that function as modifiers of DM1-associated RNA toxicity8–10, and the demonstration that in DM1 mouse models changes are detected in mRNA transcript levels and in transcript splicing not observed in mbnl1 knockout mice11,12, suggest the involvement of additional unknown factors and mechanisms in expanded CUG repeat pathogenesis. Here we find that many of the mammalian toxic features of non-coding expanded CUG repeats can be recapitulated in C. elegans muscle. Analysis of C. elegans muscle function defects caused by expanded CUG repeats, together with cell biological analysis of these aberrant RNAs in wild type and in strains with a library of genes inactivated, identified gene inactivations that modify expanded CUG repeat toxicity and CUG repeat foci accumulation, the hallmark of DM disorders. These modifiers of expanded CUG repeat toxicity include the nonsense-mediated mRNA decay (NMD) pathway, which targets CUG repeat-containing transcripts for degradation. NMD regulation of CUG repeat foci accumulation is a conserved mechanism present in both C. elegans and human cells. Recognition of these CUG repeat-containing transcripts for degradation by NMD is dependent on repeat-sequence composition.
Results
Expanded CUG repeats cause C. elegans muscle defects
We generated a set of C. elegans reporter genes expressing GFP with 3′UTR containing various lengths of CTG repeats in body wall muscle cells, using the myo-3 muscle-specific promoter (Fig. 1A). Reporter constructs without any CUG repeats in the 384-nt 3′UTR from the let-858 gene (0CUG) displayed strong GFP fluorescence at all developmental stages, with a modest decline during adulthood. Analogous constructs with eight CUG repeats showed similar results with mild changes in GFP fluorescence (data not shown). In contrast, the presence of 123 CUG repeats in the 3′UTR (123CUG, a pathogenic repeat length in mammalian myocytes) resulted in a sharp decline in GFP fluorescence as animals developed to adults. Western blotting analyses revealed a sharp decrease in GFP protein levels in 3 day (3d) old adult stage animals of the 123CUG strain (12% compared to protein levels at the L2 larval stage). The 3d adult stage animals of control 0CUG strain showed 50% of the GFP levels in L2) (Fig. 1B). We used the decline in adult stage GFP fluorescence in123CUG transgenic animals for RNAi screens to identify genes that influence toxicity of expanded CUG repeats.
We investigated the function of C. elegans muscle expressing CUG repeats by assessing locomotion phenotypes of these animals. We quantified motor defects by determining the percentage of animals that reached an attractant E. coli food ring (2cm radius) on an agar plate in 90min (Fig 1C and Supplementary Fig. 1A). The 123CUG strains exhibited severe motility deterioration at 6d adulthood, moving about five fold slower than wild type or control transgenic animals carrying 8CUGs or 0CUG constructs, which were similar to wild type. We also observed earlier locomotion defects in synchronized populations of 123CUG animals at the 2d adult stage (Supplementary Fig. 1B) and at the L4 stage (data not shown), whereas strains’ bearing 8CUG or 0CUG repeats showed no motility defects. Thus, expanded CUG repeats cause progressive muscle dysfunction as C. elegans ages, as in other organisms including mammals13–15.
Because nuclear inclusions of expanded CUG repeat RNAs are characteristic of DM, we investigated whether 123CUG RNA transcripts formed nuclear foci in C. elegans muscle cells. We used single molecule RNA fluorescence in situ hybridization (SM-FISH) that has higher sensitivity and specificity than traditional FISH16 (see Supplementary Notes). The repeat-containing region of the expanded RNA transcript is known to interact inappropriately with RNA-binding proteins17; therefore we chose RNA probes complementary to the GFP sequence expected to be accessible in SM-FISH. SM-FISH detected the accumulation of expanded mRNA transcripts in foci as ‘large’, often amorphous, bright fluorescent structures, with 123CUG repeats mRNAs causing the accumulation of 2 to 5 nuclear foci per cell (Fig. 1D). Many individual fluorescence spots, likely corresponding to individual mRNAs, were also observed in the nucleus in the 123CUG strain (Fig. 1D). In contrast, animals expressing 0CUG or 8CUG repeat RNA transcripts lacked multiple bright nuclear foci, and exhibited a predominantly cytoplasmic distribution of RNA ‘single’ transcripts (Fig. 1D).
For a systematic analysis of all SM-FISH data to quantify foci formation and nuclear versus cytoplasmic RNA distribution for 123CUG repeats vs controls, we developed an algorithm that analyzed pixel intensity and cellular distribution in SM-FISH images (Supplementary Fig. 1C–E and Supplementary Notes). We examined the SM-FISH images collected for the nuclear versus cytoplasmic distribution of CUG repeat RNA transcripts as foci or as ‘concentrated single transcripts’ (high RNA density areas) (Supplementary Fig. 1C–E). Consistent with the SM-FISH images (Fig. 1D), the analysis of multiple 123CUG images showed a higher nuclear fluorescence intensity, corresponding to nuclear foci and ‘single’ RNA transcripts (Fig. 1E), clearly distinct from the control 0CUG samples. The quantitative analysis also distinguished the 8CUG from the 0CUG samples, indicating that there are fewer RNA transcripts in the nucleus of 8CUG animals compared to 0CUG strains.
The mammalian splicing protein MBNL1 binds to RNA transcripts containing expanded CUG repeats6, and in myotonic dystrophy is sequestered by expanded CUG foci18. We examined by SM-FISH and by mosaic analysis in vivo (see Supplementary Notes) whether the C. elegans MBNL1 orthologue, MBL-119, bound the 123CUG foci detected in muscle cells. Expression of mbl-1 in a 123CUG background caused a marked increase in foci size relative to the 123CUG strain alone (Fig. 1F, G, Supplementary Fig. 2A–D). Mosaic analysis showed that MBL-1 caused the retention of expanded CUG repeat RNA transcripts in large nuclear foci disrupting transport to the cytoplasm and GFP translation (Supplementary Fig. 2E and Supplementary Notes). These effects were not observed with GFP mRNAs with 0CUG in a strain expressing MBL-1. Thus, as in other organisms, MBL-1 interacts in vivo with expanded CUG transcripts in C. elegans, and MBL-1 association with expanded CUG repeat transcripts decreases mRNA export to the cytoplasm and translation. Down-regulation of mbl-1 by RNAi did not disrupt or enhance 123CUG transcript foci accumulation (Supplementary Fig. 2F). MBL-1 down-regulation, as we show below, can affect the levels of expanded CUG transcript available for translation. These data suggested that additional regulatory factors contribute to expanded CUG foci accumulation and toxicity. Taken together our results support the hypothesis that the RNA aggregated transcripts identified by SM-FISH correspond to the key foci characteristic of DM.
Screen for modifiers of expanded CUG-mediated toxicity
To identify genes that mediate expanded CUG repeat RNA pathogenesis, we used RNAi to reveal gene inactivations that can modify expanded CUG repeat RNA toxicity. A two-step screen was performed, with an initial fluorescent-based RNAi screen, followed by a secondary motility-based screen on hits from the primary screen (Supplementary Fig. 3A). For the fluorescent-based screen, we assayed for gene inactivations that disrupt the late stage down-regulation of GFP fluorescence specific to the 123CUG strain. We screened an RNAi library of 403 clones targeting genes that encode RNA-binding proteins and factors implicated in small RNA pathways20. This type of sub-library was expected to have a high representation of genes involved in expanded CUG repeat toxicity. Of the 403 genes tested, after re-screening in triplicate, 84 gene inactivations were selected that induced an increase in late developmental stage GFP fluorescence specifically in the 123CUG strain without affecting the control 0CUG strain (Figure 2A, Supplementary Fig. 3B, Supplementary Table 1). We tested each of the 84 gene inactivations identified for their ability to modulate the motility defect observed in 123CUG animals (see Online Methods). The 123CUG animals on the control RNAi showed a severe loss in motility, with a median velocity of ≈17μm/sec, compared to the 0CUG strain on the same control RNAi at ≈100μm/sec (Fig. 2B) similar to wild type animals. We identified 14 gene inactivations that significantly (p<0.01 using the two-sample Kolmogorov-Smirnov test) increased or decreased the velocity of 123CUG animals without affecting the control (0CUG) animals (Fig. 2B, Table 1).
Table 1.
Gene inactivation | Gene | Molecular Function | Class | Human ortholog | Motility | Relative Velocity as a percentage of 123CUG on ctrl vector | RNA foci relative to 123CUG alone | |
---|---|---|---|---|---|---|---|---|
|
||||||||
Toxicity Enhancer | K10C9.6 | str-67 | G-protein coupled receptor | Signaling | OR4F5 | improved | 148 | decrease |
C16C2.3 | ocrl-1 | inositol-1,4,5-triphosphate 5-phosphatase | Signaling | OCRL | improved | 184 | mild decrease | |
C06A1.6 | uncharacterized | Cytoskeleton homology | KRTAP5-7 | improved | 187 | decrease | ||
|
||||||||
Toxicity Suppressor | R05A10.1 | uncharacterized | Signaling | ADCY4 | worsened | 78 | increase | |
K09B11.2 | nol-9 | polynucleotide 5′hydroxyl-kinase (nucleolar protein) | RNA Processing | NOL9 | worsened | 74 | increase | |
Y48G8AL.6 | smg-2 | helicase | RNA Processing and Degradation | UPF1 | worsened | 75 | increase | |
Y54E5A.4 | npp-4 | nuclear pore complex protein | RNA Transport | NUPL1 | worsened | 70 | increase | |
R74.5 | asd-1 | alternative splicing family member | RNA Processing | FOX2 | worsened | 82 | mild increase | |
F47A4.2 | dpy-22 | mediator complex subunit transcriptional mediator of RNA | Transcription | MED12L | worsened | 62 | mild increase | |
C08B11.2 | hda-2 | histone deacetylase | Transcription | HDAC1 | worsened | 66 | decrease | |
Y41C4A.14 | mrt-2 | conserved DNA-damage checkpoint protein | DNA Repair and Recombination | RAD1 | worsened | 65 | decrease | |
F29C4.7 | grld-1 | RNA-binding protein (splicing) | RNA Processing | RBM15B | worsened | 66 | mild decrease | |
R06C1.4 | uncharacterized | RNA Processing and Degradation; Translation | CSTF2T | worsened | 66 | mild decrease | ||
D1046.1 | cfim-2 | cleavage and polyadenylation factor | RNA Processing and Degradation | CPFS7 | worsened | 76 | no change | |
F48E8.6 | ribonuclease | RNA processing and Degradation | DIS3L2 | worsened | 75 | no change |
The list of genetic modifiers of expanded CUG toxicity identified can be categorized into the following three major classes: genes involved in transcription, signaling, and RNA processing and degradation (Table 1). Some of the genes identified had been previously implicated in polyglutamine (polyQ) repeat disorders: the hda-2, mrt-2 and smg-2 genes21–23, corresponding to a histone deacetylase, a RAD1 911 complex DNA damage checkpoint protein, and a RNA helicase part of the nonsense-mediated decay pathway, respectively. smg-2 was included in the final list as an additional gene inactivation that affected both the 123CUG repeat transgene and the 0CUG control transgene; smg-2 gene inactivation caused a mild decrease in motility of the 0CUG strain, but caused a much stronger loss of motility for 123CUG repeat strain and was the strongest hit from the fluorescent screen for suppression of the 123CUG-specific decline in GFP fluorescence. The identification in our screen of common regulators of expanded repeat diseases supports the view that repeat-associated disorders, where repeats occur in either coding or non-coding regions, share several protein cofactors.
CUG toxicity modulators affect nuclear foci accumulation
We examined whether any of the 15 gene inactivations that modulated expanded CUG repeat toxicity changed RNA foci accumulation of 123CUG transcripts. One prediction is that gene inactivations that improve the motility of animals expressing 123CUG RNAs would also cause a decrease in foci size or number and similarly, gene inactivations that caused further motility impairment would lead to an increase in foci size or number (Table 1). Of the 15 genes identified, inactivation of ocrl-1/inositol-1,4,5-triphosphate 5-phosphatase, str-67/GPCR chemoreceptor and C06A1.6, led to an improvement of motility in strains expressing 123CUG repeats in muscle (Table 1). Examination of GFP mRNA localization by SM-FISH in 123CUG muscles revealed a significant reduction in the number of nuclear foci when these three genes are inactivated (Fig. 3A, B, Supplementary Fig. 4, 5). The suppression of 123CUG foci was particularly striking for C06A1.6 gene inactivation, where 123CUG foci were now few and small, with SM-FISH signals close to 0CUG control levels (Fig. 3A, Supplementary Fig. 4). However, distribution of expanded RNA ‘single’ transcripts was still observed preferentially in the nucleus versus the cytoplasm for all 3 gene inactivations, suggesting a role for ocrl-1, str-67 and C06A1.6 in foci formation rather than in cellular distribution of RNA. No significant changes in RNA localization, and no foci accumulation, were found in the control 0CUG strain, when these 3 genes were inactivated (Fig. 3A, Supplementary Fig. 4, 5). Together, these data support a model in which ocrl-1, str-67 and C06A1.6 gene activities normally enhance the toxicity of expanded CUG repeats by contributing to 123CUG foci formation, and inactivation of these genes results in decreased toxicity.
For the 12 gene inactivations that further reduced motility in 123CUG animals, six gene inactivations caused an increase in foci size present in the nucleus of 123CUG body wall muscle cells. These genes are npp-4/nuclear pore complex protein, asd-1/alternative splicing regulator, smg-2/nonsense-mediated decay (NMD) factor, nol-9/polynucleotide 5′-hydroxyl-kinase, dpy-22/transcriptional mediator protein and R05A10.1 (Fig. 3, Table 1, Supplementary Fig. 4, 5). For some genes, such as npp-4, a change in RNA localization was observed, with transcript enrichment in the nucleus relative to the cytoplasm (Supplementary Fig. 4, 5). For all these genes, except smg-2, no significant changes in transcript distribution were observed for the control 0CUG mRNA. smg-2 gene inactivation in the control 0CUG led to a slight increase in transcript signal, in both the nucleus and cytoplasm, without affecting nuclear to cytoplasm RNA distribution or leading to foci formation. Inactivation of the other 6 genes either caused a reduction in foci sizes or did not cause a significant change in aggregate size or number (Table 1). The reduction of foci number associated with an increase in toxicity suggested that, in certain conditions, the accumulation of non-aggregated CUG-expanded RNAs can be a major contributor of cellular dysfunction. Similar to what was previously suggested24, these ‘free’ toxic RNAs would have the potential to affect the activity of a wider range of RNA-binding proteins than when in an ‘aggregated’ state.
To further establish that the genes identified were involved in the regulation of expanded CUG-mediated toxicity, we overexpressed npp-4/nuclear pore complex component and asd-1/alternative splicing regulator as mCherry fusion proteins in body wall muscle cells in C. elegans. Down-regulation of npp-4 and asd-1 by RNAi caused an increase in nuclear expanded CUG RNA foci sizes (Table 1). C. elegans expressing these proteins fused to the fluorophore mCherry in either 123CUG or control 0CUG backgrounds, were analyzed by SM-FISH for a change in accumulation of 123CUG RNA in nuclear foci. Overexpressing either of these genes led to a decrease in foci number in a 123CUG background relative to the 123CUG parental strain (Supplementary Fig. 6A). In contrast, overexpression of these proteins in the 0CUG strain had no effect on GFP mRNA transcript distribution. Expression of mCherry alone (Fig. 1F), or a different protein, such as RNP-2, had no effect on 123CUG foci size or number (Supplementary Fig. 6A). Thus some of the genes identified are dosage sensitive components of the CUG repeat toxicity pathway.
Nonsense-mediated decay targets 3′ UTRs with CUG repeats
smg-2 RNAi in 123CUG animals caused an increase in nuclear RNA foci sizes, an increase in muscle cell toxicity with loss of motility and increase in GFP fluorescence signal relative to the control. smg-2 gene inactivation on control 0CUG strains had no effect on nuclear foci, and the mild increase in toxicity detected was not comparable to that observed in the 123CUG strain. In addition, smg-2 acts as a common regulator of expanded repeat-containing disorders by also suppressing protein aggregation caused by expanded CAG repeats in the coding regions of the Huntingtin gene, associated to Huntington’s disease21.
smg-2 encodes an RNA helicase and is a conserved component of the nonsense-mediated mRNA decay (NMD) pathway. The NMD pathway is an evolutionary conserved surveillance mechanism that detects mRNAs containing premature stop codons, preventing toxic expression of truncated proteins25. The identification of smg-2 as a modulator of expanded CUG toxicity suggested that the NMD pathway may recognize and target for degradation RNA transcripts with expanded CUG repeats, even in the 3′ UTRs of non-truncated open reading frames. We analyzed the effects of mutations in NMD components on GFP transcripts bearing 123CUG repeats or control 0CUG in muscle cells using smg-1(r861), smg-2(qd101) and smg-6(r896) mutants. We observed that 123CUG animals in the background of any of the smg mutants showed a strong increase in GFP fluorescence signal relative to the parental strain (Fig. 4A). No such change in fluorescence was observed for the control 0CUG animals (Fig. 4A). Quantitative RT-PCR showed that, mRNA levels of gfp bearing 123CUG repeats were increased by several fold: ≈5.3 fold in smg-1(r861), ≈7.8 fold in smg-2(qd101) and ≈10.1 fold in smg-6(r896) backgrounds, compared to wild type (Fig. 4B). However, no significant change was observed in the levels of gfp mRNA without any CUG repeats in the 3′UTR in the different smg mutant backgrounds compared to the wild type (Fig. 4B). Thus the NMD pathway targets the mRNA transcripts containing the expanded CUG repeats for degradation.
We analyzed by SM-FISH and computational image analysis the gfp RNA transcript accumulation in 123CUG and the control 0CUG strains in the different smg mutant backgrounds. Disruption of NMD pathway in 123CUG animals caused an increase in foci size and number in the nucleus (Fig. 4C, Supplementary Fig. 6B) and in most cells the accumulation of foci-like structures in the cytoplasm as well (Fig. 4C, D, Supplementary Fig. 6B). Conversely, in the smg mutant animals expressing the control 0CUG we observed a uniform distribution of RNA transcripts with a large number present preferentially in the cytoplasm (Fig. 4C, and Supplementary Fig. 6B). Thus the NMD pathway recognizes RNA transcripts containing expanded CUG repeats and disruptions in NMD cause the accumulation of expanded CUG toxic RNA species in the nucleus, leading to cellular dysfunction (Supplementary Fig. 8A).
To examine whether the skewed sequence composition of expanded CUG repeat sequences targets them for the NMD pathway, we explored the influence of GC composition on NMD. 3′UTRs are typically A/U rich (≈65–70% AT-rich), exhibiting a nucleotide composition distinct from coding (≈50–55% AT-rich) or intergenic regions26,27. The let-858 3′ UTR to which the 123 CUG repeat was added is 384 nucleotides and 30% GC. The added CUG repeat elements are rich in G and C nucleotides (≈66%) that may contribute to the recognition by the NMD pathway. We generated expression plasmids in which the 3′UTR (CTG)n sequence was substituted by a non-repeat sequence with either a 66% or 34% GC nucleotide content (Supplementary Fig. 7A, B). The DNA sequences used were cloned from non-C. elegans organisms or from entirely synthetic nucleotide sequences bearing similar GC percentages to avoid a possible recognition of endogenous signal sequences (Supplementary Notes). GFP reporter genes bearing GC-rich 3′ UTR elements from non-C. elegans organisms exhibited weaker GFP fluorescence, or no fluorescence at all in the case of synthetic sequences, compared to those bearing the corresponding AT-rich elements (Fig. 5, Supplementary Fig. 7B). Strains expressing GC-rich elements from a non-C. elegans genome placed in the 3′UTR of the GFP reporter gene showed a significant increase in fluorescence when either smg-1 or smg-2 were inactivated by RNAi, whereas no change in GFP intensity was detected for AT-rich (Fig. 5, Supplementary Fig. 7A, B). Fusion genes engineered with synthetic, random high GC percentage sequences showed a stronger increase in fluorescence in the smg-2 background relative to two regulators of smg-2 phosphorylation smg-1 or smg-6 (Supplementary Fig. 7A, B). These data demonstrate that the results observed for the GC-rich versus AT-rich sequences were not due to a sequence-specific endogenous 3′ UTR identity signal present in the sequence used. These results further establish that the increase in distance between the stop codon and the polyA signal due to the addition of the CUG repeat sequence does not contribute to NMD recognition, since no repression was observed for AT-rich transcripts. These data support a model in which mRNAs, containing CUG repeats in their 3′UTR, are NMD substrates. Furthermore, it reveals that the NMD recognition of CUG-containing mRNA is dependent on nucleotide composition, either due to the presence of a GC-rich sequence in a region usually A/U-rich, or due to the formation of specific secondary structures associated to the presence of these nucleotides. While both the GC-rich 3′ UTR element and the 123CUG repeat element reporter genes are responsive to disruption of the NMD pathway, none of the 15 gene inactivations that strongly disable 123CUG repeat repression in muscle disrupt the repression conferred by GC-rich element. Thus, the detection and localization to foci of 123CUG repeats by these genes is distinct from the detection and degradation of GC rich elements by the NMD system.
To establish whether NMD recognition of expanded CUG repeats is a conserved cellular mechanism, we analyzed the nuclear RNA foci phenotype of NMD gene inactivations in human DM1 patient fibroblast cells expressing 2000 CUG repeats in the DMPK1 mRNA, as well as in control fibroblasts expressing a DMPK1 mRNA with 7 to 35 such CUG repeats. We tested for changes in foci number when the human orthologue of smg-2, UPF1, was inactivated by RNAi. We used SM-FISH for RNA foci detection, with 5 probes complementary to the CUG repeat region and 23 probes complementary to the last three exons of DMPK1 which are not composed of CUG repeats. UPF1 was down-regulated using siRNAs in DM1 and in normal fibroblasts and these cells were analyzed by SM-FISH 24h post siRNA-transfection. For both control fibroblasts and fibroblasts isolated from DM1 patients, UPF1 siRNAs decreased UPF1 protein levels by 35%–40% compared to scrambled siRNAs (Supplementary Fig. 7C, D). There was lower cell recovery after UPF1 knockdown, suggesting that knockdown of NMD components may cause a loss of cell viability, deflating the measured level of UPF1 knockdown. But even with the modest UPF1 knockdown, SM-FISH analysis revealed an increase in the number of nuclear foci in DM1 cells treated with UPF1 siRNAs compared to untreated DM1 cells or DM1 cells treated with mock siRNAs (Fig. 6A). In contrast, normal fibroblast cells bearing just a few CUG repeats in the DMPK gene exhibited no nuclear foci in both untreated or treated with UPF1 siRNAs (Fig. 6A). The number of foci present in the DM1 cells was quantified (see Online Methods) and UPF1 down-regulation caused a significant increase in the percentage of cells containing a higher number of foci (Fig. 6B). Our data supports a conserved role for NMD in the identification of transcripts bearing GC-rich sequences in their 3′UTR. Furthermore, our results support the function of NMD as an important element in the toxicity of expanded CUG repeat transcripts in myotonic dystrophy 1.
Discussion
The gene inactivations that modulate phenotypes of expanded CUG RNA repeats comprise multiple pathways, beyond splicing dysregulation. We demonstrate the involvement of a number of previously unknown genes as modulators of expanded CUG toxicity and expanded CUG repeat foci formation. The demonstration that different gene inactivations, all expanded CUG repeat toxicity suppressors, have opposing effects on foci accumulation (Table 1, Supplementary Fig. 8B), supports the hypothesis that these genes act in distinct pathways. Genes where a direct correlation exists between expanded CUG repeat toxicity and foci accumulation (Supplementary Fig. 8B) include genes where modulation of expanded RNA toxicity can occur by: clearance of CUG-containing RNA transcripts, binding of expanded CUG RNA preventing foci formation or promotion of mRNA transport from the nucleus. Inactivation of these genes causes an increase in the toxic expanded CUG species present in the nucleus. One example is smg-2/NMD helicase inactivation. Another class of suppressor gene inactivations do not correlate with an increase in foci formation (Supplementary Fig. 8B); these proteins may detect cellular damage or bind to expanded CUG repeats.
The identification in our screen of additional splicing factors, such as the asd-1 and grld-1 genes, that when inactivated caused an increase in expanded CUG toxicity was reasonable (Table 1). Unlike MBL1 overexpression (Fig. 1F, Supplementary Fig. 2D), ASD-1 overexpression led to a decrease in expanded CUG nuclear foci accumulation (Supplementary Fig. 6). ASD-1 is an alternative splicing factor and belongs to the Fox-1 splicing family. In vertebrates, MBNL genes are silenced by Fox-1/2 splicing factors28. Two mechanisms for ASD-1 suppression of expanded CUG repeat toxicity emerge: 1) ASD-1 regulates functional MBNL1 levels available by modulating splicing variants; 2) ASD-1 may bind directly or indirectly to expanded CUG repeats and affect toxicity.
Most of the gene inactivations identified make the response to expanded CUG repeats more toxic and promote the accumulation of larger RNA foci in the nuclei, suggesting that these genes constitute a CUG repeat detoxification pathway that blunts their toxicity.
Commonalities have been suggested in degenerative pathways between repeat-based RNA-mediated disorders, and protein-mediated disorders1. RNA toxicity has been implicated in polyQ expansion disorders, and MBNL1 functions as a modulator of polyQ toxicity through its interaction with CAG-containing RNA transcripts29. A subset of the genes identified in our screen as modifiers of expanded CUG toxicity are modulators of polyQ aggregation or toxicity, hda-2, mrt-2 and smg-2 genes21,23. npp-4, although not previously linked to repeat expansion disorders, is part of the nuclear pore complex together with npp-8, and npp-8 had been identified as a modulator of polyQ aggregation30. The identification of pathways that function as common regulators to a broad class of triplet nucleotide pathogenic expansions supports the model of common toxic mechanisms for coding and non-coding triplet repeat disorders.
The NMD pathway is a conserved mechanism of mRNA surveillance that regulates the expression of 5–10% of the human, D. melanogaster and yeast transcriptomes31,32. We find that in addition to its expected target transcripts, NMD modulates the abundance of transcripts containing CUG repeats in their 3′UTR, reducing the accumulation and nuclear foci formation of these toxic RNA species (Supplementary Fig. 8) in both C. elegans and human cells (Fig. 4, 6 and Supplementary Fig. 6B). Sequence composition is key in the recognition by NMD of RNA transcripts containing 3′UTR CUGs; a similar G/C-rich (≈66%) sequence, when present in the 3′UTR, is also recognized by NMD, whereas an A/T-rich sequence is not.
With the identification of NMD genes as modulators of expanded CAG repeat protein-based disorders21, our results suggest broader surveillance roles for the NMD pathway. RNA transcripts containing expanded CAG repeats, also GC-rich, are likely to form secondary structures that may directly or indirectly trigger the NMD pathway33. Additionally, NMD has been mapped to nuclear surveillance leading to nuclear RNA degradation as well as cytoplasmic degradation. Our data showing a striking accumulation of nuclear RNA foci and cytoplasmic RNA foci in NMD mutants suggests a role for NMD not only in the cytoplasm but also in nuclear clearance of expanded RNA repeat transcripts.
Modulation of the NMD pathway may offer a therapeutic approach for myotonic dystrophy patients as well as other repeat-based degenerative disorders. Pharmacological compounds that increase NMD pathway activity may clear CUG-containing RNA toxic species, with the potential to significantly ameliorate DM-related symptoms. A comparable approach, applied to distinct disorders, is currently being tested in clinical trials using compounds that promote NMD read-through34. NMD efficiency varies across tissues35 and between individuals36,37, with significant clinical implications38. These variations in NMD efficiency may have significant implications for trinucleotide repeat disease onset or progression.
Online Methods
Plasmids and Constructs
Mammalian CTG repeat sequences were amplified from plasmids pR26eGFP+100 and pR26eGFP+20039 using Extended High Fidelity from Roche in 6% DMSO and 1M betaine (Sigma). CTG repeats were cloned into the C. elegans pPD118.20 vector bearing the myo-3 body wall muscle-specific promoter, GFP, and the let-858 3′ UTR. The mbl-1 and rnp-2 genes were amplified from C. elegans N2 genomic DNA, and asd-1 and npp-4 from cDNA, using Phusion polymerase (Finnzymes). These genes were cloned into the C. elegans vectors pPD49.26 and pPD30.38 (Addgene) bearing the unc-54 body wall muscle-specific promoter. The GC-rich and AT-rich nucleotide sequences were cloned from the coding region of the 1,4-alpha-glucan branching enzyme gene of Pseudomonas aeruginosa (glgB) and the 3′utr region of the Arabidopsis thaliana myb domain protein 51 gene (myb51), respectively. The synthetic GC-rich and AT rich sequences were synthesized (GenScript). The GC-rich and AT-rich sequences were amplified and cloned into the C. elegans pPD118.20 (Addgene) vector bearing the myo-3 body wall muscle-specific promoter. Detailed cloning information, including primers used, is indicated in Supplementary Notes.
C. elegans Strains
Nematodes were handled using standard methods40 and experiments were performed at 20°C, unless otherwise indicated. The C. elegans N2 Bristol strain was used as wild-type strain. Strains generated for this study are indicated in Supplementary Table 2. Transgenes containing gfp fused to different CTG lengths were integrated by exposing animals to UV irradiation and strains were outcrossed 5 times. Several independent strains were obtained carrying the different GFP transgenes and the different strains generated exhibited similar length-dependent phenotypes, as described in the Results section. The remaining transgenic strains expressed their transgenes as extrachromosomal arrays.
RNA Fluorescence In Situ Hybridization (RNA FISH)
Oligonucleotide probes were designed and SM-FISH was performed as described in Raj et al41. SM-FISH was performed in 3d adult animals, and in human fibroblast cells 24h post siRNA transfection, using probes synthesized by BioSearch Technologies. Two probe sets were used for C. elegans samples, each with thirty-four probes complementary to gfp. One set of probes used was labeled with the dye CAL Fluor Red 590, and the other set with Quasar 670. A distinct probe set was used for the fibroblast cell samples, comprised of twenty-eight probes, labeled with the CAL Fluor Red 590 dye and targeting the CUG repeat region and the 3′ region of the dmpk mammalian gene (see Supplementary Notes). DAPI was used for nuclear staining and SM-FISH images were collected with an Olympus FV-1000 confocal microscope with an Olympus PlanApo 60 3 Oil 1.45 NA objective at 4 zoom, and a 559 nm (mCherry/CALFluor probe), 635nm (Quasar probe) and 405nm (DAPI) diode laser.
SM-FISH computational image analysis
To analyze SM-FISH images, an algorithm was developed to quantify the RNA intensity pixel by pixel in the image. Based on its intensity, each pixel was categorized into one of three RNA populations present in the cell: ‘single’ RNAs (low RNA density), several RNA transcripts (high RNA density), and RNA foci structures (Supplementary Fig. 1E). Pixel intensity corresponding to fluorescence intensity correlates with the number of RNA transcripts present41. DAPI staining was used to identify the nucleus in each cell. Because the accumulation of foci in DM is characterized by its nuclear localization (asymmetric cellular foci distribution), we used the cytoplasmic region in each image to normalize for variations in staining. This approach would allow also the detection of changes in nuclear foci accumulation. This algorithm allowed us to calculate for each nucleus the percent of foci (pixels) and of “high density RNA” (pixels) from the total pixel population. The data was plotted where each ‘dot’ represents a nucleus, with the Y axis representing the percentage of foci pixels and the X axis indicating the percentage of pixels with ‘high density’ RNA. For an example, see Supplementary Notes.
C. elegans Fluorescence Imaging
For in vivo imaging, animals were mounted on a 2% agar pad on a glass slide and immobilized in 1mg/ml levamisole (Sigma). Fluorescence imaging was done on a Zeiss AxioImager.Z1 Microscope.
RNAi screens
RNAi-mediated gene inactivation was by feeding42 in a 12-well plate RNAi bacterial culture 2x concentrated. Animals were synchronized by NaOCl bleaching and overnight hatching in M9. Twenty to thirty L1 larval stage animals (≈24h after synchronization) were aliquoted onto agar plates containing a 48h culture of RNAi bacteria expressing double-stranded RNA, and allowed to develop to adulthood. The drug 5-fluorodexoyuridine was added at the L4-larval stage to a final concentration of 0.1mg/ml, to inhibit progeny production. Each 12-well plate contained the empty L4440 control vector as a negative control. Animals were analyzed either as 3d and as 4d old adults for the GFP fluorescence screen, or at 2d old adults for the locomotion-based toxicity screen. The RNAi clones identified as positives from the screen were verified by sequencing of the insert. Additional information on the RNAi screens is provided in the Supplementary Notes.
C. elegans locomotion assays
The locomotion assay on plates with a ring of OP50 food attractant was performed as previously described43. The percentage of age-synchronized animals that reached the OP50 food in 90min was determined. The second locomotion assay, with analysis of animal velocity, was performed at room temperature and off food. Each experiment performed contained a control corresponding to 123CUG and 0CUG animals fed on control vector (L4440). The locomotion behavior was recorded on a Zeiss Discovery Stereomicroscope using Axiovision software. The center of mass was recorded for each animal on each video frame using object-tracking software in Axiovision. Imaging began 30min after animals were removed from food and recordings were 30sec long. For each assay, 20–45 2d old age-synchronized animals were recorded. The motility data was analyzed using the two-sample Kolmogorov-Smirnov test to compare the distributions of the values in the two data vectors x1 and x2. The null hypothesis is that x1 and x2 are from the same continuous distribution. This test was applied in two different ways 1) using the median velocities of all experiments obtained from all the 123CUG or 0CUG animals fed on control vector and 2) using the experimental internal control corresponding to the median velocity of the 123CUG or 0CUG on control vector. RNAi clones were only considered positive if strongly significant on both analyzes.
qRT-PCR
Total RNA was isolated from synchronized 2d old C. elegans adults using Trizol (Invitrogen) followed by chloroform extraction and isopropanol precipitation. Samples were DNase treated with Turbo DNA-free (Invitrogen) and cDNA was synthesized from 1μg total RNA using Retroscript (Invitrogen). Quantitative RT-PCR assays of mRNA (SYBR Green, Bio-Rad) levels were done according to Bio-Rad recommendations. Three independent biological samples were used for all strains analyzed for gfp levels, and we used rpl-32 levels for normalization across samples. The 2−ΔΔct method was used for comparing relative levels of mRNAs. Primers are listed as Supplementary information.
Protein blot assays
Proteins were extracted from synchronized animals and actin levels were used for normalization across samples. Three independent biological samples were used for all strains analyzed. Harvested C. elegans samples were boiled for 10min in Laemmli buffer, spun and the supernatant collected. Proteins were resolved on 4–12% Bis-Tris SDS polyacrilamide gels, transferred to nitrocellulose membranes and probed with GFP and actin antibodies (Roche, Cat#11814460001; Abcam, ab3280). Protein levels were quantified on a Typhoon phosphoimager using the ImageQuant TL software (GE Healthcare Life Sciences). p values were calculated using Student’s t test.
Mammalian Cell Culture
Human lymphoblast cell lines were obtained from the Coriell Cell repository corresponding to cells from unaffected individuals (GM07492) and fibroblast from DM1-affected individuals (GM03989). Cells were maintained in high glucose EMEM (Lonza) supplemented with 15% fetal bovine serum, 1x antibiotic-antimycotic (Gibco) and 1x non-essential amino acids solution (Sigma), at 37°C, 5% CO2.
siRNA knockdown of UPF1 in human cells
Fibroblast cells were transfected with UPF1 ON-TARGETplus SMARTpool siRNA (Thermo Scientific, cat. No. J-011763-05), or nontargeting siRNA as control (Thermo Scientific, cat. No. D-001810-01) for 24h, using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. The final siRNA concentration used was 100nM. Cells were fixed after transfection for analysis by FISH as described in Raj et al41 (see Supplementary Notes). Knockdown efficiency was monitored by Western Blotting with a UPF1 (kindly provided by Dr. Lykke-Andersen) and GAPDH specific antibodies.
Foci quantification in human fibroblasts
Nuclear foci in DM1-affeceted fibroblasts were quantified using the CellProfiler software (http://www.cellprofiler.org), and specifically a script in CellProfiler, “Speckle Counting’ that allows the identification of individual cells, their nuclei, together with the number of foci present (see Supplementary Notes). The percentage of DM1 cells containing different numbers of nuclear foci was plotted and the p value calculated using two sample t-test function in the Matlab package.
Supplementary Material
Acknowledgments
We thank Prof. M. Mahadevan for providing plasmids bearing the CUG repeats, S. Fischer and J. Kim for the RNAi library used in the screen and D. Kim for the smg-2(qd101) strain, and Lykke-Andersen for the UPF1 mammalian antibody. We are grateful to the J. Kaplan laboratory for the use of the Olympus FV-1000 confocal microscope, and the B. Seed lab for the use of their tissue culture facilities. We are also thankful to S. Djonovic and S. Choi for reagents and technical assistance; J. Bai for reagents, technical assistance and helpful discussions; A. Connery for help with the CellProfiler software and J. Urbach for help editing the manuscript. We thank the Caenorhabditis elegans Genetics Center for strains; the American Heart Association for funding S.M.D.A.G and NIH AG043184 for funding GR.
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