Human Molecular Genetics 2006 15(Review Issue 2):R162-R169; doi:10.1093/hmg/ddl181
© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
RNA-dominant diseases
Robert J. Osborne and
Charles A. Thornton*
Department of Neurology, University of Rochester Medical Center, Rochester, NY 14642, USA
* To whom correspondence should be addressed at: Department of Neurology, University of Rochester Medical Center, PO Box 673, 601 Elmwood Avenue, Rochester, NY 14642, USA. Tel: +1 5852752542; Fax: +1 5852731255; Email: charles_thornton{at}urmc.rochester.edu
Received July 6, 2006; Accepted July 12, 2006
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ABSTRACT
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Several examples have come to light in which mutations in non-protein-coding
regions give rise to a deleterious gain-of-function by non-coding
RNA. Expression of the toxic RNA is associated with formation
of nuclear inclusions and late-onset degenerative changes in
brain, heart or skeletal muscle. In the best studied example,
myotonic dystrophy, it appears that the main pathogenic effect
of the toxic RNA is to sequester binding proteins and compromise
the regulation of alternative splicing. This review describes
some of the recent advances in understanding the pathophysiology
of RNA-dominant diseases.
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INTRODUCTION
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The fraction of the human genome that is transcribed far exceeds
the fraction that encodes protein (reviewed in
1). Considering
the vast repertoire of non-coding transcripts and the diverse
structural, regulatory and catalytic properties of RNA, mutations
in non-protein-coding regions theoretically have the potential
to produce RNAs that have a deleterious gain-of-function similar
to the effects of some mutant proteins. Presently, the clearest
examples that this can occur involve repetitive sequences in
non-coding RNA (ncRNA). In this review, we will discuss two
prototypical RNA-dominant diseases, myotonic dystrophy and fragile
X tremor ataxia syndrome (FXTAS), with a focus on molecular
pathogenesis. We will also briefly discuss other diseases which
may involve RNA-mediated pathogenesis.
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MYOTONIC DYSTROPHIES
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Myotonic dystrophy [dystrophia myotonica (DM)] is of particular
interest to geneticists because it produces an extremely wide
range of clinical features. A partial listing would include
muscle wasting, cataracts, insulin resistance, testicular atrophy,
slowing of cardiac conduction, cutaneous tumors and effects
on cognition. The classical form of DM, which is now called
DM type 1 (DM1), is caused by an expansion of CTG repeats in
the 3'-untranslated region (UTR) of
DMPK, a gene encoding a
cytosolic protein kinase (
2). More recently, a second form of
DM, DM type 2 (DM2), was shown to result from expansion of a
CCTG repeat in the first intron of
ZNF9 (
3), a gene encoding
a nucleic acid binding protein. Clinical signs in DM1 and DM2
are similar, although there are some distinguishing features.
Both of these mutations are unstable in the germline and in
somatic cells, with a bias towards further expansion. Because
of the instability, the expansions in affected tissues are usually
several thousand repeats in length (
3–
5).
After the DM1 mutation was discovered in 1992, unraveling the disease mechanism was a slow process because the expansion mutation has complex effects on gene expression at the DM1 locus. For example, there is bidirectional transcription through the DMPK 3'-UTR on both WT and mutant alleles and conversion of these transcripts to 21 nt RNAs (6). Patterns of histone methylation and HP1 binding indicate a localized region of heterochromatin centered on the CTG/CAG repeat. Expansion of the CTG repeat is associated with spread of the heterochromatin and reduced expression of the adjacent SIX5 gene (6–8). The repeat expansion also affects the expression of DMPK itself, but at a post-transcriptional level. As discussed below, transcripts containing an expanded CUG repeat (CUGexp) are retained in the nucleus (9,10), which leads to reduced expression of DMPK protein (11). However, reduced expression of DMPK and SIX5 probably has a minor role, at most, in the pathogenesis of DM1 (reviewed in 12). Instead, the main pathogenic effect is a deleterious gain-of-function by the mutant mRNA. The main evidence for an RNA-dominant mechanism is that characteristic clinical and biochemical features of DM1 are reproduced by expression of CUGexp RNA in transgenic mice and flies. This is observed when the CUGexp element is expressed in the natural context of DMPK (13) or when it is inserted in the 3'-UTR of an unrelated transcript (14,15).
Fortunately, the situation in DM2 is more straightforward, and had the order of discovery of DM1 and DM2 mutations been reversed, it is likely that recognition of RNA-dominant disease would have come much sooner. In rare individuals who are homozygous for the DM2 mutation, the clinical findings are similar to those of heterozygous siblings (16) and the cytoplasmic levels of ZNF9 mRNA and protein are normal (17). These findings argue that enormous expansions of the intronic CCTG repeat, which reach sizes of up to 44 kb, have no effect on transcription, processing or export of the ZNF9 mRNA. RNA-mediated disease in DM2, therefore, is not confounded by abnormal expression of ZNF9 or flanking genes. Similar to DM1, the expanded CCUG RNA (CCUGexp) in DM2 accumulates in nuclear foci that are spatially distinct from the site of transcription (Fig. 1); however, unlike DM1, the sequence flanking the expanded repeat in the ZNF9 transcript is not detectable in DM2 foci (9,17). These results indicate that the pathogenic RNA in DM2 is a trapped decay intermediate of an excised intron, composed mainly if not entirely of CCUGexp RNA that is stripped of its flanking intronic sequences. This suggests that CCUGexp RNA is partially resistant to exonucleolytic degradation, presumably due to secondary structure in the transcript, extensive interaction with RNA-binding proteins or both. The implication of these findings for DM pathogenesis is that features common to DM1 and DM2, which include most features of the disease, can be attributed to the CUGexp or CCUGexp elements themselves. An important exception is that very large expansions of the DM1 CTG repeat are associated with a severe, congenital-onset phenotype. This aspect of DM1 is poorly understood and it does not occur in DM2 (18).
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Figure 1. Sequestration of MBNL1 protein in nuclear foci of CUGexp or CCUGexp RNA. Shown are high power views of individual nuclei in transverse sections of DM1 (upper panel) or DM2 (lower panel) skeletal muscle. Nuclear foci are demonstrated by immunofluorescence for Mbnl1 (green, left panels) combined with fluorescence in situ hybridization for repeat expansion RNA (red, center panels). Nuclei are counterstained in blue. Scale bar indicates 5 µm.
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MODEL OF RNA-DOMINANT DISEASE IN DM
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A current model of the disease process in DM is that interaction
of CUG
exp or CCUG
exp RNA with binding proteins leads to abnormal
regulation of alternative splicing for a selected group of pre-mRNAs
(
19,
20). However, the specific proteins mediating these effects
have been a topic of debate. Attention was initially focussed
on CELF proteins, a family of RNA-binding proteins that regulate
several aspects of mRNA biogenesis, including alternative splicing,
transport and stability (reviewed in
21). CUG-BP1, the founding
member of the family, was first identified for binding to (CUG)
8 oligonucleotides
in vitro (
22), but it does not bind efficiently
to CUG repeats that are pathologically expanded (
23) nor does
it colocalize with nuclear foci of CUG
exp in cells (
24,
25).
Muscleblind (MBNL) proteins were first identified for binding
(CUG)
90 in preference to (CUG)
11 in vitro (
20). These proteins
are heavily recruited into nuclear foci in both types of DM
(
26). Muscleblind was initially described as a protein required
for photoreceptor and muscle development in
Drosophila (
27,
28),
and subsequently the mammalian homologs were shown to function
as regulators of alternative splicing (
29). Of the three mammalian
MBNL genes, MBNL1 and MBNL2 are expressed in skeletal muscle,
heart and brain, and MBNL3 is expressed mainly in placenta (
30,
31).
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SPLICEOPATHY IN DM
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In their studies of alternative splicing of cardiac troponin
T (cTnT) pre-mRNA, Cooper and coworkers (
19) noted the presence
of short CUG repeats in a group of intronic splice enhancers,
and they postulated that in DM1 the CUG
exp RNA may compete with
these elements for binding of factors that regulated cTnT splicing.
Studies confirmed that alternative splicing of cTnT was abnormally
regulated in DM1 cardiac cells and that alternative splicing
of cTnT was regulated by CUG-BP1 (
19). However, against the
hypothesis of CUG-BP1 sequestration on CUG
exp RNA (
22), the
perturbation of cTnT splicing in DM1 would predict an excess,
rather than deficiency, of CUG-BP1 activity (
19). This work
led to the recognition of the major biochemical defect so far
identified in DM, misregulation of alternative splicing. Although
many human genetic disorders ultimately can be traced to the
effects of mutations on RNA splicing, in nearly all cases, these
are
cis-acting effects that affect splicing of a single pre-mRNA,
and they lead to aberrantly spliced transcripts that encode
a non-physiological, mutant protein (reviewed in
32). DM1 was
the first example of a human genetic disease resulting from
spliceopathy, i.e. a
trans-effect on the alternative splicing
of many RNAs, which does not result in production of mutant
protein but leads to expression of splice products that are
developmentally inappropriate for a particular tissue.
Table 1 lists the genes and exons known to be affected by spliceopathy in DM1. Although the full range of affected genes is unknown, and is likely to include tens to hundreds of genes, there are several provisional conclusions about the nature of the spliceopathy. First, it does not reduce the general fidelity of RNA processing or weaken the recognition of constitutive exons (14,33). Instead, it selectively affects a group of exons that are normally subject to alternative splicing. Secondly, the spliceopathy targets pre-mRNAs that share a common pattern of developmental regulation. For example, in transgenic mice that express CUGexp in skeletal muscle, the spliceopathy affects exons that normally undergo a splicing switch during the first 3 weeks of postnatal development (33). The end result is that mature muscle fibers in DM1 express a group of splice products that are normally found in fetal or neonatal tissue (19,33,34). Thirdly, as discussed subsequently, the spliceopathy in skeletal muscle selectively targets exons that require MBNL1 for normal regulation.
In addition to their role in splicing regulation, CELF and MBNL
proteins also influence transport, stability and translation
of mRNA (
35–
37). Although the specific implications for
pathogenesis are uncertain, it seems likely that effects of
DM will extend beyond spliceopathy to include a more general
disturbance of post-transcriptional control.
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SPLICEOPATHY LEADS TO A COMPOSITE PHENOTYPE
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Because of the wide range of disease manifestations in DM, it
was difficult to generate a hypothesis about a unitary biochemical
mechanism that underlies so many distinct and seemingly unconnected
clinical features. It now seems likely that DM is a composite
phenotype in which different facets can be parsed to independent
effects of spliceopathy on different transcripts. In skeletal
muscle, for example, the core features of DM are myotonia, insulin
resistance and muscle wasting. Single-gene disorders can produce
each of these features in isolation. In DM, however, they are
combined, and they likely reflect the effect of spliceopathy
on several different pathways. For instance, myotonia is a disturbance
of muscle excitability that causes repetitive action potentials
and delayed relaxation. Recessive myotonia is caused by loss-of-function
mutations in the muscle-specific chloride channel,
CLCN1 (
38).
In DM1, loss of CLCN1 function results instead from production
of
CLCN1 splice products that have premature termination codons
(PTCs) (
39,
40). Notably, similar PTC-containing transcripts
are normally expressed in neonatal mice, but there is a transition
to splice products encoding full-length CLCN1 and an increase
in CLCN1 channel activity, during early postnatal development
(J. Lueck, R. Dirksen, C. Thornton, unpublished data). MBNL1
is required for the postnatal splicing switch in CLCN1. Reversion
to the neonatal splicing pattern in DM, therefore, results in
loss of chloride conductance and electrical instability of the
muscle membrane. In the case of insulin receptor, the predominant
isoform expressed in DM1 is the exon 11 skipped, non-muscle,
splice isoform that has lower signaling capacity (
41). This
effect of spliceopathy may explain the unusual pattern of insulin
resistance observed in DM, in which sensitivity to insulin is
reduced in skeletal muscle but not in liver (
42). However, for
most of the exons listed in Table
1, the functional consequences
of splicing misregulation are unknown. A key question at present
is whether the cumulative effects on splicing of many different
transcripts can explain the muscle wasting and other degenerative
aspects of DM.
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STRUCTURE OF POLY(CUG) RNA
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The X-ray crystal structure of (CUG)
6 is a hairpin (
43), confirming
previous biochemical analyses of poly(CUG) RNA (
23,
44,
45). In
the stem of the hairpin, G-C and C-G base pairs are separated
by a periodic U–U mismatch. Interestingly, the U–U
pairs caused minimal distortion of the A-form helix, and the
stem structure was very similar to a duplex formed by two complementary
RNAs (
43). Under the conditions of crystallization, short (CUG)
6 repeats formed a stem–loop structure, but under more physiological
conditions, poly(CUG) RNA probably does not form a highly stable
hairpin unless the repeat is pathologically expanded (
44,
45).
This can explain why CUG
exp is recognized by double strand (ds)
RNA-binding proteins, whereas non-expanded CUG repeats are not
(
45), and why the RNA-binding domains in CUG-BP1, which are
of the RRM-type that generally recognize single-strand RNA,
bind to short (CUG)
8 oligonucleotides but not to dsRNA in the
CUG
exp stem (
23). Considering that CUG
exp RNA is predicted to
form extended regions of duplex RNA
in vivo (
44,
45), it is curious
that dsRNA-binding proteins do not colocalize with foci of CUG
exp (
26).
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MBNL1 SEQUESTRATION
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Initial skepticism about protein sequestration as a mechanism
for RNA dominance has given way to recognition that the level
of CUG
exp expressed in DM1 is sufficient to markedly alter the
cellular distribution of MBNL1 (Fig.
1). In brain, skeletal
muscle and heart, MBNL1 is recruited into ribonuclear foci to
such an extent that it is markedly depleted elsewhere in the
nucleoplasm (
25,
33,
46). MBNL proteins are the only factors known
to show this effect, out of more than 20 proteins examined (
25,
33,
47).
Swanson and coworkers (
48) have demonstrated that disruption
of the MBNL1 gene in mice reproduces not only a myotonic myopathy
similar to DM1, but also DM1-like cataracts and cardiac disease
(R. Kanadia, S. Swanson, personal communication). Furthermore,
the spliceopathy in skeletal muscle of MBNL1 knockout mice is
very similar to, if not indistinguishable from, that induced
by expression of CUG
exp, and the concordance of splicing defects
in human DM1 or DM2 skeletal muscle with MBNL1 knockout mice
is also remarkably high (
33). Taken together, these findings
indicate that sequestration of MBNL1 protein on repeat expansion
RNA has a pivotal role in both types of DM. Although strong
overexpression of CUG-BP1 is also capable of inducing DM-like
effects on alternative splicing in muscle (
19,
49), it is uncertain
whether CUG-BP1 expression is increased in DM1 tissues to levels
that are high enough to achieve these effects. Notably, the
splicing defect in DM1 myoblasts is not reversed by depletion
of CUG-BP1 (
50), and the spliceopathy in CUG
exp-expressing or
MBNL1-deficient mice is not accompanied by an increase of CUG-BP1
protein (
33).
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FRAGILE X TREMOR ATAXIA SYNDROME
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In the course of their work with families affected by fragile
X syndrome (FXS), Hagerman
et al. (
51) observed late-onset
neurological symptoms in maternal grandfathers of children with
FXS. Although FXS is a developmental syndrome of X-linked mental
retardation, the grandfathers had an entirely different phenotype
of impaired motor control and cognitive dysfunction beginning
after age 50, which is now known as FXTAS (
52). Both disorders
result from expansion of a CGG repeat in the 5'-UTR of
FMR1,
a gene encoding an RNA-binding protein, but the lengths of the
expansion and the effects on
FMR1 expression differ. FXS results
from loss of FMR1 protein, due to transcriptional silencing
of
FMR1 by expansions of >200 CGG repeats, or, less often,
point mutations in
FMR1 that eliminate its function. In contrast,
FXTAS is associated with ‘intermediate’ expansions
of 70–120 repeats (
53), and levels of
FMR1 mRNA are increased
2–8-fold (
54,
55).
FMR1 alleles in the general population
have fewer than 55 CGG repeats. The CGG expansions in FXTAS
are unstable and tend to increase in size with intergenerational
transmission, which explains the occurrence of FXS and FXTAS
in the same family.
A growing body of evidence supports the proposal that FXTAS results from an RNA-dominant disease mechanism (52). A complicating feature, however, is that FXTAS patients have the simultaneous occurrence of increased FMR1 mRNA and decreased FMR1 protein (54,55). The modest decrease of FMR1 protein can be explained by reduced translational efficiency of CGG-expanded FMR1 transcripts (56,57). The increase in FMR1 mRNA is thought to reflect increased transcription (54,55). Several observations indicate that toxicity of CGGexp RNA is more important than partial deficiency of FMR1 protein in causing the neurodegeneration of FXTAS. In FXS, complete loss of FMR1 protein does not produce tremor, ataxia or parkinsonism, clinical signs that are characteristic of FXTAS, nor does it cause age-dependent neurodegeneration (52). Loss of FMR1 in FXS also does not lead to the brain pathology or imaging features that are characteristic of FXTAS, such as nuclear inclusions in neurons and astrocytes (58,59) and abnormal MRI signal in the cerebellar white matter (60). In contrast, neurodegeneration and inclusion formation are provoked by expression of a non-coding CGGexp in transgenic flies (61). Similarly, targeted insertion of 98 CGG repeats in the 5'-UTR of the FMR1 gene in mice causes neuronal nuclear inclusions and late-onset neurobehavioral abnormalities (62,63). Importantly, these knockin mice display elevated levels of FMR1 mRNA, whereas levels of FMR1 protein remain normal (62).
The mechanism whereby these relatively small expansions of CGG repeats in ncRNA cause neurodegeneration in FXTAS is unknown. The nuclear inclusions in FXTAS are labeled by antibodies to ubiquitin and heat shock proteins (58,64), suggesting the presence of aggregated or misfolded proteins. However, there is no other evidence for dysfunction of the ubiquitin–proteasome system. CGG-expanded FMR1 transcripts are a component of the nuclear inclusions (65), and it is possible that they may nucleate the formation of inclusions, but the size and staining properties (58) suggest that FXTAS inclusions are composed mainly of protein. Also, findings of normal FMR1 protein levels in the CGGexp-knockin mice (62), and analogy with the size threshold for nuclear retention of CUGexp transcripts (66), would argue against significant nuclear retention of the mutant FMR1 mRNA. In comparison, nuclear foci in DM1 neurons are smaller, they are not ubiquitinylated and they are not visualized by conventional histological or ultrastructural preparations (25) (C. Thornton, unpublished data). Mass spectrometric analysis of inclusions purified from FXTAS brain tissue showed more than 20 different proteins (64), including MBNL1, but presently there is no evidence for protein sequestration or spliceopathy in FXTAS. Finally, lamin A/C is a component of FXTAS inclusions (64), and transient expression of CGGexp transcripts in neural-derived cells led to disorganization of lamin A/C at the nuclear membrane and altered nuclear morphology (67). Taken together, these results are early indications that mechanisms for RNA-dominant disease in FXTAS and DM may differ.
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SPINOCEREBELLAR ATAXIA TYPE 8 AND HUNTINGTON'S DISEASE-LIKE 2 (HDL2)
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CAG repeat expansions in protein-coding regions of several genes
lead to different types of spinocerebellar ataxia (SCA) or Huntington's
disease (reviewed in
68). The expansion lengths usually range
in size from 42 to 200 repeats, overlapping with the length
of repeat expansions in FXTAS. In common with CUG
exp and CGG
exp,
CAG
exp RNA also has a propensity to form hairpins (
69), activate
the dsRNA-activated protein kinase, PKR (
70), and cause MBNL1
redistribution in cells (
71), raising the possibility that CAG
exp RNA in protein-coding regions may have a role in pathogenesis
(reviewed in
72). However, evidence from transgenic mouse and
fly models does not support a pathogenic role of CAG
exp RNA
except to serve as a template to direct the synthesis of expanded
polyglutamine proteins (
73,
74).
In the late-onset neurodegenerative disease SCA type 8 (SCA8), an expanded CTG repeat is expressed in a ncRNA, making this disorder a prime candidate for RNA-dominant disease (75). A toxic gain-of-function by mutant protein was considered unlikely because there was no apparent reading frame, in either orientation, for expression of protein with a reiterated codon. Moseley et al. (76) derived lines of transgenic mice that have integrated a large genomic fragment containing the entire SCA8 locus. Transgenic mice that express a mutant (CTG)107 SCA8 allele developed late-onset progressive gait impairment and a general lack of motor control, whereas mice expressing a WT (CTG)11 allele did not. The motor impairment, though ultimately lethal, was accompanied by reduced inhibitory synaptic input on neurons in cerebellar cortex, a region of the brain that is closely involved with coordination of movement, but there was no histopathological evidence of cerebellar degeneration. The CUGexp transcript was expressed in this region, but it did not form nuclear foci (consistent with data concerning the size threshold for nuclear retention of CUGexp). An unexpected finding, however, was bidirectional transcription of the repeat tract. Furthermore, despite the apparent absence of a suitable reading frame, the CAG-repeat transcript was translated as a polyglutamine protein, and the mutant protein formed nuclear inclusions in neurons. Subsequent examination of postmortem specimens from patients with SCA8 showed similar polyglutamine-containing inclusions in the cerebellum. Remarkably, the predicted translation product from the CAG strand consisted only of an initiator methionine followed by a polyglutamine tract, punctuated by two arginine residues near the C-terminus. On the basis of these findings, Ranum and coworkers proposed that CUGexp RNA and polyglutamine protein are both involved in the pathogenesis of SCA8 (76).
HDL2 is another neurogenetic disease that blurs the borders between RNA and protein gain-of-function. The clinical features and histopathological lesions in HDL2 are quite similar to conventional Huntington's disease (77). This disorder is caused by an expanded CTG repeat in the junctophilin 3 (JPH3) gene (78). However, depending on alternative splicing of the JPH3 transcript, the expanded CUG repeat could lie in an intron or in the 3'-UTR, or it could encode a polyleucine or polyalanine tract. To add further confusion, antibodies detect the presence of polyglutamine in nuclear inclusions (77), raising the possibility that bidirectional transcription through the expanded CTG/CAG repeat tract, similar to that observed in DM1 and SCA8, may be a general phenomenon.
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CONCLUSION
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Current examples of RNA-dominant disease involve an unusual
mutation mechanism, expansion of an unstable microsatellite
repeat, and a limited number of sequence motifs. Does this mean
that only a few types of mutations and sequences are capable
of mediating
trans-dominant RNA disease? The answer is probably
no. More likely, it is the propensity of CNG or NCNG repeat
sequences to form intramolecular hairpins that underlie both
the genetic instability and the RNA gain-of-function steps of
pathogenesis. In other words, formation of DNA hairpins may
facilitate the genetic instability that generates the mutation
(reviewed in
79), whereas formation of RNA hairpins (
69) may
stabilize a secondary structure that confers pathogenicity or
slows RNA decay. Furthemore, whereas RNA has the capacity to
perform a wide range of structural and catalytic functions,
the most common non-template function of RNA probably is to
interact with binding proteins. In the expansion mutations,
it is perhaps not surprising that protein interaction is the
main function implicated in RNA toxicity. Expansions of a repetitive
element create a sink for RNA-binding proteins by increasing
the mass of target RNA per nucleus and also by increasing the
avidity of RNA–protein interaction due to a high local
concentration of binding sites in each mutant transcript. Indeed,
the MBNL–CUG
exp interaction may actually trigger the formation
of nuclear RNA foci (
50), perhaps in a manner analogous to the
formation of immunoprecipitates by the interaction of antibodies
with polyvalent antigens.
There are indications, however, that mutations in non-repetitive sequence can also give rise to RNA-dominant effects. Of course, the first ncRNAs were the ‘infrastructure’ RNAs, rRNA and tRNAs. Point mutations in tRNAs of the mitochondrial genome are well recognized as a cause of dominantly inherited mitochondrial disease. Also, mutations in single-copy sequences of the nuclear genome are associated with RNA-mediated pathogenesis in mice by mechanisms that may involve competition for limiting binding proteins or nucleation of protein aggregates (80,81). For some genes, the stoichiometric balance of transcript and specific binding proteins may be particularly delicate. For mutations that create or eliminate elements for recognition by RNA-binding proteins or microRNAs, there may be no need for sequence repetition (e.g. repeat expansion) if the mutation occurs in a transcript that is highly abundant. These instances may be hard to recognize because in the absence of a polyvalent RNA-binding site, RNA foci may not occur, the pathogenic transcript may become dispersed throughout the cytoplasm, and the cellular distribution of the sequestered factor may appear normal.
Recent insights into the mechanism of RNA-dominant disease offer hope for effective treatment. Among genetic disorders, the therapeutic opportunities in RNA-mediated disease may be unusually favorable. In DM, for example, the mutation does not directly cause the absence of an essential protein or a dysfunctional protein. The main effect is to induce maldistribution of splicing factors in cells, and the consequence is reversion to a pattern of alternative splicing that is characteristic of immature tissue. Furthermore, in the early to middle stages of disease, many of the symptoms may reflect functional deficits rather than cell degeneration, and these should be reversible. For example, in transgenic flies that express CUGexp, overexpression of human MBNL1 can prevent the muscle degeneration (15), and in transgenic mice that express CUGexp, AAV-mediated overexpression of MBNL1 can reverse spliceopathy and myotonia that have already developed (82). Alternatively, gene therapy approaches for production of antisense or siRNA have been used to accelerate degradation of the mutant DMPK mRNA (83,84).
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ACKNOWLEDGEMENTS
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This work comes from the University of Rochester Senator Paul
D. Wellstone Muscular Dystrophy Cooperative Research Center
(NIH/NS48843) with support from NIH/NIAMS (AR46806, AR48143),
the Muscular Dystrophy Association and the Saunders Family Neuromuscular
Research Fund.
Conflict of Interest statement. No conflict of interest to report.
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ABSTRACT
INTRODUCTION