Human Molecular Genetics, 2010, Vol.

19, Review Issue 1 R77–R82

doi:10.1093/hmg/ddq132
Advance Access published on April 4, 2010

Partners in crime: bidirectional transcription

in unstable microsatellite disease
Ranjan Batra, Konstantinos Charizanis and Maurice S. Swanson ∗

Department of Molecular Genetics and Microbiology and the Genetics Institute, University of Florida,
College of Medicine, Gainesville, FL, USA

Received March 11, 2010; Revised and Accepted April 2, 2010

Nearly two decades have passed since the discovery that the expansion of microsatellite trinucleotide
repeats is responsible for a prominent class of neurological disorders, including Huntington disease and
fragile X syndrome. These hereditary diseases are characterized by genetic anticipation or the intergenera-
tional increase in disease severity accompanied by a decrease in age-of-onset. The revelation that the vari-
able expansion of simple sequence repeats accounted for anticipation spawned a number of pathogenesis
models and a flurry of studies designed to reveal the molecular events affected by these expansions. This
work led to our current understanding that expansions in protein-coding regions result in extended homopo-
lymeric amino acid tracts, often polyglutamine or polyQ, and deleterious protein gain-of-function effects. In
contrast, expansions in noncoding regions cause RNA-mediated toxicity. However, the realization that the
transcriptome is considerably more complex than previously imagined, as well as the emerging regulatory
importance of antisense RNAs, has blurred this distinction. In this review, we summarize evidence for bidir-
ectional transcription of microsatellite disease genes and discuss recent suggestions that some repeat
expansions produce variable levels of both toxic RNAs and proteins that influence cell viability, disease
penetrance and pathological severity.

INTRODUCTION transmission (6 – 11). Alternatively, RNA gain-of-function

Microsatellites are inherently polymorphic and variations in
the lengths of some of these repeats result in more than 30
neurological and neuromuscular diseases (1,2). For disorders
where the expansion mutation occurs in the coding region,
such as Huntington disease (HD) and six spinocerebellar
ataxias (SCA1, 2, 3, 6, 7 and 17), (CAG)n expansions
(CAGexp) result in the synthesis of polyglutamine (polyQ)
tracts in their respective proteins, which accumulate in intra-
cellular, and often ubiquinated, inclusions (3,4). A similar
scenario has been described for coding region GCGexp
mutations, polyalanine (polyA) expression and the presence
of intranuclear filamentous inclusions in oculopharyngeal
muscular dystrophy (5). Toxicity is thought to ensue by
a protein gain-of-function mechanism. For example, polyQ-
containing proteins have been reported to disrupt various
cellular pathways, including transcriptional regulation, the
ubiquitin proteasome system, autophagy and synaptic
∗
To whom correspondence should be addressed at: Department of Molecular Genetics and Microbiology, Cancer Genetics Research Complex,
University of Florida, 2033 Mowry Rd, Gainesville, FL, USA 32610-3610, USA. Tel: +1 3522738076; Fax: +1 3522738284; Email: mswanson@
ufl.edu
models have been proposed for diseases where microsatellite
expansions are positioned in noncoding regions. In the case
of myotonic dystrophy type 1 (DM1), transcription of
CTGexp mutations in the 3′ -untranslated region (3′ -UTR) of
the DMPK gene gives rise to stable RNA hairpin structures
that serve as both scaffolds and triggers which alter the activi-
ties of certain alternative splicing factors, including MBNL1
and CUGBP1, respectively (12,13). Lately, this distinction
between coding and noncoding regions has become blurred
by the discovery of pervasive transcription across the human
genome and the functional relevance of natural antisense
RNAs to gene expression (14– 16).
Antisense regulatory issues
Recent evaluations of genome-wide transcription have demon-
strated that antisense transcripts can be detected for many
mammalian genes, including the wild-type alleles associated

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R78 Human Molecular Genetics, 2010, Vol. 19, Review Issue 1

Table 1. Bidirectional transcription in microsatellite disease genes

Disease Gene(s) Repeat normal/expanded Current pathogenic Sense/antisense

mechanisma readsb

Dentorubral pallidoluysian atrophy (DRPLA) DRPLA (CAG)3 – 36/49 – 88 Protein GOF (polyQ) 741/27
Fragile X syndrome (FRAXA) FMR1 sense (CGG)6 – 52/230 to .2000 Protein LOF (FMRP) 227/1
Fragile X-associated tremor/ataxia syndrome (FXTAS) ASFMR1, FMR4 antisense (CGG)6 – 52/60 – 200 RNA GOF (CGGexp)
Friedreich ataxia (FRDA) FXN (GAA)6 – 32/.200 Protein LOF (frataxin) 102/3
Huntington disease (HD) HTT (CAG)6 – 35/36 – 121 Protein GOF (polyQ) 2554/43
Huntington disease-like 2 (HDL2) JPH3 (CAG)6 – 28/40 – 59 RNA GOF (CUGexp) 31/188
Myotonic dystrophy type 1 (DM1) DMPK (CTG)5 – 37/50 to .3500 RNA GOF (CUGexp) 483/27
Myotonic dystrophy type 2 (DM2) ZNF9 (CCTG)10 – 26/75 to 11 000 RNA GOF (CCUGexp) 2091/0
Oculopharyngeal muscular dystrophy (OPMD) PABPN1 (GCG)10/12 – 17 Protein GOF (polyA) 292/1
Spinal and bulbar muscular atrophy (SBMA) AR (CAG)9 – 36/40 – 55 Protein GOF (polyQ) 103/3
Spinocerebellar ataxia type 1 (SCA1) ATXN1 (CAG)6 – 39/39 – 81 Protein GOF (polyQ) 2541/59
Spinocerebellar ataxia type 2 (SCA2) ATXN2 (CAG)13 – 33/.34 Protein GOF (polyQ) 759/13
Spinocerebellar ataxia type 3 (SCA3) ATXN3 (CAG)13 – 44/.55 Protein GOF (polyQ) 177/20
Spinocerebellar ataxia type 6 (SCA6) CACNA1A (CAG)4 – 18/20 – 29 Protein GOF/LOF (?) 242/48
Spinocerebellar ataxia type 7 (SCA7) ATXN7 (CAG)4 – 35/37 – 306 Protein GOF (polyQ) 1313/34
Spinocerebellar ataxia type 8 (SCA8) ATXN8 sense (CTG),50/74 – 1300 Protein GOF (polyQ)sense 95/4
ATXN8OS antisense RNA GOF (CUGexp)antisense
Spinocerebellar ataxia type 10 (SCA10) ATXN10 (ATTCT)10 – 29/800 – 4500 Unknown 1560/9
Spinocerebellar ataxia type 12 (SCA12) PPP2R2B (CAG)4 – 32/51 – 78 Unknown 264/35
Spinocerebellar ataxia type 17 (SCA17) TBP (CAG)25 – 42/47 – 63 Protein GOF (polyQ) 66/2
Spinocerebellar ataxia type 31 (SCA31) TK2 (TGGAA)0/≥110 RNA GOF (UGGAAexp) 412/4

Twenty microsatellite expansion diseases are listed. For a complete list see Ref. (1).
a
Either protein or RNA gain-of-function (GOF) or protein loss-of-function (LOF) with the toxic (GOF) or affected (LOF) moiety indicated in parentheses (e.g.
polyQ). For SCA8 and FRAXA/FXTAS, the corresponding antisense and sense genes are indicated (superscript).
b
Cumulative number of sequencing reads from ASSAGE study of non-expanded alleles from normal peripheral blood mononuclear cells (PBMC) and four cell
lines (Jurkat, T cell leukemia; HCT116, colorectal cancer; MiaPaCa2, pancreatic cancer; MRC5, normal lung fibroblast) from Ref. (14). Since none of these cells
have a neuronal origin, it is important to note that antisense expression of these genes in the CNS may vary significantly from these values.

with microsatellite expansion diseases [Table 1 (14,17– 20)].
In most cases, the major sense strand is protein coding and
is generally present at a higher copy level than the correspond-
ing antisense transcript(s) which is noncoding and only par-
tially overlaps the sense transcriptional unit, often in the
promoter and 3′ terminal regions. Antisense transcription has
been suggested to regulate gene expression at multiple
levels, including (i) transcriptional interference of the sense
strand due to potential collisions between RNA polymerase
II holoenzyme complexes transcribing opposite strands; (ii)
modulation of gene expression by recruitment of various chro-
matin remodeling complexes which mediate histone modifi-
cations and DNA methylation; (iii) masking splice and
polyadenylation sites of pre-mRNAs transcribed from sense
strands leading to alternative splice, and poly(A), site selec-
tion; (iv) duplex formation between sense and antisense tran-
scripts that induces RNA editing and modulates RNA
interference, stability and translation (21– 23). The expansion
of microsatellite repeats could disrupt these regulatory steps
by altering the relative levels of sense versus antisense tran-
scripts or by affecting transcriptional start or termination sites.
In the following sections, we focus on three neurological
disorders where bidirectional transcription has been implicated
in disease pathogenesis and progression although antisense
RNAs have been reported at other microsatellite expansion
loci (2,13). First, we review progress on spinocerebellar
ataxia type 8 (SCA8) where both a polyQ protein produced
from the sense strand and CUGexp antisense RNAs have
been suggested to play significant roles in pathogenesis. In
contrast to SCA8, fragile X syndrome (FRAXA) and fragile
X-associated tremor/ataxia syndrome (FXTAS) are two dis-
eases caused by variations in a CGG†CCG expansion at the
FMR1/ASFMR1,FMR4 locus. While loss of FMR1 expression
clearly underlies FRAXA, potential roles for the antisense
ASFMR1 and FMR4 transcripts in FXTAS will be reviewed.
Finally, we evaluate the curious molecular etiology of
HD-like 2 (HDL2), where the current evidence indicates that
this disease is associated with a CTG expansion in the JPH3
gene and CUGexp RNA toxicity. Given the clinical similarity
of HDL2 to HD and the overwhelming evidence for a
primary role of polyQ toxicity in the latter disease, it is sur-
prising that HDL2 CAGexp antisense transcripts have not
been detected in the brain.
Prior to beginning our discussion, it is important to note that
any potential pathological impacts of antisense transcription
likely depend on several parameters, including the genomic
organization of the disease locus, spatial and temporal
expression patterns of the affected genes, repeat sequence
and repeat length together with location in the gene(s). In
addition, the expression of neighboring genes may also be
altered which could be particularly relevant to disease loci
where gene density is high, such as the DMPK gene in DM1
(24 –27).
SCA8: a toxic cocktail of polyQ protein and CUGexp RNA?
For the majority of unstable microsatellite disease genes, it is
unknown if antisense RNAs are transcribed through the
expanded repeat regions (14,20). An exception is SCA8, a
dominant SCA with reduced penetrance, where there is evi-
 Human Molecular Genetics, 2010, Vol. 19, Review Issue 1
dence for bidirectional transcription that results in the pro-
duction of toxic noncoding RNAs from the antisense transcrip-
tional unit (ATXN8OS) and a nearly pure polyQ protein, ataxin
8, from ATXN8 (18). Unlike other forms of spinocerebellar
ataxia that contain CAGexp in the coding region of the sense
strand, SCA8 was initially thought to be an exclusively RNA-
mediated disease, similar to DM1, because the only detectable
RNAs were noncoding ATXN8OS transcripts containing a
CUGexp near the 3′ end (28). As mentioned previously,
RNA toxicity in the neuromuscular disease DM1 is caused
by transcription of a noncoding CTGexp in the DMPK gene.
Transcription results in CUGexp RNAs that effectively trap
the MBNL1 splicing factors and alter alternative exon use
during RNA processing to favor the production of fetal iso-
forms (12,13). These CUGexp RNA – MBNL1 complexes
accumulate in nuclear RNA foci that may be the more toxic
entity or simply a protective response (29).
The possibility that SCA8 is also caused by this type of
CUGexp toxicity mechanism is supported by several studies.
Transgenic expression of either the wild type or an expanded
human SCA8 gene in Drosophila causes progressive retinal
neurodegeneration which is enhanced by muscleblind (the fly
homologue of MBNL1) loss-of-function mutations (30).
Moreover, CUGexp foci are detectable in the cerebellar
cortex (Purkinje cells, Bergmann glia, molecular layer inter-
neurons) of both SCA8 patients and transgenic mice that
express a human AXTN8/ATXN8OS gene with a (CTG)116
expansion mutation (SCA8 BAC-exp) (31). These mutant
mice develop a progressive neurological phenotype, striking
gait disturbances and a decrease in cerebellar-cortical inhi-
bition (18,31). While both low copy SCA8 BAC-exp and het-
erozygous Mbnl1 DE3/+ knockout mice are overtly normal,
crosses between these lines result in progeny with enhanced
rotarod deficits. If the toxic RNA-mediated mechanism is
similar to DM1, then the reduction in cerebellar inhibition
could reflect altered splicing and subsequent mis-expression
of a protein involved in cerebellar inhibitory pathways.
Indeed, this appears to be the case since the levels of the
GABA transporter GAT4/GABT4 are elevated in both
human SCA8 and mouse SCA8 BAC-exp brains and this has
been linked to MBNL1-regulated alternative splicing of
GAT4 pre-mRNA. These data are consistent with a model in
which mutant ATXN8OS CUGexp transcripts trap MBNL1
proteins which in turn causes mis-splicing of MBNL target
RNAs that encode proteins important for GABAergic
inhibition.
On the sense strand, ATXN8 transcription was initially missed
in human brain samples perhaps due to CAGexp amplification
problems (18). Fortunately, ATXN8 RNAs are detectable by
strand-specific RT–PCR using primers immediately down-
stream of the expansion in the mouse SCA8 BAC-exp model.
Surprisingly, and unlike other CAGexp expansion diseases, this
RNA is predicted to encode a nearly pure polyQ protein (18).
Cerebellar polyQ inclusions, detectable by monoclonal antibody
(mAb) 1C2 that recognizes expanded polyQ tracts, are observed
in Purkinje cells in human SCA8 and SCA8 BAC-exp mouse
brains and brain stems, indicating that antisense transcription
occurs through these microsatellite repeats. Thus, SCA8 is the
first candidate for a microsatellite disease in which both
RNA-mediated (RNA foci formation and mis-splicing) and
R79
Figure 1. Potential roles for bidirectional transcription in SCA8 pathogenesis.
Transcription of the sense (ATXN8) strand (blue ribbon) results in the synthesis
of the ataxin8 polyQ (PolyQ) protein from the CAGexp transcript (blue line).
While a direct role for ataxin8 has not been demonstrated, polyQ (green)
expression in other microsatellite expansion disorders, such as Huntington
disease, disrupts multiple regulatory pathways (transcription, ubiquitin protea-
somal system, autophagy, synaptic transmission). In contrast, ATXN8OS tran-
scription produces a toxic CUGexp RNA (red line, RNA stem-loop with U –U
mismatches shown as bulged triangles) that reverts RNA splicing patterns to a
fetal pattern due to MBNL1 sequestration and CELF1 hyperphosphorylation.
Also shown is transcriptional interference induced by divergently transcribing
RNA polymerase II complexes (Pol II).
protein-mediated (polyQ intranuclear inclusions) events have
been documented (Fig. 1). Nevertheless, the pathogenic picture
is incomplete. For sense transcription, it is not clear whether
the levels of the ATXN8 transcription and polyQ protein, which
cannot be detected by protein blot analysis, are sufficient to
cause cellular dysfunction although pure polyQ proteins appear
to be highly toxic, so susceptible cell populations may rapidly
be eliminated (32). For antisense transcription, expression of
CUGexp RNAs leads to RNA gain-of-function effects on RNA
splicing and loss of cerebellar GABAergic inhibition but the
basis for the cerebellar atrophy observed in SCA8 remains an
enigma since these neurodegenerative changes are not recapitu-
lated in the SCA8 BAC-exp transgenic model. An additional
complication is that another gene, KLHL1, is also transcribed
in an antisense direction to the ATXN8OS gene and the 5′ ends
of these genes overlap in a head-to-head configuration (33).
Because antisense transcription could theoretically down-
regulate expression of the opposite strand, ATXN8OS CTGexp
transcripts might decrease KLHL1 mRNA levels in SCA8. In
agreement with this possibility, Klhl1 knockout mice show
mild atrophy of the molecular layer in the cerebellum, a charac-
teristic feature of SCA8 patients that is missing in the transgenic
SCA8 BAC-exp model. Confirmation of this antisense
loss-of-function model, which requires that KLHL1 gene
expression is reduced in SCA8 brains, has not been reported.
One of the puzzling features of SCA8 is the low penetrance
associated with this disease and reports that some unaffected
individuals harbor ATXN8/ATXN8OS alleles with as many as
R80 Human Molecular Genetics, 2010, Vol. 19, Review Issue 1
1000 CTG repeats (34 –36). Perhaps bidirectional transcrip-
tion through repeats leads to silencing of the genes at this
locus in some individuals significantly lowering the levels of
toxic RNAs and proteins. However, there are no reports of het-
erochromatin formation at this locus. Are ATXN8 and
ATXN8OS coexpressed in the same cells and, if they are,
what are their relative expression levels? Laser microdissec-
tion of specific cell types and strand-specific RT –PCR could
be used to address this important question. Moreover,
additional cell and animal model systems need to be devel-
oped to determine the relative toxicity thresholds of
rCUGexp RNA and polyQ.
Bidirectional transcription in FXTAS
In DM1, the most severe form of the disease is present at birth
and is caused by very large CTGexp (usually .1000 repeats)
mutations while shorter expansions are associated with the
adult-onset degenerative disease. A similar scenario occurs
for CGGexp mutations in the 5′ -UTR of the FMR1 gene (37).
FMR1 expansions cause two distinct diseases depending
upon repeat length (38). In the normal population, FMR1
alleles contain 6 – 52 CGG repeats and larger expansions
(.200 repeats) cause FMR1 transcriptional silencing and the
neurodevelopmental disorder fragile X syndrome. Although
longer repeats (55 – 200) were originally considered to be pre-
mutations, these intermediate expansions are now known to
cause a late adult-onset disorder, fragile X tremor/ataxia syn-
drome (FXTAS) (39). Clinical manifestations of FXTAS
include cerebellar degeneration with loss of Purkinje cells,
spongiosis of the deep cerebellar white matter and axonal
swelling. The primary neuropathological hallmark is eosino-
philic ubiquitin-positive intranuclear inclusions disseminated
through the brain and spinal column.
Several lines of evidence suggest that RNA-mediated protein
sequestration events similar to those observed in DM1 and
SCA8 may be important for FXTAS pathogenesis. FMR1
mRNA levels increase 2–10-fold in FXTAS and these RNAs
can be detected in the ubiquitin-positive intranuclear inclusions
that have been reported to also contain an array of proteins,
including lamin A, Pura, hnRNPA2/B1, MBNL1, HSP40 and
the 20S proteasome complex (40,41). In support of the idea
that some of these proteins are effectively trapped by
rCGGexp inclusions, overexpression of hnRNP A2/B1 or Pura
suppresses neurodegeneration in a Drosophila (CGG)90-EGFP
transgenic model and Pura knockout mice develop tremors
and seizures by 4 weeks of age, suggesting an essential role
of Pura in brain development and function (42–44).
FXTAS-associated CGGexp repeats are also pathogenic inde-
pendent of gene context. Mouse Fmr1 (CGG)98 and
(CGG)120 knockin models have been reported which repro-
duce several features of FXTAS (45,46). Recently, transgenic
mice have been generated in which expression of a (CGG)90
repeat upstream of either Fmr1 or EGFP is driven by the Pur-
kinje cell-specific L7 promoter (47). In contrast to control
lines expressing the corresponding transgenes minus CGGexp,
both Fmr1- and EGFP-driven (CGG)90 lines develop ubiqui-
nated intranuclear inclusions, Purkinje cell axonal swelling
and increased neuronal loss. Interestingly, hnRNP A2/B1 and
Pura do not colocalize with these CGGexp inclusions.
Antisense transcription may also play a role in FXTAS
pathogenesis (19). A promoter located in the intron 2 of
FMR1 drives expression of the antisense ASFMR1 gene
through the CCG repeat region. ASFMR1 transcript levels
increase in blood cells from FXTAS patients and the
ASFMR1 transcript is spliced, polyadenylated, transported to
cytoplasm and may be translated into a polyproline protein
using an initiation codon upstream of the CUGexp although
there is no evidence that this occurs (19). Multiple alterna-
tively spliced forms of ASFMR1 RNA exist and the CGGexp
mutation alters the splicing pattern. Another noncoding
gene, FMR4, initiates upstream of FMR1 start site (48).
These two genes appear to share a bidirectional promoter
since both transcriptional units are silenced in fragile X, but
upregulated in FXTAS, patient leukocytes. Of course, these
antisense repeats could also fold into RNA hairpins that
sequester rCCG-binding proteins.
HDL2: one frame is not enough
Huntington disease-like 2 (HDL2) is another autosomal domi-
nant and progressive neurodegenerative disease caused by a
CTG†CAG expansion mutation which, like HD, results in
prominent loss of striatal neurons (49– 51). In contrast to
HD, the HDL2 sense transcript, encoded by the Junctophilin-3
(JPH3) gene, contains a CTGexp that is located in a 3′ -UTR or
in a coding region to produce either a polyalanine or polyleu-
cine protein depending on the splicing of JPH3 exon 2A that
contains three alternative 3′ splice sites upstream of the CTG
repeat. HDL2 expansions are relatively short (40 – 59 repeats)
which probably reflects the location of the repeats in both
coding and noncoding regions. While a combination of
protein- and RNA gain-of-function mechanisms could
explain HDL2 pathogenesis, the location of the CTGexp in a
noncoding region suggests an RNA-mediated mechanism.
Indeed, CUGexp RNA foci are detectable in cortical neurons
of HDL2 postmortem brain and these foci colocalize with
MBNL1 (50). Mis-splicing of several MBNL1 targets,
MAPT exon 2 and APP exon 7, in the HDL2 brain has also
been reported.
Because of the similarities between HD and HDL2, protein
gain-of-function mechanisms have also been evaluated. One
obvious candidate would be a polyQ protein expressed from
JPH3 antisense CAGexp transcripts. In support of this possi-
bility, the 1– 2 mm ubiquitin-positive peri-nucleolar inclusions
observed in HDL2 brains are also recognized by the mAb 1C2,
which was originally elicited against the TATA-box-binding
protein (TBP) that contains a polyQ tract (51). An argument
against a role for JPH3 antisense transcription is that it has
not been possible to detect CAGexp transcripts from HDL2
brains using RT – PCR strategies and mAb 1C2 has been
reported to cross-react with polyalanine and polyleucine,
which are encoded on the sense transcript (50,51). However,
another study failed to confirm that 1C2 recognizes either
polyalanine or polyleucine (18) and anti-JPH3 antibodies do
not detect expanded polyalanine or polyleucine proteins in
the HDL2 brain (51). Another confounding factor is that
TBP is detectable in HDL2 intranuclear inclusions (51).
Similar to SCA8, these 1C2-positive inclusions do not gener-
ally colocalize with nuclear RNA foci in the HDL2 brain so
 Human Molecular Genetics, 2010, Vol. 19, Review Issue 1
sense or antisense transcripts may not be expressed at similar
levels in the same cell populations or the combination of toxic
RNA and protein leads to under-representation due to rapid
cell death. Nevertheless, the issue of JPH3 antisense transcrip-
tion in the brain should be re-investigated using RNA-seq
technologies particularly in light of a recent asymmetric
strand-specific analysis of gene expression (ASSAGE)
report. Using normal blood mononuclear cells as well as
several cell lines, this study demonstrated that JPH3 antisense
transcripts exist and indeed are more abundantly expressed
(6-fold more sequencing reads) than sense RNAs [Table 1
(14,20)].
Perspective
While contemporary studies on SCA8, FXTAS and HDL2
have revealed the potential for bidirectional transcription to
play a formidable role in the pathogenesis of these diseases,
many fundamental questions remain. Does repeat length influ-
ence bidirectional expression of the affected locus? Is it poss-
ible that toxic RNAs and proteins are coordinately produced
from the same expansion mutation or do some cell types pre-
ferentially synthesize one over the other? Are cells that
produce both toxic RNAs and proteins particularly susceptible
to expansion-mediated cell death? Answers to these and many
related questions require the development of a new generation
of cell- and animal-based models that are designed to specifi-
cally evaluate the potential for combinatorial protein and RNA
toxicity induced by bidirectional transcription.
Conflict of Interest statement. None declared.
FUNDING
Our research is supported by the NIH (AR046799, NS048843,
NS058901) and the MDA.
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