Download Stochastic and reversible aggregation of mRNA with expanded CUG

1703
Research Article
Stochastic and reversible aggregation of mRNA with
expanded CUG-triplet repeats
Emmanuelle Querido, Franck Gallardo, Mélissa Beaudoin, Catherine Ménard and Pascal Chartrand*
Department of Biochemistry, Université de Montréal, 2900 Edouard-Montpetit, Montréal, QC H3C 3J7, Canada
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 5 January 2011
Journal of Cell Science 124, 1703-1714
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.073270
Summary
Transcripts containing expanded CNG repeats, which are found in several neuromuscular diseases, are not exported from the nucleus
and aggregate as ribonuclear inclusions by an unknown mechanism. Using the MS2–GFP system, which tethers fluorescent proteins
to a specific mRNA, we followed the dynamics of single CUG-repeat transcripts and RNA aggregation in living cells. Single transcripts
with 145 CUG repeats from the dystrophia myotonica-protein kinase (DMPK) gene had reduced diffusion kinetics compared with
transcripts containing only five CUG repeats. Fluorescence recovery after photobleaching (FRAP) experiments showed that CUGrepeat RNAs display a stochastic aggregation behaviour, because individual RNA foci formed at different rates and displayed different
recoveries. Spontaneous clustering of CUG-repeat RNAs was also observed, confirming the stochastic aggregation revealed by FRAP.
The splicing factor Mbnl1 colocalized with individual CUG-repeat transcripts and its aggregation with RNA foci displayed the same
stochastic behaviour as CUG-repeat mRNAs. Moreover, depletion of Mbnl1 by RNAi resulted in decreased aggregation of CUG-repeat
transcripts after FRAP, supporting a direct role for Mbnl1 in CUG-rich RNA foci formation. Our data reveal that nuclear CUG-repeat
RNA aggregates are labile, constantly forming and disaggregating structures, and that the Mbnl1 splicing factor is directly involved
in the aggregation process.
Key words: CUG-repeat RNA, MS2–GFP, Live-cell microscopy, FRAP, Mbnl1
Introduction
Unlike protein aggregates, which have been extensively studied
using biochemical and cytological approaches, little is known about
the mechanism of RNA aggregation. More and more RNA
aggregates or foci have been identified in different pathologies,
such as Myotonic Dystrophy type 1 (DM1) (Davis et al., 1997) and
type 2 (DM2) (Liquori et al., 2001), Fragile X-associated
tremor/ataxia syndrome (FXTAS) (Tassone et al., 2004),
Spinocerebellar ataxia type 8 (SCA8) (Daughters et al., 2009) and
Huntington’s disease-like 2 (HDL2) (Rudnicki et al., 2007). All
these diseases are characterized by microsatellite expansions of
CNG or CCTG repeats in specific genes, leading to the
accumulation of their transcripts as nuclear RNA foci (Ranum and
Cooper, 2006).
The well-studied RNA-mediated disease, Myotonic Dystrophy,
is characterized by progressive muscle weakness, muscle wasting
and myotonia (Harper, 2001; Ranum and Day, 2004). Myotonic
dystrophy type 1 (DM1) is caused by an expansion of CTG
trinucleotide repeats in the 3⬘ untranslated region (UTR) of the
protein kinase gene DMPK (Brook et al., 1992; Buxton et al.,
1992; Fu et al., 1992; Harley et al., 1992; Mahadevan et al., 1992).
This long tract of CUG repeats in the 3⬘UTR has been shown to
cause the retention of this mRNA in the nucleus, where it aggregates
as foci (Davis et al., 1997; Taneja et al., 1995). This is thought to
be a causative event of DM1. The toxic RNA hypothesis posits that
these nuclear foci sequester essential proteins and disrupt their
normal function in the cell (Kuyumcu-Martinez and Cooper, 2006;
Miller et al., 2000; Nykamp and Swanson, 2004; Ranum and Day,
2004). In support of the toxic RNA hypothesis, a mouse model
reproduced key DM1 features by expressing an unrelated mRNA
with 250 CUG repeats (Mankodi et al., 2000).
The muscleblind splicing regulatory factor Mbnl1 has been
directly implicated in the causation of DM1 because it binds CUG
repeats, colocalizes with CUG-repeat RNA foci and regulates the
alternative splicing of genes mis-spliced in DM1 patients (Fardaei
et al., 2002; Ho et al., 2004; Kino et al., 2009; Mankodi et al.,
2001; Osborne et al., 2009). Altered mRNA splicing has been
reported in more than 20 genes in DM1 patients (Kalsotra et al.,
2008; Lin et al., 2006). In skeletal and heart muscle cells, and
neurons of DM1 patients, the recruitment of Mbnl1 into CUGrepeat RNA foci is so extensive that it is depleted from the
nucleoplasm (Cardani et al., 2006; Fardaei et al., 2001; Jiang et al.,
2004; Mankodi et al., 2005; Mankodi et al., 2003; Miller et al.,
2000; Wheeler et al., 2007). In support of the Mbnl1-sequestration
hypothesis, disruption of the Mbnl1 gene in mice reproduces both
the spliceopathy found in DM1 and the characteristic symptoms of
myotonia and myopathy (Kanadia et al., 2003).
The mechanisms that underlie the nuclear retention of mRNA
containing CUG-triplet repeats and its aggregation into nuclear
foci are not currently well understood. CUG-repeat tracts form
hairpins that could affect the processing of the mutant DMPK
mRNA and render it incompetent for export. However, it is thought
that DMPK mutant mRNA is properly spliced, capped and
polyadenylated (Davis et al., 1997; Hamshere et al., 1997). DM1
foci were found to be adjacent to SC35 speckles, suggesting that
DMPK mutant mRNA could be blocked from entry into speckles
and restricted at this step of nuclear export (Holt et al., 2007; Smith
et al., 2007). Mbnl1 has been implicated in the nuclear retention
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and aggregation of CUG-triplet repeat mRNAs, because depletion
of Mbnl1 by RNAi decreases foci accumulation in DM1 myoblast
cells (Dansithong et al., 2005). However, it is unclear whether
Mbnl1 has a direct role in the aggregation of mRNAs with CUGtriplet repeats.
To explore the mechanism of CUG-repeat RNA foci formation,
we directly followed these RNA foci in living myoblast cells using
the MS2–GFP system, which tethers fluorescent proteins to
transcripts containing MS2 stem-loops (Bertrand et al., 1998;
Querido and Chartrand, 2008). With this system, we have
successfully tracked the dynamic motion of CUG-repeat transcripts,
which show reduced diffusion kinetics compared with transcripts
that have five CUG triplets. Our study of Mbnl1 dynamics,
colocalization with CUG-repeat transcripts and depletion by RNAi
supports the hypothesis that Mbnl1 directly participates in the
aggregation of these mRNAs into larger foci. This work
demonstrates that CUG-repeat ribonuclear inclusions are labile,
constantly forming and disaggregating structures, which display
stochastic aggregation and disaggregation behaviour.
Results
Journal of Cell Science
The MS2–GFP system for visualization of transcripts in
living cells
The MS2–GFP system is designed to study the dynamic movement
and localization of RNAs in living cells (Bertrand et al., 1998;
Querido and Chartrand, 2008). A tract of 24 MS2 stem-loops was
shown to recruit an average of 33 molecules of MS2–GFP out of
a maximum possible number of 48 (the MS2 coat protein binds the
MS2 RNA stem-loop as a dimer), and this construction was
sufficient to detect single transcripts in living cells (Fusco et al.,
2003; Shav-Tal et al., 2004). The dynamics and localization of
transcripts containing 24 MS2 repeats have been studied and none
have found a tendency of these mRNAs to aggregate or become
retained in the nucleus. The reporter mRNA used in this study is
described in Fig. 1A, and contains the open reading frame of lacZ,
24 MS2 stem-loop sequences, and the 3⬘UTR of DMPK containing
either five or 145 CUG-triplet repeats (lacZ–MS2–5CUG or lacZ–
MS2–145CUG). Although five CUG repeats are insufficient to
promote RNA aggregation, 145 CUG repeats are known to promote
RNA foci formation in DM1 patients (Botta et al., 2008). We chose
this CUG-repeat expansion size so that the transcripts would not
all be aggregated and we would be able to detect and track single
CUG-rich mRNAs (see below).
Stable murine myoblast C2C12 cell lines were established with
each construct; lacZ–MS2 mRNA expression was detected after
infection of a retroviral vector expressing MS2–GFP. Large MS2–
GFP foci were observed in the nucleus of the lacZ–MS2–145CUG
cell line, whereas no nuclear aggregates were visible in parental
C2C12 Tet-Off cells or the lacZ–MS2–5CUG cell line (Fig. 1B).
Fluorescent in situ hybridization (FISH) with probes against the
lacZ sequence was used to confirm the colocalization of the MS2–
GFP foci with lacZ mRNA in our model system. As seen by FISH
on lacZ–MS2–5CUG cells, the majority of lacZ transcript filled
the cytoplasm (Fig. 1B, middle panel). In lacZ–MS2–145CUG
cells, the lacZ transcript was mostly retained in the nucleus, forming
large aggregates that colocalized perfectly with MS2–GFP foci
(Fig. 1B, bottom panel). It is known that nuclear RNA foci observed
in cells of DM1 patients range in diameter from 0.2 to 2 m (Jiang
et al., 2004). We observed a similar size range in foci formed with
the MS2–GFP system in the lacZ–MS2–145CUG cell line,
suggesting that this transcript behaves similarly to the endogenous
DMPK mutant mRNA in forming ribonuclear inclusions. As
expected, -galactosidase was more abundant in cells expressing
the mostly cytoplasmic lacZ mRNA with 5CUG repeats compared
with those expressing the nuclear retained lacZ mRNA with 145
CUG repeats (supplementary material Fig. S1A). The three cell
lines were also infected with retroviral vector for GFP–Mbnl1 to
confirm that the lacZ mRNA foci colocalized with Mbnl1 in the
lacZ–MS2–145CUG cells (supplementary material Fig. S2, bottom
panel).
Fig. 1. The MS2–GFP system for the visualization
of RNA in living cells. (A)Schematic representation
of the pRevTRE retroviral vectors used to stably
integrate a cDNA containing the lacZ coding
sequence, followed by 24 repetitions of MS2 stemloops fused to the 3⬘UTR of DMPK containing either
5 or 145 uninterrupted CTG repeats. The lacZ
transcript produced does not contain an intron and
uses the polyadenylation signal in the 3⬘LTR. Murine
myoblast C2C12 clonal lines were derived to express
the lacZ–MS2–5CUG or lacZ–MS2–145CUG
transcripts inducible with the Tet-Off system.
(B)Fluorescent in situ hybridization (FISH) with a
lacZ probe on cells expressing MS2–GFP. The cell
lines are identified on the left. Scale bars: 10 m.
Stochastic aggregation of CUG-repeat RNA
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Table 1. Velocity and diffusion coefficient (D) of 5CUG and 145CUG RNA particles
Particles
5CUG single diffusive
5CUG single corralled
5CUG single static
145CUG single diffusive
145CUG single corralled
145CUG single static
145CUG aggregated diffusive
145CUG aggregated corralled
145CUG aggregated static
Frequency (%)
No. of
tracks
No. of
displacements
Min.
Max.
Mean
D
(m2/second)
58
38
4
55
33
12
23
43
34
<200
<200
<200
<200
<200
<200
200–1250
200–1250
200–1250
48
31
3
31
19
7
17
32
25
670
525
60
423
377
114
302
676
541
0.137
0.081
0.038
0.082
0.023
0.022
0.054
0.026
0.018
0.889
0.887
0.310
0.629
0.287
0.195
0.491
0.337
0.168
0.439
0.356
0.120
0.293
0.131
0.097
0.217
0.146
0.067
0.054
0.043
0.002
0.025
0.006
0.001
0.014
0.005
0.001
MS2–GFP particle dynamics reveal that single mRNAs
with 145 CUG triplets diffuse less rapidly than those
containing 5 CUG repeats
Journal of Cell Science
Particle velocity (m/second)
Diameter
(nm)
To analyze the dynamics of the CUG-rich RNA foci in living cells,
the lacZ–MS2–5CUG and lacZ–MS2–145CUG cell lines were
infected with retroviral vector expressing MS2–GFP, and live-cell
microscopy was performed at a rate of three images per second
using a spinning disk confocal microscope, to visualize the
dynamics of MS2–GFP tagged RNA particles (see supplementary
material Movies 1 and 2). Table 1 summarizes the results obtained
from single particle tracking analysis. lacZ–MS2–5CUG transcripts
were observed in the nucleus and their particle velocity was
analyzed after classifying the particles according to their type of
movement. A previous publication revealed that mRNAs follow
three patterns of movement in the nucleus: diffusive, corralled and
static (Shav-Tal et al., 2004). Diffusive and corralled particles both
move by simple diffusion, but corralled particles are constrained
in the distance they reach. Stationary or static particles have
extremely confined movements (representative graph in
supplementary material Fig. S3). As shown in Table 1, the majority
of 5CUG transcripts tracked (58%) showed a diffusive movement
pattern, with a mean velocity of 0.44 m/second. A proportion of
38% of 5CUG particles moved in a corralled fashion, with a mean
velocity of 0.36 m/second, and only 4% of particles were static.
The diffusion coefficient (D) of the diffusive 5CUG particles was
0.054 m2/second and 0.043 m2/second for corralled particles
(Table 1), which is consistent with previously published results of
0.045 m2/second for CFP–MS2–-globin transcripts in the nucleus
(Shav-Tal et al., 2004).
The 5CUG single mRNA particles we tracked had an apparent
diameter of less than 200 nm, and their mean speed and diffusion
coefficients were consistent with the single mRNA particles
described by Shav-Tal and colleagues (Shav-Tal et al., 2004). Thus
we assigned these <200 nm particles as representing single mRNPs.
Although cells expressing the lacZ–MS2–145CUG mRNA had
large nuclear RNA foci, as shown in Fig. 1B, they also contained
single particles of <200 nm diameter with the same fluorescence
intensity as 5CUG single mRNA particles. Therefore, we could
track CUG-repeat mRNA molecules that were not yet incorporated
into large aggregates. These 145CUG transcripts had a mean
velocity of 0.29 m/second for diffusive particles and 0.13
m/second for corralled particles, which was considerably slower
than velocities measured for 5CUG mRNPs. Their diffusion
coefficients were also strongly reduced, decreasing to 0.025
m2/second for diffusive particles and 0.006 m2/second for single
145CUG corralled mRNAs (Table 1).
The movements of large aggregates of lacZ–MS2–145CUG
mRNA, which ranged in size from 200 nm to 1250 nm in diameter,
were also tracked. The average velocity of diffusive aggregates
was 0.22 m/second, whereas corralled aggregates had a mean
velocity of 0.14 m/second. It is interesting that the mean velocity
of 145CUG particles was very similar, both when single particles
or RNA aggregates were measured, indicating that single transcripts
already had a reduced diffusion velocity, even when not aggregated
with other CUG-repeat mRNAs. A third of the nuclear 145CUG
foci were static, because they displayed very little measurable
displacement. These data demonstrate that the speed and the
diffusive movements of single CUG-rich mRNPs are greatly
restricted in the nucleus, compared with that of single 5CUG
transcripts or mRNA particles characterized previously (Shav-Tal
et al., 2004).
FRAP on RNA foci of CUG-triplet repeats uncovers a
stochastic RNA aggregation mechanism
One hypothesis for the role of RNAs with expanded CUG triplets
in the etiology of DM1 is that these RNAs sequester splicing
factors in nuclear RNA foci, affecting the alternative splicing of
several transcripts in DM1 patients (Ranum and Day, 2004).
However, it is not yet clear how these RNA foci are formed and
whether they constitute irreversible aggregates. To determine
whether these RNA foci are stable structures, we performed
fluorescence recovery after photobleaching (FRAP) experiments
on RNA foci tagged with MS2–GFP. The MS2 coat protein and
RNA stem-loop used in this study are variants that interact with
extremely high affinity and have a very low exchange rate, so this
complex has a half-life of several hours (Boireau et al., 2007).
Previous studies have shown that one can measure the dynamics
of the MS2-tagged RNA itself, and not the exchange rate of the
MS2–GFP molecule on the MS2 stem-loop, by using FRAP
(Boireau et al., 2007; Mollet et al., 2008; Shav-Tal et al., 2004).
With this system, we were thus able to perform FRAP analysis on
the CUG-repeat RNA foci to get a representation of the aggregation
of this RNA in living cells.
Three-dimensional time-lapse microscopy was performed on a
spinning disk confocal microscope equipped with a photobleaching
device at an acquisition rate of two image stacks per minute. After
the prebleach series, a 2-m-diameter region was positioned over
the foci and bleached. Recovery of MS2–GFP RNA in the foci was
then quantified for 20 minutes. A FRAP experiment on a lacZ–
MS2–145CUG foci tagged with MS2–GFP is shown in Fig. 2A
(see supplementary material Movie 3). For each photobleached
foci, the fluorescence recovery curve was traced. Fig. 2B shows
the FRAP curves of five individual MS2–GFP foci of various
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Fig. 2. FRAP analysis of MS2–GFP tagged RNA foci. (A)lacZ–MS2–145CUG RNA foci labelled with MS2–GFP were bleached and the recovery of
fluorescence was monitored for 20 minutes. A maximum intensity projection of the image stack at the indicated time points is shown for one experiment. The
bleached focus is indicated by the arrowhead. Scale bar: 10m. (B)Recovery of fluorescence for six individual MS2–GFP-labelled RNA foci after FRAP. The
prebleach average fluorescence of each focus was set at 1, and the recovery is displayed as relative fluorescence intensity. The prebleach diameter of each focus is
indicated in the legend. The 590 nm focus is the one shown in A. (C)Graph of the half-time of fluorescence recovery after FRAP plotted against the prebleach
diameter for all the foci analyzed by FRAP (n34). (D)Graph of the percentage of fluorescence recovery of each focus according to its prebleach diameter.
prebleach diameters. The recovery curves of these different foci
were not averaged as usually done with this type of analysis,
because they were not consistent. Interestingly, each individual
focus behaved differently in terms of the percentage and half-time
of fluorescence recovery (Fig. 2B). Indeed, although some foci
showed almost no recovery, an individual focus was observed to
recover to 350% of its initial value (Fig. 2D). Although there was
no consistent result for half-time of recovery from FRAP analysis
of MS2–GFP-labelled RNA foci, the median half-time of recovery
was 2.7 minutes. Moreover, there was no relationship between the
half-time of recovery and the initial prebleach diameter of the
MS2–GFP foci (Fig. 2C), and this was also true for the percentage
of recovery achieved (Fig. 2D). Altogether, this wide range of
values suggests an aggregation mechanism that is stochastic in
nature. We also performed fluorescence loss in photobleaching
(FLIP) experiments, in which we repeatedly photobleached the
free nucleoplasmic lacZ–MS2–145CUG RNA tagged with MS2–
GFP, and measured the effect on the fluorescence of MS2–GFPlabelled RNA foci (supplementary material Fig. S4). We observed
a decrease in the fluorescence of MS2–GFP-labelled RNA foci,
suggesting an exchange between the free nucleoplasmic and
aggregated lacZ–MS2–145CUG RNA. Moreover, variability in
the rate of fluorescence loss during FLIP confirmed the results
obtained by FRAP, corroborating a stochastic aggregationdisaggregation model for CUG-repeat mRNA foci.
Heterogeneity in the FRAP recovery might occur because some
of the photobleached MS2–GFP foci correspond to lacZ–MS2–
145CUG RNA transcription sites, which would have different
kinetics of recovery compared with that of RNA aggregates. To
determine the number of lacZ–MS2–145CUG transcription sites in
this C2C12 cell line, DNA FISH was performed using a mixed
LNA-DNA probe complementary to the MS2 antisense sequence.
With this assay, only one lacZ–MS2–145CUG transcription site
was observed in this cell line (supplementary material Fig. S5).
Because more than 10 MS2–GFP foci with a diameter larger than
400 nm were observed per nucleus on average, this means that less
than 10% of the foci studied by FRAP correspond to transcription
sites.
Spontaneous aggregation and disaggregation of CUGtriplet-repeat RNA supports a stochastic mechanism
When analyzing time-lapse 3D live cell microscopy films, we
were able to observe the spontaneous formation or disappearance
of lacZ–MS2–145CUG foci labelled with MS2–GFP. One can
observe the formation of such a foci in isosurface view to represent
the 3D image (Fig. 3A and supplementary material Movie 4). Fig.
3B displays a graph of corresponding total fluorescence intensity
of six foci. Because the acquisitions were performed in 3D, we
were able to eliminate the possibility that these foci might simply
come from out-of-focus preformed foci. The half-time of
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Stochastic aggregation of CUG-repeat RNA
Fig. 3. Spontaneous aggregation and disaggregation of MS2–GFP-tagged RNA foci. (A)3D time-lapse microscopy of the appearance and subsequent
disaggregation of a lacZ–MS2–145CUG RNA focus labelled with MS2–GFP. An isosurface view of the 3D image of the MS2–GFP RNA focus was rendered with
the MetaMorph software. (B)Graph of the total fluorescence intensity over time for six spontaneous foci aggregation events. The focus shown in A is Aggr1.
(C)Comparison of the half-times of recovery for all the MS2–GFP-labelled RNA foci analyzed by FRAP (n34) and the half-times of aggregation to first plateau
for all the spontaneous aggregation events observed (n16). The median value for each data set is shown on the graph. (D)3D time-lapse microscopy of an RNA
focus that splits in two. Isosurface view as in A. (E)3D time-lapse microscopy of several RNA foci. Two smaller RNA aggregates indicated by arrows can be
observed to fuse with the large focus. Total fluorescence intensity of the large focus shown at bottom left. Scale bars: 1m.
spontaneous aggregation events was measured (Fig. 3C) and also
showed a large variability, with a median half-time of 2.1 minutes,
consistent with the half-times measured for FRAP recovery of
MS2–GFP foci. Spontaneous disaggregation of foci was also
observed within the time frame of imaging. These disappearances
were not due to acquisition photobleaching, because neighbouring
foci in the same nucleus remained stable throughout the acquisition
time. Time-lapse imaging allowed us to observe that RNA
aggregates can split into smaller foci (Fig. 3D), and also coalesce
and fuse with other foci (Fig. 3E). Aggregates of CUG-repeat RNA
displayed a globular joining and disjoining dynamic similarly to
that of a viscous liquid, rather than a solid aggregate. Altogether,
these results confirmed that CUG-rich RNA foci are dynamic and
follow a stochastic behaviour of aggregation-disaggregation.
Live-cell multicolor imaging shows binding of Mbnl1 to
single CUG-triplet repeat transcripts
The splicing factor Mbnl1 has been shown to have a role in
accumulation of CUG-rich mRNA foci in DM1 myoblasts
(Dansithong et al., 2005). However, it is not clear whether Mbnl1
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Journal of Cell Science 124 (10)
fluorescent proteins were inverted by tagging MS2 with mCherry,
and using a GFP–Mbnl1 retroviral vector (Fig. 4C). Colocalization
analysis showed that GFP–Mbnl1 could be detected in 100% of
MS2–mCherry foci larger than 200 nm in diameter, and that only
7% of single particles smaller than 200 nm in diameter lacked
GFP–Mbnl1 (Fig. 4D). In contrast to the colocalization observed
in foci containing lacZ–MS2–145CUG and MS2–GFP, no
colocalization with mCherry–Mbnl1 was observed in foci
containing lacZ–MS2–5CUG and MS2–GFP (supplementary
material Fig. S6). This live-cell analysis reveals the association of
Mbnl1 with single nucleoplasmic CUG-repeat mRNA and not only
with RNA aggregates.
FRAP analysis reveals a stochastic aggregation of Mbnl1
with foci of CUG-repeat RNAs
To study the role of Mbnl1 in aggregation of CUG-repeat RNA,
we also looked at Mbnl1 dynamics using FRAP. The C2C12 cell
line expressing lacZ–MS2–145CUG RNA was infected with
retroviral vector encoding GFP–Mbnl1. To ensure that we did not
overexpress GFP–Mbnl1, we compared the rate of photobleaching
of GFP–Mbnl1 and MS2–GFP in the nucleoplasm in regions
distinct from CUG-repeat foci. Unlike the free, fast diffusive MS2–
Journal of Cell Science
directly participates in foci formation or if it has a role in stabilizing
RNA aggregates once they have formed. Is Mbnl1 associated with
single CUG-rich mRNAs or does it bind only to RNA aggregates?
To explore this question, we used our capacity to image single
lacZ–MS2–145CUG mRNA particles and investigated whether
these single mRNAs colocalized with Mbnl1 protein in living
cells. Cells expressing lacZ–MS2–145CUG mRNAs were coinfected with retroviral vectors expressing MS2–GFP and mCherry–
Mbnl1, allowing the detection of both Mbnl1 protein and CUG-rich
mRNA in vivo (Fig. 4A). To characterize the colocalization of
these signals, all the MS2–GFP-labelled lacZ–MS2–145CUG
particles were identified and sorted according to particle diameter
in each nucleus, and the mCherry–Mbnl1 images were then overlaid
to detect the presence of a corresponding Mbnl1 particle in the
same location. As shown in Fig. 4B, 100% of MS2–GFP foci that
had a diameter larger than 400 nm were found to contain mCherry–
Mbnl1. Smaller 145CUG foci that were between 200 and 400 nm
in diameter had detectable mCherry–Mbnl1 in 94% of cases,
whereas <200-nm-diameter single MS2–GFP particles had Mbnl1
in 73% of cases. To eliminate the possibility that mCherry–Mbnl1
could not be detected in some of the MS2–GFP particles because
mCherry is not as bright as EGFP (Shaner et al., 2005), the
Fig. 4. Recruitment of Mbnl1 to CUG-triplet-repeat mRNAs in living cells. (A)Live-cell multicolour imaging of MS2–GFP and mCherry–Mbnl1 in cells
expressing the lacZ–MS2–145CUG transcript. The top row images are MS2–GFP (a), mCherry–Mbnl1 (b) and overlay (c) from one nucleus. Scale bar: 10m.
Bottom row images are MS2–GFP (d), mCherry–Mbnl1 (e) and overlay (f) from the boxed region in image c. Scale bar: 1m. Arrowhead indicates a focus of
<200 nm. (B)Percentage of nuclear MS2–GFP foci in which mCherry-tagged Mbnl1 protein could be detected. Intervals were set according to the diameters of the
MS2–GFP foci. 279 MS2–GFP foci were analyzed. (C)Reversal of the fluorophores. Live-cell multicolour imaging of MS2–mCherry and GFP–Mbnl1 in lacZ–
MS2–145CUG cells. The top row images are MS2–mCherry (a), GFP–Mbnl1 (b) and overlay (c) from one nucleus Scale bar: 10m. Bottom row images are
MS2–mCherry (d), GFP–Mbnl1 (e) and overlay (f) images from the boxed region in image c. Scale bar: 1m. Arrowhead indicates a focus of <200 nm in
diameter. (D)Percentage of nuclear MS2–mCherry highlighted transcripts in which GFP–Mbnl1 could be detected. Intervals were set according to the diameters of
the MS2–mCherry foci. 364 MS2–mCherry foci were analyzed.
Stochastic aggregation of CUG-repeat RNA
with MS2–GFP FRAP (Fig. 5C). The percentage of recovery of
GFP–Mbnl1 foci was charted in Fig. 5D, showing that 60% of foci
had 0–50% fluorescence recovery after photobleaching and 40%
had more than 50% recovery. A very similar distribution was
observed with MS2–GFP FRAP. As in the case of MS2–GFPtagged RNA foci, there was no consistent correlation between halftime of recovery and prebleach diameter of the GFP–Mbnl1 foci
(Fig. 5E). Altogether, these results show that association of Mbnl1
with the CUG-rich RNA foci follows a stochastic process, as
observed with lacZ–MS2–145CUG RNA.
Depletion experiments support a direct role for Mbnl1 in
CUG-repeat RNA foci aggregation
To further explore the role of Mbnl1 in CUG-rich RNA foci
formation, we designed a short hairpin RNA interference (shRNA)
retroviral vector targeting Mbnl1. The shMbnl1 vector achieved
Journal of Cell Science
GFP protein, which was quickly photobleached, GFP–Mbnl1
photobleached less rapidly, suggesting that most GFP–Mbnl1 in
the nucleus was associated with proteins or transcripts (either
endogenous or 145CUG RNAs) (supplementary material Fig. S7).
Photobleaching was performed on lacZ–MS2–145CUG RNA foci
tagged with GFP–Mbnl1 with the same 2-m-diameter bleach
region used on the MS2–GFP foci (Fig. 5A). For GFP–Mbnl1
FRAP, 20 image stacks per minute were acquired and fluorescence
recovery was measured for 5 minutes (see supplementary material
Movie 5). Fig. 5B shows relative fluorescence recovery curves for
five GFP–Mbnl1 foci with various prebleach diameters.
Interestingly, similarly to the MS2–GFP curves, they could not be
averaged, as they diverged too widely between individual foci. The
median half-time for GFP–Mbnl1 fluorescence recovery was faster
than the recovery of MS2–GFP foci (1.2 minutes vs 2.7 minutes)
and the half-times of recovery varied less widely than they did
1709
Fig. 5. FRAP analysis of foci containing GFP–Mbnl1. (A)lacZ–MS2–145CUG RNA foci labelled with GFP–Mbnl1 were bleached and the recovery of
fluorescence was monitored for 5 minutes. A maximum intensity projection of the image stack at the indicated time points is shown for one experiment. The
bleached focus is indicated by the arrowhead. Scale bar: 10m. (B)Recovery of fluorescence for five individual GFP–Mbnl1 foci after FRAP. The prebleach
average fluorescence of each focus was set at 1, and the recovery is displayed as relative fluorescence intensity. The prebleach diameter of each focus is indicated in
the legend. The 927 nm focus is the one shown in A. (C)Comparison of the half-time of recovery for all the MS2–GFP (n34) and GFP–Mbnl1 foci (n13)
analyzed by FRAP. The median value for each data set is shown on the graph. (D)Comparison of the percentage of fluorescence recovery for all the MS2–GFP and
GFP–Mbnl1 foci analyzed by FRAP. The proportion of all foci which had 0–50%, or more than 50% recovery after photobleaching are shown. (E)Graph of the
half-time of recovery for each GFP–Mbnl1 foci versus its prebleach diameter.
Journal of Cell Science
1710
Journal of Cell Science 124 (10)
efficient depletion of Mbnl1 protein in cells, whereas the control
shMbnl1mut vector containing two point mutations in the shRNA
did not change Mbnl1 levels (Fig. 6A). To determine the effect of
Mbnl1 depletion on CUG-rich RNA foci formation, a FISH
experiment with a Cy3-labelled (CAG)10 probe was performed on
lacZ–MS2–145CUG cells stably expressing shMbnl1 or control
vector. Using quantitative fluorescence microscopy, the nuclear
surface area covered by RNA foci was measured, which revealed
a decrease of 2.5 times the median value of the surface area
occupied by RNA foci between Mbnl1-depleted cells versus control
cells (Fig. 6B). Measurement of fluorescence intensity of the RNA
foci instead of their surface area also resulted in a two times
decrease in RNA foci accumulation in Mbnl1-depleted cells versus
control cells (data not shown). Compared with control cells,
shMbnl1 cells displayed higher -galactosidase activity without a
change in lacZ–MS2–145CUG mRNA levels, suggesting an
increased cytoplasmic accumulation and translation of this transcript
(supplementary material Fig. S8). To determine whether depletion
of Mbnl1 affects aggregation of foci containing CUG-rich RNA,
FRAP analysis was performed on MS2–GFP-labelled RNA foci in
Mbnl1-depleted cells. As a positive control for the inhibition of
CUG-rich RNA foci formation, cells were treated with the RNA
polymerase II inhibitor a-amanitin. Depletion of another splicing
factor, hnRNP A1, by shRNA was also performed to control for the
specificity of Mbnl1 knockdown in the RNA aggregation process
(Fig. 6A). The recovery of 145CUG RNA foci after FRAP was
reduced in Mbnl1-depleted cells, because the median half-time of
recovery was two times lower, indicating that aggregation time
was shorter than in control cells (Fig. 6C). Moreover, the percentage
of foci recovery after photobleaching was altered towards less
recovery in Mbnl1-depleted cells compared with control cells (Fig.
6D). Cells treated with a-amanitin had less lacZ–MS2–145CUG
mRNA because transcription was inhibited before FRAP analysis,
and they displayed even lower recovery of foci after FRAP than
Mbnl1-depleted cells. In both Mbnl1-knockdown and a-amanitintreated cells, RNA aggregation after FRAP did not last as long as
it did in WT cells, leading to shorter half-times of recovery. A
mutated shRNA against Mbnl1 or depletion of hnRNP A1 had no
effect on half-time of recovery and percentage of foci recovery
(Fig. 6C,D). These results strongly suggest that the nuclear retention
and foci formation of CUG-rich mRNA is not simply a selfaggregation of the RNA, but is based, in part, on a protein
machinery that involves the Mbnl1 protein.
Discussion
CUG-repeat transcripts do not diffuse freely in the
nucleoplasm
Unlike protein aggregates, RNA aggregates have been poorly
studied, despite the fact that they are now implicated in several
pathologies (DM1, DM2, HDL2, SCA8, FXTAS) (Ranum and
Cooper, 2006). Thus, the question of the role of RNA aggregates
in the etiology of these diseases is very relevant. The current
model posits that these aggregates can sequester RNA-binding
proteins and disrupt their normal biological functions (Ranum and
Day, 2004). Because most studies on these RNA foci are based on
fluorescent in situ hybridization in fixed cells, it is still unclear
whether the foci behave as insoluble, static aggregates or whether
they are dynamic structures.
To answer this question, we used the MS2–GFP system to
follow the dynamics and aggregation of mRNAs with expanded
Fig. 6. Mbnl1 depletion reduces formation of nuclear
foci containing CUG-triplet RNA. (A)Western blot
showing Mbnl1 depletion in cells stably expressing a
short hairpin vector against Mbnl1. Cells infected with
the empty MSCV vector, or a mutant shMbnl1 vector,
are shown for comparison. The experiment was
repeated three times and an average depletion of 95% of
endogenous Mbnl1 was measured. Western blot against
hnRNPA1 shows depletion of the protein in cells stably
expressing shRNPA1. a-tubulin (Tub) was detected as a
loading control. (B)Quantification of CUG-triplet
mRNA accumulation in the nucleus of cells expressing
MSCV vector or shMbnl1. The lacZ–MS2–145CUG
RNA was detected by FISH with a (CAG)10 Cy3labelled probe, and the surface area covered by CUGtriplet RNA in each nucleus was quantified and graphed
on a log scale. The median value for each data set is
shown on the graph. The experiment was repeated three
times, and an average decrease of 2.5 times for the
median value in the shMbnl1 cells was obtained.
***P<0.0001, using unpaired t-test. (C)Comparison of
the half-time of recovery for all the MS2–GFP foci
analyzed by FRAP in normal conditions (n34), Mbnl1depleted cells (shMbnl1; n17), a-amanitin-treated
cells (n10), control shRNA (shMbnl1mut; n15) or
hnRNPA1-depleted cells (shRNPA1; n14). **P<0.005;
***P<0.0001, using unpaired t-test. The median value
for each data set is shown on the graph. (D)Comparison
of the proportion of all RNA foci which had 0–50%, or
more than 50% recovery after photobleaching are
shown for all the MS2–GFP foci.
Stochastic aggregation of CUG-repeat RNA
CUG triplets in living cells. Using single-particle tracking, we
observed that even single non-aggregated mRNPs with 145CUG
triplets are considerably slowed in their diffusive movement
compared with mRNPs with 5CUG. Indeed, single mRNPs
containing 145 CUG-triplet repeats had an average diffusion
coefficient of 0.025 m2/second for diffusive single transcripts,
and 0.014 m2/second for diffusive aggregated mRNPs, compared
with 0.054 m2/seconds for diffusive single transcripts containing
only 5CUG. This is probably not due to the increased size of the
mRNA caused by the addition of 140 CUG-triplets (420
nucleotides), because this represents an increase in size of only 6%
for this long lacZ transcript. According to the Stokes–Einstein law
of diffusion, the decreased diffusion coefficient might reflect a
change in the radius or shape of the mRNP (i.e. the CUG triplets
adopt long stem-loop structures) and/or the mass of the mRNP has
increased (we show that Mbnl1 binds single 145CUG transcripts
and not the 5CUG). This reduced diffusion of the CUG-triplet
repeat mRNPs might provide an explanation for the failure of the
majority of these mutant transcripts to reach the nuclear pores and
be exported to the cytoplasm.
Journal of Cell Science
CUG-repeat transcripts aggregate and disaggregate
stochastically
Using FRAP and FLIP, we showed that the MS2–GFP-labelled
CUG-rich mRNAs form dynamic aggregates, which does not
support the hypothesis that these foci are insoluble and static. The
FRAP and FLIP experiments also revealed that fluorescence
recovery and loss of the various RNA foci vary and do not follow
a predictable pattern, suggesting that the aggregation process of
these RNA foci is stochastic. Surprisingly, we observed spontaneous
aggregation-disaggregation events during 4D imaging, which shows
that these RNA foci are unstable. The observation of spontaneous
aggregation, suggesting the presence of a lag phase before
aggregation, and the stochasticity in the aggregation process, support
a nucleation model in which the formation of a stable aggregate
nucleus would be the rate-limiting step. Interestingly, similar kinetics
has been reported for the aggregation of huntingtin and -amyloid
peptide (Colby et al., 2006; Hortchansky et al., 2005).
Mbnl1 is recruited to nuclear CUG-repeat transcripts and
promotes RNA aggregation
We used FRAP to study the dynamics of Mbnl1 recruitment to foci
containing CUG-rich RNA. The recovery that can be observed in
the GFP–Mbnl1 foci might be due to three possible mechanisms:
(1) exchange of free nucleoplasmic GFP–Mbnl1 with the GFP–
Mbnl1 in CUG-rich RNA foci; (2) aggregation of single transcripts
of 145CUG RNA bound by GFP–Mbnl1; and (3) aggregation with
neighbouring foci containing GFP–Mbnl1. The last two
mechanisms are stochastic in nature, as we discussed above.
Because (1) the FLIP experiment shows a direct exchange between
CUG-rich RNA foci and the free nucleoplasmic 145CUG RNA,
(2) these nucleoplasmic 145CUG RNA are associated with Mbnl1,
and (3) similarly to the MS2–GFP-tagged RNA foci, GFP–Mbnl1
FRAP recoveries were stochastic, these results are consistent with
the hypothesis that a large portion of the GFP–Mbnl1 signal
recovers as the protein is carried along and aggregates with mRNAs
into foci in a stochastic process.
A previous study in which FRAP was performed on GFP–
Mbnl1 foci in cells expressing a CUG-triplet repeat transgene and
in DM1 cells reported very different results from ours (Ho et al.,
2005). The authors measured a half-time of recovery of 3.5 seconds
1711
for GFP–Mbnl1 foci, in contrast to the median half-time of recovery
of 1.2 minutes that we observed. In addition, the recovery curves
and measured percentages of recovery reported by Ho and
colleagues were very consistent between foci, whereas we observed
a heterogeneous recovery of GFP–Mbnl1 foci. The differing results
might be explained by the level of expression of GFP–Mbnl1 in
our two systems. Using FRAP on free GFP–Mbnl1 in the
nucleoplasm, Ho and co-workers measured a fast recovery (~3
seconds), suggesting the presence of a considerable amount of free
diffusive GFP–Mbnl1. In our study, we expressed low levels of
GFP–Mbnl1 compared with endogenous Mbnl1 in the C2C12
myoblasts (supplementary material Fig. S7). We also used FLIP to
measure the rate of GFP–Mbnl1 photobleaching in the nucleoplasm
and observed little free diffusing GFP–Mbnl1 (supplementary
material Fig. S7), suggesting that most of it was associated with
low-diffusing mRNPs or proteins. Therefore, the data from Ho and
colleagues might reflect the fast exchange between unbound GFP–
Mbnl1 in the nucleoplasm and GFP–Mbnl1 bound to RNA
aggregates. Our FRAP data are not consistent with that model, but
better fit with the hypothesis that recovery of GFP–Mbnl1 signal
occurs as the protein is carried with newly aggregating CUG-rich
mRNAs. However, the faster half-times of recovery measured with
GFP–Mbnl1 compared with MS2–GFP (median of 1.2 minutes
versus 2.7 minutes, respectively) suggest that some exchange of
free GFP–Mbnl1 also participates in the recovery.
The depletion of Mbnl1 had previously been shown to affect the
accumulation of CUG-rich RNA foci in the nucleus (Dansithong
et al., 2005). However, this study looked at RNA foci accumulation
by FISH and did not assess the role of Mbnl1 in the RNA
aggregation process. We observed that the depletion of Mbnl1
decreased by 2.5 times the accumulation of CUG-repeat RNA foci,
as measured by the quantification of nuclear lacZ–MS2–145CUG
RNA foci by FISH. The observations that Mbnl1 was already
associated with single 145CUG transcripts in the nucleus and that
the depletion of Mbnl1 reduced the recovery of CUG-rich RNA
foci after FRAP suggest a direct role of Mbnl1 in RNA aggregation.
Mbnl1 oligomerization on CUG stems might directly cause RNA
aggregation (Yuan et al., 2007). However, this role is not essential
because the depletion of Mbnl1 causes only a partial defect in
CUG-rich RNA foci formation. Therefore, other proteins might
also be involved in this process. For instance, the Mbnl1 paralogues
Mbnl2 and Mbnl3 are expressed in C2C12 myoblasts (Holt et al.,
2009), and they might, in part, be complementing the function of
Mbnl1 in RNA aggregation (Fardaei et al., 2002; Miller et al.,
2000). Other RNA-binding proteins, such as hnRNP H, have been
shown to be recruited to DM1 foci, and might also contribute to
the aggregation of CUG-rich RNA (Kim et al., 2005; Paul et al.,
2006).
Implications for the treatment of DM1 and other RNAdominant diseases
Our data support a model in which Mbnl1 is directly involved in
the aggregation of CUG-rich mRNA transcripts into large nuclear
foci. In DM2, HDL2, SCA8 and FXTAS RNA gain-of-function
pathologies, Mbnl1 has been found to colocalize with the nuclear
RNA foci (Daughters et al., 2009; Iwahashi et al., 2006; Mankodi
et al., 2001; Tassone et al., 2004). In SCA3, Mbnl1 has been shown
to increase CAG-repeat RNA toxicity in a Drosophila model for
the disease (Li et al., 2008). It will be interesting to investigate
whether Mbnl1 also has a direct role in RNA aggregation in these
diseases, similarly to its role in DM1.
1712
Journal of Cell Science 124 (10)
This work demonstrates that nuclear aggregates of CUG-repeat
RNA are labile, constantly forming and disaggregating structures.
This strongly suggests that it will soon be possible to inhibit or
reverse (CUG)n RNA foci formation in patients. Indeed, recent
studies used (CAG)25 oligos in mouse models of myotonic
dystrophy to cause the disaggregation of RNA foci and achieved
reversal of symptoms (Mulders et al., 2009; Wheeler et al., 2009).
A thorough understanding of the aggregation mechanism of toxic
RNA should help achieve effective therapies for DM1 and other
RNA gain-of-function diseases.
Materials and Methods
Journal of Cell Science
Retroviral vectors
The pRevTRE-lacZ–MS2–145CUG retroviral vector was constructed by subcloning
a fragment of the plasmid pR26EGFP+200 (Amack and Mahadevan, 2001) containing
EGFP and the DMPK 3⬘UTR into the pRev TRE plasmid (Clontech Laboratories,
Mountain View, CA). The EGFP sequence was replaced with the coding sequence
of lacZ followed by 24 MS2 stem-loops from the RSV-Z–MS2–24 plasmid (Fusco
et al., 2003). The MS2–GFP fusion protein without an NLS was sub-cloned into the
pBabe puro retroviral vector (Morgenstern and Land, 1990). The MS2–mCherry
fusion protein was constructed by cloning the mCherry sequence into the pcDNA3HA–MS2–linker plasmid. The MS2 coat protein used is the V29I-dIFG double
mutant, with increased affinity for the MS2 stem-loop (Lim and Peabody, 1994). The
GFP–Mbnl1 fusion protein was subcloned from the pDEST53–GFP–Mbnl1-42kDa
plasmid (Dhaenens et al., 2008) into the pBabe puro vector. To make the mCherry–
Mbnl1 fusion protein, a linker sequence (GGGGSGGGGS) was inserted between
mCherry and the Mbnl1 sequence from pDEST53–GFP–Mbnl1-42kDa.
Cell culture
Cell lines expressing the lacZ–MS2–5CUG or 145CUG transcripts were generated
by infecting C2C12 Tet-Off cells with the desired pRevTRE plasmid and selecting
for stable clones. The cell line 145–8, which expresses high levels of lacZ–MS2–
145CUG mRNA (supplementary material Fig. S1), was used throughout this study.
All the experiments were performed 48 hours after removal of doxycycline to fully
induce transcription from the Tet-response element (TRE) promoter. For live-cell
microscopy, cells were plated on 35 mm glass-bottom plates (Fluorodish, World
Precision Instruments) and transiently infected with retroviral vector(s) expressing
the desired fluorescent fusion protein(s). The spinning disk confocal microscopes
were equipped with environment chambers for the control of CO2 concentration,
temperature (chamber and objective heater) and humidity, adjusted to maintain a
reading of 37°C in the cell medium. The a-amanitin treatment was done by adding
100 g/ml of drug to the medium. Live-cell experiments were performed from 1.5
to 3 hours after adding a-amanitin (A2263 Sigma).
Single-particle tracking
Time-lapse microscopy was performed at McGill University on the Cell Imaging
and Analysis Network (CIAN) SD2 spinning disk confocal microscope assembled
by Quorum Technologies (Guelph, Ontario, Canada) on a Leica DMI6000B platform.
Images were acquired using a 100⫻ oil objective NA 1.40 and the Hamamatsu
C9100-12 EMCCD camera (Hamamatsu, Higashi Japan). Exposure time was set at
333 ms at maximum speed and a single confocal image was acquired per time point.
Tracking was done with the MetaMorph object-tracking module, using the Threshold
Result algorithm, which detects the center of the intensity peak for each object and
determines which object is closest in the preceding image. Tracks were visually
inspected to ensure that the particle could be not be confused with neighbouring
particles. The mean square displacement (MSD) for each tracked particle was
calculated with the formula:
i
MSD = ∑ ( DTOi − DTOi = 1 )2 .
i= 0
Distance to origin (DTO) is the straight-line distance between the particle’s current
position and the first point in the track, calculated by the MetaMorph object tracking
module. The diffusion coefficient (D) for each particle was then derived by measuring
the average slope of the MSD (m2) over time. To determine particle diameters, four
measures of the diameter were taken at different angles and averaged.
Live-cell multicolor imaging
The lacZ–MS2–5CUG or 145CUG cells were co-infected with retrovirus for MS2–
GFP and mCherry–Mbnl1, or with MS2–mCherry and GFP–Mbnl1 to reverse the
fluorophores. Live cells were visualized by microscopy on a spinning disk confocal
microscope (either the CIAN SD2 or Perkin-Elmer UltraView Vox). GFP and
mCherry images were acquired sequentially, and the images from the two channels
were then overlaid for analysis. The absence of bleed-through between GFP and
mCherry was verified by visualizing cells infected with only one of the fluorescent
fusion proteins. The RNA particles were first identified in the image of the MS2
fluorophore, their diameter was measured and the MS2 RNA particles were then
scored for the detection of an overlapping peak in the Mbnl1 signal by applying a
linescan through both red and green channels with the MetaMorph software.
Additional particles could be observed in the Mbnl1 channel, which did not colocalize
with any MS2 signal. These were not included in the analysis because they could
represent endogenous transcripts bound by Mbnl1.
FRAP and FLIP experiments
3D time-lapse confocal microscopy was performed on an UltraView Vox spinning
disk confocal microscope equipped with a PhotoKinesis device for FRAP (Perkin
Elmer, Massachusetts USA). Images were acquired using a 100⫻ NA 1.40 oil
objective and the Hamamatsu C9100-50 EMCCD camera. Exposure time was set at
50 ms and a Z-stack sufficient to cover all foci in the nucleus was acquired for each
cell, with a slice thickness of 0.25 m. Two image stacks were acquired per minute.
To minimize phototoxicity and photobleaching, the camera was set to a bin of two
and the 488 nm laser power was reduced to 8% of maximum. After a prebleach
series, a 2-m-diameter bleach area was positioned over the chosen foci and bleached
for 20 iterations at 100% laser power. Data acquisition was continued for 20 minutes
to measure recovery. For fluorescence loss in photobleaching (FLIP), fields were
chosen where two adjacent cells containing MS2–GFP RNA foci were visible.
Exposure time was kept at 50 ms, but time intervals were set to 3 seconds. The 2m-diameter bleaching area was positioned in a region of the nucleus that did not
contain foci. A prebleach series was obtained and then, every 3 seconds, a new image
stack was acquired, followed by 20 iterations of bleaching at 100% laser power in
the bleach area. Total duration of data acquisition for FLIP was 5 minutes. For the
observation of the CUG-repeat mRNA foci with the GFP–Mbnl1 protein, exposure
time was set at 100 ms and 488 nm laser power was boosted to 12%. Time intervals
were set to 3 seconds, and the 2 m circular bleach area was positioned on the
chosen GFP–Mbnl1 foci immediately before bleaching. GFP signal in neighbouring
cells was monitored to control for incidental photobleaching during image acquisition;
no decrease in fluorescence intensity was observed with these settings.
The resulting films were analyzed using the MetaMorph program. The 3D images
were thresholded to remove signal from MS2–GFP or GFP–Mbnl1 molecules not
sequestered in CUG-repeat RNA aggregates. An isosurface rendering of the RNA
foci was performed to define them as singular objects, and the total fluorescence
intensity was obtained for each object at every time point. FRAP recovery curves
were analyzed with the GraphPad Prism program (GraphPad Software, La Jolla,
CA). The majority of the FRAP recovery curves fit the one-phase exponential
association equation, indicating a single population of RNA. The recovery curves
that did not fit this equation occurred when there was no recovery, and their halftime and % recovery were set as zero. The percentage of fluorescence recovery and
the half-times of recovery were calculated by the GraphPad Prism software. For
MS2–GFP foci analyzed by FLIP, 18 out of 22 foci fit a one-phase exponential decay
equation. The half-lives of the foci were calculated using GraphPad Prism software.
Fluorescent in situ hybridization
Fluorescent in situ hybridization (FISH) on RNA was performed according to the
protocol described previously (Querido and Chartrand, 2008). Briefly, cells were
fixed in PBS containing 4% paraformaldehyde, dehydrated for 2 hours in 70%
ethanol, and incubated overnight with 10 ng of probe in 2⫻SSC, 40% formamide at
37°C. To visualise the localization of lacZ mRNA, five Cy3-labelled probes designed
to hybridize in different regions of the lacZ coding sequence were used, as described
previously (Long et al., 1995). To visualize foci of CUG-triplet-repeat mRNA, a
single probe with the sequence (CAG)10 was labelled with Cy3 or Cy5 and used for
FISH, as originally described (Taneja et al., 1995). A control Cy3-labelled probe
with the sequence (CTG)10 gave no hybridization signal in cells expressing lacZ–
MS2–145CUG mRNA (data not shown). For simultaneous DNA and RNA FISH,
cells were fixed and dehydrated as above. Chromatin was denatured by treatment
with 70% formamide, 2⫻SSC at 70°C for 10 minutes, followed by two washes in
ice-cold 2⫻SSC. Hybridization with 50 ng of LNA-DNA probe complementary to
the MS2 antisense sequence and 10 ng (CAG)10 probe was performed overnight at
37°C in 50% formamide, 2⫻SSC, 10% dextran sulfate, 1 mg/ml BSA, 20 mM VRC.
Three washes were performed with 50% formamide, 2⫻SSC at 42°C for 5 minutes
each, followed by three washes with 2⫻SSC at 42°C for 5 minutes each. Images for
FISH were acquired on a transmitted light Nikon TE2000U microscope with 100⫻
oil objective of 1.40 NA and the CoolSNAPfx CCD camera (Photometrics, Tucson,
AZ) using the MetaMorph software (Molecular Devices). To quantify the CUG
repeat RNA foci visualized by FISH, a MetaMorph script was designed to first
outline the nuclei using the DAPI-stained images, then threshold the CUG-repeat
RNA foci in the nuclei to define them as objects, and finally measure and sum the
total surface area covered by RNA foci in each nucleus or the total fluorescence
intensity of the nuclear RNA foci.
We thank Mani S. Mahadevan (University of Virginia, VA) for
providing the DMPK 3⬘UTR plasmids, Nicolas Charlet-Berguerand
(Université de Strasbourg, France) for GFP–Mbnl1 plasmids, Stephen
W. Michnick (Université de Montréal, Canada) for the mCherry
plasmid, Charles Thornton (University of Rochester, NY) and Glenn
Stochastic aggregation of CUG-repeat RNA
E. Morris (Wolfson Centre for Inherited Neuromuscular Disease, UK)
for Mbnl1 antibodies, and Benoit Chabot (Université de Sherbrooke,
Canada) for hnRNP A1 antibody. We are grateful to P. Lapointe from
Perkin-Elmer Canada and G. Hickson for access to the UltraView Vox
microscope. We thank J. Lacoste from the Cell imaging and analysis
network (CIAN) at McGill University for helpful discussions. We are
also thankful to M. Vasseur of the Université de Montréal for expert
technical assistance. E.Q. was supported by a development grant from
the Muscular Dystrophy Association USA (MDA). F.G. was supported
by a fellowship from the Terry Fox foundation of the National Cancer
Institute of Canada. This research was funded by grants from the
Canadian Institute of Health Research (NDS62501) to P.C., and the
MDA (E.Q.). P.C. is a Senior Scholar from the Fonds de la Recherche
en Santé du Québec.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/124/10/1703/DC1
Journal of Cell Science
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