1540
Research Article
A regulatory role for CRM1 in the multi-directional
trafficking of splicing snRNPs in the mammalian
nucleus
Judith Sleeman
Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK
e-mail: [email protected]
Journal of Cell Science
Accepted 26 February 2007
Journal of Cell Science 120, 1540-1550 Published by The Company of Biologists 2007
doi:10.1242/jcs.001529
Summary
Distinct pathways of ribonucleoprotein transport exist
within the nucleus, connected to their biogenesis and
maturation. These occur despite evidence that the major
mechanism for their movement within the nucleus is
passive diffusion. Using fusions of Sm proteins to YFP, CFP
and photoactivatable GFP, I have demonstrated that
pathways with uni-directional bulk flow of complexes can
be maintained within the nucleus despite multi-directional
exchange of individual complexes. Newly imported splicing
small nuclear ribonucleoproteins (snRNPs) exchange
between Cajal bodies (CBs) within a nucleus and access the
cytoplasm, but are unable to accumulate in speckles. By
contrast, snRNPs at steady-state exchange freely in any
direction between CBs and speckles, but cannot leave the
nucleus. In addition to these surprising qualitative
observations in the behaviour of nuclear complexes,
sensitive live-cell microscopy techniques can detect subtle
Introduction
The nucleus of the mammalian cell contains many distinct
structures,
enriched
in
different
proteins
and
ribonucleoproteins (RNPs) (Carmo-Fonseca, 2002; Matera and
Shpargel, 2006; Misteli, 2005). The nucleus is a stable
structure formed despite the constant, often rapid, flux of
molecules through structures within it. Proteins and RNAs
have been demonstrated to move within the nucleus by
apparently random diffusion (Dundr et al., 2004; Kruhlak et
al., 2000; Misteli, 2005; Misteli et al., 1997; Phair and Misteli,
2000; Politz and Pederson, 2000; Snaar et al., 2000). Even
proteins involved in pathological formation of nuclear
aggregates are in constant exchange between the aggregates
and the nucleoplasm (Tavanez et al., 2005). The sub-nuclear
structures formed by this constant dynamic exchange are,
however, amenable to isolation for biochemical and proteomic
analyses, suggesting that the intermolecular interactions taking
place within them are not exclusively transient (Andersen et
al., 2002; Lam et al., 2002; Saitoh et al., 2004).
The U1, U2, U4, U5 and U6 small nuclear
ribonucleoproteins (snRNPs) are essential splicing factors.
Each snRNP consists of a uridine-rich small nuclear RNA
(snRNA) and seven common (Sm) proteins (B/B⬘, D1, D2, D3,
E, F and G), which form a ring-like structure around the
snRNA. In addition to these core proteins, each snRNP also
quantitative disturbances in nuclear dynamics before they
have had an effect on overall nuclear organization.
Inhibition of the nuclear export factor, CRM1, using
leptomycin B results in a change in the dynamics of
interaction of newly imported snRNPs with CBs. Together
with the detection of interactions of CRM1 with Sm
proteins and the survival of motor neurons (SMN) protein,
these studies suggest that the export receptor CRM1 is a
key player in the molecular mechanism for maintaining
these pathways. Its role in snRNP trafficking, however,
appears to be distinct from its previously identified role in
small nucleolar RNP (snoRNP) maturation.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/120/9/1540/DC1
Key words: Cajal body, snRNPs, Nucleus, CRM1
contains particle-specific proteins. At steady state, snRNPs
show a complex localization within the cell. The majority of
snRNPs localize to speckles, with a small amount localizing to
Cajal bodies (CBs). The localization pattern of snRNPs also
comprises a diffuse nucleoplasmic component and, sometimes,
a diffuse cytoplasmic component. The biogenesis and
maturation of splicing snRNPs is a complex process
(Lührmann et al., 1990; Will and Luhrmann, 2001), with
different stages occurring at different physical locations within
the cells. The snRNA is transcribed within the nucleus and then
exported to the cytoplasm as part of an export complex
containing CRM1 (XpoI), PHAX, CBC and Ran-GTP
(Fornerod et al., 1997; Izaurralde et al., 1997; Izaurralde et al.,
1995; Ohno et al., 2000). The core Sm proteins are assembled
onto the snRNA in the cytoplasm, forming a seven-membered
ring around the snRNA (Kambach et al., 1999a; Kambach et
al., 1999b; Lehmeier et al., 1994; Raker et al., 1996). The
survival of motor neurons (SMN) protein is required for
cytoplasmic addition of Sm proteins onto snRNAs (Liu and
Dreyfuss, 1996; Pellizzoni et al., 1999). Insufficient expression
of SMN results in the inherited neurodegenerative disease,
Spinal Muscular Atrophy (SMA) (Lefebvre et al., 1995). The
association of Sm proteins with snRNA is a prerequisite for 3⬘
end trimming and hypermethylation of the cap to form the
characteristic 5⬘ trimethylguanosine (TMG) cap (Will and
Journal of Cell Science
Nuclear snRNP trafficking
Luhrmann, 2001). The Sm domain and the TMG cap form a
bipartite nuclear localization signal (NLS) that is required for
re-import of the newly assembled core snRNP into the nucleus
(Fischer et al., 1993; Hamm et al., 1990; Mattaj, 1986).
Splicing snRNP import requires snurportin 1, importin-␤ and
SMN (Massenet et al., 2002; Narayanan et al., 2004;
Narayanan et al., 2002; Shpargel and Matera, 2005; Sleeman
et al., 2001). There is increasing evidence from animal models
of SMA that a defect in snRNP processing may be responsible
for the cellular pathology of SMA (Eggert et al., 2006; Winkler
et al., 2005).
Once re-imported, newly assembled snRNPs accumulate
preferentially in CBs (Carvalho et al., 1999; Sleeman and
Lamond, 1999). Spliceosomal snRNAs contain numerous
pseudouridines and 2⬘-O-methyl groups (Massenet et al.,
1998). Guide RNAs termed scaRNAs (small CB RNAs) have
been demonstrated to direct such modifications of the snRNA
(Darzacq et al., 2002; Jady et al., 2003) within CBs. The CB
has also been implicated as the cellular location for the addition
of the snRNP-specific heterotrimeric splicing factor SF3a to
U2 snRNPs, completing their maturation (Kramer et al., 2005;
Nesic et al., 2004), suggesting that snRNPs leave CBs fully
mature. Labelling of newly imported snRNPs with fluorescent
protein (FP)-tagged Sm proteins shows a delay of between 6
and 24 hours during which GFP-Sm proteins can be detected
in the nucleoplasm and CB, where they colocalize with SMN,
but not in nuclear speckles (Sleeman et al., 2001; Sleeman et
al., 2003). Later, labelled snRNPs are seen also in speckles,
suggesting that the CB phase is a necessary and rate-limiting
step in the nuclear snRNP biogenesis pathway. The delays seen
experimentally are not a direct measure of the length of time
required for each individual snRNP to be matured, but reflect
the time needed for a sufficient proportion of the FP-tagged Sm
proteins to be assembled into snRNPs and accumulated in
speckles to a level that can be detected. Further investigation
of the dynamics of snRNP maturation and of the pathways
controlling it is vital because of its potential involvement in
SMA, as well as for its implications for the further
understanding of the formation and maintenance of
mammalian nuclear structure.
Results
snRNPs that accumulate in the nucleus after short
expression times contain fully assembled Sm cores
Previous studies using FP-tagged Sm proteins have shown that
they are incorporated into snRNPs and localize correctly within
the nucleus (Sleeman et al., 1998; Sleeman et al., 2001;
Sleeman and Lamond, 1999). There is a period of time when
cells have recently begun to express FP-tagged Sm protein
during which snRNPs tagged with FP-Sm proteins are present
diffusely within the cytoplasm and nucleoplasm but
concentrate only in CBs, not in splicing speckles. Both the
diffuse nucleoplasmic signal and the nucleolar accumulation
are more prominent in cells expressing higher levels of the
tagged Sm proteins (J.E.S., unpublished), leading to the
suggestion that these components of the localization may
represent Sm proteins not assembled onto snRNA cores and
result from overexpression of the FP-tagged Sm proteins. To
address this, acceptor-bleaching fluorescence resonance energy
transfer (FRET) experiments were performed using cells
expressing YFP-SmB and CFP-SmD1 for different lengths of
1541
time. In a FRET experiment, if two fluorophores have
overlapping spectra, are close to each other (<10 nm) and in a
favourable orientation, then one (the donor, CFP) can transfer
energy to the other (the acceptor, YFP) in addition to emitting
energy to be detected as a fluorescent signal (Chen et al., 2003;
Day et al., 2001; Wouters et al., 2001). This energy transfer
can be detected in several ways. In acceptor-bleaching FRET
experiments (Chusainow et al., 2005), a laser is used to bleach
the population of acceptor (YFP) molecules in a region of
interest in a living cell so that the donor molecules can no
longer transfer energy to them. This results in a transient
increase in the measured fluorescence intensity of the donor
(CFP).
Living HeLa cells expressing YFP-SmB and CFP-SmD1
were used for acceptor-photobleaching FRET experiments.
The order of assembly of Sm proteins onto the snRNA core of
snRNPs is known (Kambach et al., 1999a; Kambach et al.,
1999b; Raker et al., 1996) (Fig. 1A). Only after formation of
the complete core domain will SmB and SmD1 be found in
close proximity, so FRET will only occur between CFP-SmD1
and YFP-SmB in a fully assembled snRNP Sm core (Fig. 1B).
Because each Sm protein is only represented once in each core
assembly, coexpression of CFP-SmB and YFP-SmB provides
a negative control. HeLa cells were transfected with plasmids
to express FP-Sm fusion proteins, and used for FRET
experiments after 48 hours, when both tagged proteins
accumulated in CBs and speckles and, in some cells, the
nucleolus. Cells were selected that showed low levels of
expression of the tagged proteins, with a similar intensity seen
for each of the two. Cells with high expression levels show
cytoplasmic accumulations of Sm proteins and were not
analysed. In each of the selected cells, a 200-msecond
stationary laser pulse at 532 nm was used to bleach the YFPSmB signal in a region of interest, and the effect of this
bleaching on the intensity of the CFP-SmD1 signal was
measured over time. Representative plots of signal intensity
against time are shown in Fig. 2. A transient increase in the
intensity of CFP-SmD1 (donor) was seen on bleaching of YFPSmB (acceptor) in CBs, nucleoli and speckles, demonstrating
that fully assembled Sm cores are present in each of these
locations. The FRET efficiencies calculated for each region
were similar and approximately 25% of the maximum value
obtained using a control construct in which CFP and YFP are
separated by only seven amino acids. FRET experiments were
also performed in HeLa cells 24 hours after transfection, when
the fusion proteins were in CBs and a diffuse nucleoplasmic
pool, but not in speckles. Similar FRET efficiencies were
detected (Fig. 2), indicating that, although some of the
fluorescent signal may result from free Sm proteins, fully
formed Sm snRNP cores are present in all nuclear regions
examined at both timepoints.
snRNPs show unrestricted exchange between nuclear
structures
Splicing snRNPs show a pathway of entry into the nucleus,
associated with their biogenesis. A one-way nuclear pathway
has been reported for the protein NHPX that binds both to box
C/D snoRNAs and to U4 snRNA. NHPX interacts with
speckles only when newly expressed before forming a stable
interaction with the nucleolus (Leung and Lamond, 2002). It
is not clear whether newly imported snRNPs also follow a uni-
Journal of Cell Science
1542
Journal of Cell Science 120 (9)
Fig. 1. Incorporation of FPtagged Sm proteins into
snRNPs for FRET analyses.
(A) Assembly of Sm
subcomplexes D1/D2 and
E/F/G onto snRNA forms a
stable subcomplex. Subsequent
binding of the D3/B complex
completes the Sm core domain
and places SmD1 and SmB next
to each other (adapted from
Kambach et al., 1999b, with
permission from Elsevier).
(B) Transfection of cells with
different combinations of FPtagged Sm proteins is predicted
to produce complexes
containing YFP-SmB next to
CFP-SmD1, resulting in FRET
interaction, or YFP-SmB and
CFP-SmB in separate
complexes, producing no FRET
and acting as a negative control.
directional pathway with speckles the final destination. As
snRNPs are believed to leave the CB fully mature (Kramer et
al., 2005; Nesic et al., 2004), snRNPs that have reached
speckles would not be expected to return to CBs during their
maturation if the biogenesis pathway is one-way. In order to
gain more information about the directionality of the snRNP
import pathway, a fusion of the core snRNP protein, SmB to
photoactivatable-GFP (PA-GFP), was made. PA-GFP shows
very weak fluorescence at 488 nm until activated using a 406
nm laser, following which it gains fluorescence similar to that
of enhanced GFP (EGFP). PA-GFP-SmB shows a similar
temporal pathway of entry into the nucleus as YFP- and CFPSmB (data not shown). HeLa cells were transfected with PAGFP-SmB and incubated for 48 hours, until PA-GFP-tagged
Fig. 2. FRET detection of fully assembled Sm cores in speckles and CBs. (A) HeLa cells were transfected with plasmids to express YFP-SmB
and CFP-SmD1. The YFP (acceptor) fluorescence was bleached in a region of interest. A transient (~1 second) increase in fluorescence of the
donor was seen, resulting from the decrease in the intensity of acceptor fluorescence. (B) FRET efficiencies obtained in different regions of the
nucleus in HeLa cells expressing YFP-SmB and CFP-SmD1 for 24 or 48 hours. No significant difference was seen in the efficiency calculated
for each region using a one-way analysis of variance (ANOVA) test. The negative control of YFP-SmB and CFP-SmB gave a small negative
value, probably a result of some inadvertent bleaching of the CFP donor by the 532 nm laser. A positive control using a single vector with YFP
and CFP (pYFPCFP) separated by seven amino acids gives a FRET efficiency of 0.43 (data not shown). Efficiencies of less than 0.05 are
generally regarded as not significant.
Nuclear snRNP trafficking
1543
Journal of Cell Science
snRNPs were seen in CBs and speckles, identified by their
size and morphology, using a PA-GFP (405/40 nm)
excitation filter. Regions of interest of approximate
diameter 1.5 ␮m within the nuclei were then activated
using a fixed laser pulse of 200 mseconds at 406 nm and a
short time-lapse sequence taken using a standard
fluorescein isothiocyanate (FITC) filter set to visualize the
exit of photoactivated snRNPs from the region of interest
(Fig. 3). This sequence was repeated 20 times in rapid
succession. Activation of PA-GFP-SmB signal in CBs (Fig.
3A,B; see supplementary material Movies 1, 2) or speckles
(Fig. 3C; see supplementary material Movie 3) led to the
rapid redistribution of signal throughout the entire nucleus,
with no clear preference seen for uptake of tagged snRNPs
into CBs or speckles, regardless of the type of structure
centred in the activated region of interest. This argues
against a specific one-way pathway for snRNP transport
within the nucleus from CBs to speckles, and suggests that
mature snRNPs can return to CBs.
Fig. 3. Mature PA-GFP-Sm-tagged snRNPs show unrestricted exchange
between nuclear structures. Repeated activation of a region of interest
(circled) containing a CB (A,B) or a speckle (C) results in rapid
accumulation of PA-GFP-SmB in both speckles (arrowheads) and CBs
(arrows) throughout the entire nucleus. In each panel, the left-hand
image shows the cell before any laser activation, the central image is
immediately after the first activation event and the right-hand image is
immediately after the tenth activation event. Bar, 22 ␮m.
Fig. 4. Mature snRNPs do not show
a measurable export from the
nucleus to the cytoplasm. (A) HeLa
cells stably expressing YFP-SmB
were bleached repeatedly using a
region of interest including a region
of the cytoplasm of one cell and a
region of the nucleus of its
neighbour (rectangle in first image).
The cells were imaged following
each bleach event. In the cell
undergoing bleaching of the
nucleus, the entire nucleus was
rapidly depleted of YFP-SmB. By
contrast, the cell undergoing
cytoplasmic bleaching lost virtually
none of its nuclear signal. (B)
Intensity of YFP-SmB signal in the
nucleus (outside the bleached
region) for the two bleached cells
against time.
Mature snRNPs do not show a measurable export
from the nucleus to the cytoplasm
Splicing snRNPs are large complexes that are perceived as
being stable with a slow rate of turnover. If this is the case,
Sm proteins incorporated into fully mature snRNPs would
not be expected to return to the cytoplasm. To address this
question, fluorescence loss in photobleaching (FLIP)
experiments were performed using cell line YFP-SmB-E1,
a HeLa cell line constitutively expressing YFP-tagged
SmB. A region of interest was selected containing a portion
of the nucleus of one cell and a portion of the cytoplasm
of a neighbouring cell (Fig. 4). This region was repeatedly
bleached, using a 532 nm laser, with a short time-lapse
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Journal of Cell Science 120 (9)
Journal of Cell Science
sequence being taken between bleaching events to assess the
effect of the bleaching (see supplementary material Movie 4).
As anticipated from the preceding studies using PA-GFPSmB, bleaching a region within the nucleus led to a rapid loss
of YFP-SmB-tagged snRNPs from all nuclear structures (Fig.
4). By contrast, no loss of nuclear signal was seen in the
nucleus of the neighbouring cell bleached in the cytoplasm.
These data demonstrate that, although there is rapid and
unrestricted exchange of FP-Sm-tagged snRNPs within the
nucleus, there is no significant exit of nuclear snRNPs to the
cytoplasm over the 5-minute duration of this experiment.
Recently imported snRNPs are unable to accumulate in
speckles, despite the presence of complete Sm cores
Cells that have recently begun to express FP-tagged Sm
proteins show accumulation of FP-Sm in CBs but not speckles,
with a diffuse cytoplasmic and nucleoplasmic FP-Sm signal
also seen. HeLa cells were transfected with a plasmid to
express PA-GFP-SmB and used for photoactivation studies 20
hours after transfection. Repeated activation of a region of the
nucleus containing a CB demonstrated that, although PA-GFPSmB could travel across the nucleus and accumulate in other
CBs, the rest of the signal remained diffuse within the
nucleoplasm (Fig. 5; see supplementary material Movie 5). No
accumulation in speckles was seen, even at early stages of the
experiment in which there was only a weak diffuse signal that
would be insufficient to mask any localization in speckles.
Although it is likely that some of the signal represents free Sm
protein not assembled onto snRNPs, the FRET studies detailed
above (Fig. 2) confirm that snRNPs with fully assembled Sm
cores are present in both the CB and the diffuse nucleoplasmic
pool at this timepoint. These partially mature snRNPs are
clearly unable to localize to speckles, forming interactions of
sufficient duration to result in an observed accumulation only
within CBs.
At early timepoints, YFP-SmB can exit the nucleus into
the cytoplasm
To determine whether recently imported snRNPs can return to
the cytoplasm, FLIP experiments were performed as described
above. In contrast to the results obtained for steady-state
snRNPs, in HeLa cells expressing YFP-SmB for only 20 hours,
repeated bleaching of a region encompassing part of the
nucleus of one cell and part of the cytoplasm of its neighbour
led to a decrease in nuclear signal in both nuclei (Fig. 6; see
supplementary material Movies 6, 7).
This suggests that a proportion of the YFP-SmB signal seen
in the nucleus at this early timepoint comprises protein that is
not assembled into fully mature snRNPs and so can still leave
the nucleus and access the cytoplasm. The purpose of this
exchange is not clear but it may result from the absence of
some molecular signal, either an RNA modification or an
associated protein, marking mature snRNPs for retention
within the nucleus. Measurement of the loss of YFP-SmB from
CBs in cells undergoing bleaching of YFP-SmB signal from
the cytoplasm demonstrated a similar rate of loss of signal from
the CB as from the nucleus as a whole (Fig. 6). This suggests
that the YFP-SmB-containing complexes that are able to access
the cytoplasm are interchangeable with those present in the CB.
CRM1 localizes to CBs and is lost rapidly from them
following treatment with leptomycin B
Leptomycin B (LMB) is a specific inhibitor of the export factor
CRM1/exportin. CRM1 is required for the export of nascent
snRNAs from the nucleus and its inactivation by binding to
LMB has been used as an indirect way to block snRNP import
into the nucleus (Carvalho et al., 1999; Sleeman et al., 2001).
HeLa cells treated with LMB show a reduction in the
accumulation of snRNPs in CBs. Furthermore, LMB treatment
of cells at early stages of FP-Sm protein expression prevents
the accumulation of newly imported snRNPs into nuclear
speckles. The complexity of the effects seen on snRNP
distribution following LMB treatment, together with the
detection of CRM1 in CBs (Boulon et al., 2004; Ospina et al.,
2005), suggests that CRM1 may have an additional role in
transporting species between structures within the nucleus.
CRM1 has been implicated in the transport of U3 small
nucleolar RNPs (snoRNPs) from CBs to nucleoli (Boulon et
al., 2004; Watkins et al., 2004). Treatment of HeLa cells with
LMB results in a rapid loss of CRM1 from CBs (Fig. 7),
suggesting that the interaction of CRM1 with CBs is dynamic
and a result of its activity. Incubation times as short as 15
minutes cause total loss of CRM1 from CBs. Extended
incubation with LMB (6 hours or more) is required for effects
on other CB markers to be observed. These include the loss of
snRNPs from CBs and the relocalization of coilin to the
nucleolus (Carvalho et al., 1999; Sleeman et al., 2001).
CRM1 is required for the retention of snRNPs in CBs
In order to investigate the potential role of CRM1 in the
interaction between newly imported snRNPs and CBs, HeLa
cells were transfected with PA-GFP-SmB and incubated for 20
hours. The positions of 20 cells expressing PA-GFP-SmB and
showing clear CBs were marked. One CB in each of these cells
was activated using a single pulse from a 406-nm laser and
time-lapse recording of the cells made for a 30-second period.
LMB was then added to the cells at a concentration of 0.05
␮g/ml (LC Laboratories, Woburn, MA). After 2 hours of
incubation with LMB, each of the 20 cells was revisited and
the single-pulse activation experiments repeated. Where
Fig. 5. Recently imported snRNPs are unable to
accumulate in speckles. Repeated activation of a region
(circled) containing a CB results in rapid accumulation
of signal in distant CBs (arrows). No accumulation in
speckles can be detected. The left-hand image shows
the cell before any laser activation, the central image is
immediately after the first activation event and the
right-hand image is immediately after the tenth
activation event. Bar, 22 ␮m.
Journal of Cell Science
Nuclear snRNP trafficking
1545
Fig. 6. At early timepoints, YFPSmB can exit the nucleus into the
cytoplasm. (A,B) HeLa cells
expressing YFP-SmB for 24 hours
were bleached repeatedly using a
region of interest including a region
of the cytoplasm of one cell and a
region of the nucleus of its
neighbour (rectangle in first image).
The cells were imaged following
each bleach event. In the cell
undergoing bleaching of the
nucleus, the entire nucleus was
rapidly depleted of YFP-SmB. In
the cell undergoing cytoplasmic
bleaching, the nucleus also lost a
significant amount of the nuclear
signal. Bar, 22 ␮m. (C) Intensity of
YFP-SmB signal in the nucleus
(outside the bleached region) for
the two bleached cells in A against
time. (D) Intensity of YFP-SmB in
a CB and a region of the
nucleoplasm in a cell undergoing
cytoplasmic bleaching demonstrates
that signal is lost equally from the
CB and the nucleoplasm.
possible, the same CB was used. The average intensity of
signal in the CB was measured, corrected for bleaching and
image variation by subtracting the average intensity of a region
within a neighbouring, non-activated cell, and plotted against
Fig. 7. CRM1 localizes to CBs and is
lost rapidly from them following
treatment with LMB. In untreated HeLa
cells, Crm1 (A) is found in CBs (arrows
in A,B,D) where it colocalizes with
snRNPs, detected with anti-Sm
antibodies (B). Treatment of HeLa cells
with LMB causes the loss of CRM1
from CBs (arrowheads in E,F,H). At
timepoints up to 4 hours, snRNPs,
detected by anti-Sm (F,H) or anti-TMG
(J,L) antibodies, are still clearly seen in
CBs (arrowheads and arrows). The CB
marker protein, coilin, is also clearly
seen in CBs after 4 hours of LMB
treatment (I,L). Images A-H are
deconvolved sections of HeLa cell
nuclei; images I-L are threedimensional projections of deconvolved
serial sections of HeLa cell nuclei taken
at 0.2 ␮m z-intervals. Bars, 13 ␮m.
time (Fig. 8). Exponential decay curves were fitted to the data
using non-linear regression software (Prism 4; GraphPad
Software, San Diego, CA). Although measurements taken
before LMB addition fitted best to a two-phase exponential
1546
Journal of Cell Science 120 (9)
Journal of Cell Science
Fig. 8. CRM1 is required for the
retention of snRNPs in CBs. The loss of
PA-GFP-Sm from CBs was measured in
HeLa cells before and after treatment for
2 hours with LMB. (A) Average
intensity of CBs over time, corrected for
bleaching of the sample and expressed as
a percentage of the pre-bleach intensity
of each CB. The loss of PA-GFP-SmB
from CBs is more rapid in cells treated
with LMB. (B) Mean half-lives of loss
of PA-GFP-SmB from CBs under
control and LMB-treated conditions.
decay curve, measurements taken in LMB-treated cells gave a
best fit to a single-phase decay curve. To allow comparison of
the results, therefore, a simpler one-phase curve was used to
calculate an approximate half-life of fluorescence loss for each
data set. PA-GFP-SmB was lost from CBs more quickly in cells
following LMB treatment (Fig. 8A,B). This suggests that the
inhibition of CRM1 and its subsequent loss from CBs prevents
the retention of newly imported snRNPs in CBs. This change
in kinetics can be measured following 2 hours of LMB
treatment: a length of inhibition insufficient to cause any
detectable change in CB components other than the loss of
CRM1.
was able to access speckles in distant regions of the nucleus.
This demonstrates that LMB does not block the uptake of
snRNPs into speckles, suggesting that the inability of newly
imported snRNPs to accumulate in speckles in LMB-treated
cells is connected to the alterations in their kinetics within the
CB, probably resulting from a failure of maturation or
assembly.
CRM1 associates with newly imported snRNPs in vivo
To investigate whether the requirement for CRM1 in the CB
for SmB retention is mediated by interactions between CRM1
and snRNPs, protein lysates were prepared from HeLa cells
expressing YFP alone, YFP-SmB for 24 hours, YFP-SmB for
CRM1 is not required for the uptake of snRNPs into
48 hours and GFP-SmD1 for 24 hours. Immunoprecipitations
speckles
were performed using anti-GFP antibodies (Fig. 10A). AntiLMB treatment of cells newly transfected with FP-tagged
GFP beads efficiently precipitated YFP-SmB, GFP-SmD1
snRNP proteins prevents the accumulation of FP-tagged
(data not shown) and YFP with a small amount of the target
snRNPs in nuclear speckles (Sleeman et al., 2001). To
protein remaining in the unbound fraction and none
investigate the effect of LMB on the ability of snRNPs to
precipitated by control, protein-G sepharose, beads (Fig.
accumulate in speckles, cells were transfected with PA-GFP10Ai,ii). On blots immunodetected with anti-CRM1 (Fig.
SmB and incubated for 72 hours so that PA-GFP-SmB was
10Aiii-vi), the majority of CRM1 signal was in the unbound
seen in CBs and speckles. The cells were then treated with
fraction, with a band corresponding to CRM1 also detected in
LMB for 4 hours, and PA-GFP-SmB-tagged snRNPs activated
the anti-GFP-bound fractions from cells transfected with GFPin different regions of the nucleus using repeated pulses of a
SmD1 (Fig. 10Aiii) or YFP-SmB (Fig. 10Aiv) for 24 hours.
406-nm laser, as in Figs 4 and 5 above. The results obtained
No CRM1 was immunoprecipitated from cells transfected with
were indistinguishable from those seen in cells without LMB
YFP alone (Fig. 10Avii) or with GFP-SmDI (Fig. 10Av) or
(Fig. 9; see supplementary material Movie 8). Whether the
YFP-SmB (Fig. 10Avi) for 48 hours. Thus, a small amount of
activated region contained a speckle or a CB, activated signal
endogenous CRM1 can be co-immunoprecipitated with FPtagged Sm proteins expressed for short periods of
time. This suggests that CRM1 interacts with
newly imported Sm-containing snRNPs in vivo,
and that the loss of this interaction is responsible
for the defect in retention of tagged SmB in CBs
in LMB-treated cells.
To further investigate the nature of the Smcontaining complexes with which CRM1
interacts, whole-cell lysates were prepared from
cells transiently expressing CRM1-GFP (a gift
from M. Fornerod, The Netherlands Cancer
Institute, Amsterdam, The Netherlands). CRM1Fig. 9. LMB does not affect the ability of speckles to accumulate snRNPs. HeLa
GFP was immunoprecipitated using anti-GFP
cells expressing PA-GFP-SmB were treated with LMB for 4 hours. A region of the
antibodies (Fig. 10Bi). Duplicate blots were
nucleus containing a CB (circle in A, arrow in B) was repeatedly activated with a
detected with antibodies to SMN, which is
406 nm laser and short time-lapse sequences were taken between each bleach
associated with new snRNPs in the cytoplasm and
event to monitor the spread of PA-GFP-SmB in the nucleus. The signal rapidly
accompanies them to CBs (Fig. 10Bii), to Sm
accumulated in speckles throughout the nucleus (arrowheads in C). Image A shows
proteins (Fig. 10Biii) and to the U2 snRNPthe cell before any laser activation, image B is immediately after the first activation
event and image C is immediately after the tenth activation event. Bars, 11 ␮m.
specific protein U2B⬙ (Fig. 10Biv). The cellular
Nuclear snRNP trafficking
1547
HeLa cells expressing GFP-SMN (Fig. 10Ci), but not from
cells expressing the U1 snRNP-specific protein, U1A (Fig.
10Cii). These data suggest that CRM1 is associated with newly
imported snRNPs in complexes containing SMN, prior to the
addition of snRNP-specific proteins.
Journal of Cell Science
Discussion
Steady-state snRNPs show rapid multi-directional
exchange between nuclear structures
Despite evidence for a pathway of maturation of splicing
snRNPs involving sequential interaction with CBs and
speckles, snRNPs show a great deal of flexibility in their
movement between nuclear structures. In cells expressing PAGFP-SmB for 48 hours or more, the steady-state distribution
of tagged snRNPs in CBs and speckles is observed. In these
cells, regardless of the structure selected for photoactivation of
PA-GFP-tagged snRNPs, the signal moves away from the
activated region in all directions and is able to accumulate in
speckles and CBs. This flexibility of movement is also seen
using FLIP, where signal is rapidly lost from all areas of the
nucleus during photobleaching of a nuclear region. These data
favour a model of movement of mature snRNPs within the
nucleus whereby they diffuse randomly, forming interactions
with sub-nuclear structures such as speckles and CBs of
sufficient duration to result in detectable accumulation if they
encounter binding sites. They argue against the presence of a
uni-directional pathway of complexes through CBs to speckles
connected with snRNP biogenesis. The observed maturation
pathway of splicing snRNPs is formed by the ability of snRNPs
to interact only with those sub-nuclear structures applicable to
their stage of maturation. The molecular basis for this
mechanism is currently under investigation. A role for the CB
in the regeneration of U4/U6 di-snRNPs between rounds of
splicing has been proposed (Schaffert et al., 2004; Stanek et
al., 2003). The observed ability of Sm-containing complexes
activated in speckles to accumulate in CBs may well be
connected to this process.
Fig. 10. CRM1 interacts with newly imported snRNPs in vivo.
(A) Immunoprecipitations from lysates of cells expressing YFP-SmB
(i) or YFP (ii) using anti-GFP antibodies. Detection of products using
anti-GFP demonstrates efficient precipitation of YFP-SmB and YFP
(GFP Beads lane). Duplicate blots probed with anti-CRM1
demonstrate co-immunoprecipitation of endogenous CRM1 with GFPSmD1 (iii) or YFP-SmB (iv) after 24 hours of expression (GFP Beads
lanes), but not with GFP-SmD1 (v) or YFP-SmB (vi) expressed for 48
hours or YFP alone (vii) (GFP Beads lanes). (B) Immunoprecipitations
from lysates of cells expressing CRM1-GFP using anti-GFP
antibodies. Detection of products using anti-GFP demonstrates
efficient precipitation of CRM1-GFP (i) (GFP Beads lane). Duplicate
blot probed with anti-SMN (ii) or Y12 anti-Sm (iii) demonstrates coimmunoprecipitation of endogenous SMN and Sm proteins with GFPCRM1, whereas the use of anti-U2B⬙ (iv) demonstrates no coimmunoprecipitation of this marker of mature snRNPs.
(C) Immunoprecipitations from cells expressing GFP-SMN or UIAGFP using anti-GFP antibodies. GFP-SMN (i) co-immunoprecipitates
endogenous CRM1, whereas U1A-GFP (ii) does not.
location of addition of U2B⬙ to U2 snRNPs is not known, but
the addition occurs after re-import into the nucleus. CRM1GFP co-immunoprecipitated SMN and Sm proteins but not
U2B⬙. Furthermore, a small amount of endogenous CRM1 was
co-immunoprecipited using GFP antibodies from lysates of
The presence of a fully assembled Sm core is not
sufficient for localization to speckles
In contrast to the behaviour of PA-GFP-SmB-tagged snRNPs
following 48 hours of expression, newly expressed PA-GFPSmB (16 to 24 hours) shows a restricted pattern of interaction
with nuclear structures. Activated PA-GFP-SmB accumulates
in CBs in distant regions of the nucleus, but is unable to
accumulate in speckles. It cannot be ruled out that some of the
PA-GFP signal in these nuclei represents PA-GFP-SmB protein
not assembled into snRNP particles. Although newly
assembled snRNPs require both a TMG cap and an Sm core
for their import (Fischer et al., 1993; Hamm et al., 1990), it
has not been formally demonstrated that Sm proteins need to
be assembled onto snRNAs to be imported. However, FRET
analyses of cells expressing CFP-SmD1 and YFP-SmB
demonstrate the presence of fully assembled Sm cores in CBs
and the nucleoplasm of cells expressing tagged Sm proteins for
short periods. These particles are unable to accumulate in
speckles, indicating that a complete Sm core domain is
insufficient for the accumulation of snRNPs in speckles.
Whether it is further modification of the snRNA, which is
subject to pseudouridylation and 2⬘-O-methylation, the
presence of additional snRNP proteins, for example SF3a for
1548
Journal of Cell Science 120 (9)
U2 snRNP, or both of these that mediate the interaction of
snRNPs with speckles is not yet clear.
Journal of Cell Science
The ability of snRNPs to exchange between the
cytoplasm and nucleus is lost with time
FLIP demonstrates that recently imported snRNPs are able to
access the cytoplasm, indeed the cytoplasmic and CB fractions
appear to be interchangeable, because bleaching of the
cytoplasm results in loss of signal from CBs. By contrast,
bleaching of the cytoplasm has no effect on the nuclear signal
in cells stably expressing YFP-SmB. The ability to accumulate
in speckles thus appears to be gained as the ability to access
the cytoplasm is lost. This suggests that mature snRNPs are
retained exclusively within the nucleus and that, if their Sm
cores are subject to turnover or recycling, this occurs at a low
rate, or involves nuclear rather than cytoplasmic machinery.
LMB causes a rapid loss of CRM1 from CBs and affects
snRNP dynamics within them
CRM1 is required for the export of newly transcribed snRNAs,
and LMB has been used previously as an indirect inhibitor of
the snRNP import pathway (Carvalho et al., 1999; Sleeman and
Lamond, 1999). In HeLa cells, CRM1 is often found in CBs
(Boulon et al., 2004; Ospina et al., 2005). A brief incubation
with LMB (30 minutes) is sufficient to cause complete loss of
CRM1 from CBs, suggesting that CRM1 is localized in CBs
as a result of its activity. This loss of CRM1 from CBs is seen
before any major disruption of nuclear structure is observed.
Longer incubations with LMB (6 hours or more) result in the
depletion of snRNPs from CBs and relocalization of the CB
marker protein, coilin, to the nucleolus (Carvalho et al., 1999;
Sleeman et al., 2001). Short incubations with LMB are
sufficient to affect the dynamics of PA-GFP-SmB within CBs.
LMB is a specific inhibitor of the nuclear export receptor
CRM1 and functions by binding directly to the region of
CRM1 required for binding to the nuclear export signals of its
cargo (Fornerod et al., 1997). In cells maintained in normal
growth conditions, a two-phase or three-phase exponential
decay curve is required to model the loss of PA-GFP-SmB
from the CB (Fig. 8 and data not shown). The most rapid rate
of loss under these conditions may represent free PA-GFPSmB not assembled onto snRNA. In cells treated with LMB
for 2-3 hours, the dynamics of PA-GFP-SmB in CBs are less
complex, with a curve of best fit obtained using single-phase
exponential decay. Furthermore, the observed average halflives for PA-GFP-SmB loss in LMB-treated cells are shorter
than those seen in untreated cells (P=0.0006). This suggests
LMB results in disruption of the interaction of Sm-containing
complexes with CBs. Together with the interaction between
early snRNP complexes and CRM1 detected by coimmunoprecipitation, this suggests that CRM1 has a role in the
interaction of snRNPs with CBs in addition to its established
role in snRNA export. The pattern of interactions detected
between CRM1 and proteins SmB, SmD1, SMN, U2B⬙ and
U1A suggest that CRM1 associates preferentially with newly
imported snRNPs, associated with the SMN protein, prior to
the addition of snRNP-specific proteins. The presence of
CRM1 and/or the absence of snRNP-specific proteins may
form part of a signal preventing accumulation of immature
snRNPs in speckles. Furthermore, the presence of CRM1, an
export factor, in newly imported, partially mature, snRNP
complexes could explain their ability to access the cytoplasm.
The current data cannot conclusively rule out the possibility
that CRM1 interacts independently with SMN and
unassembled Sm proteins. A role for the CB in reclamation of
unassembled snRNP components has been proposed (Xu et al.,
2005). This would place the role for CRM1 in modulating
snRNP assembly earlier in the biogenesis pathway, in ensuring
that unassembled Sm proteins are available to the cytoplasmic
assembly complex. However, the rationale for the LMBsensitive retention of free Sm proteins in the CB rather than
their rapid export to the cytoplasm is unclear.
Effect of LMB highlights differences between nuclear
snoRNP and snRNP transport pathways
snoRNPs are ribonucleoprotein particles found predominantly
in the nucleolus that function in rRNA biogenesis (Bachellerie
et al., 2002; Kiss, 2002). In contrast to splicing snRNPs, their
biogenesis is thought to occur exclusively within the nucleus
with no cytoplasmic stage (Terns and Dahlberg, 1994). Like
splicing snRNPs, the box C/D snoRNPs localize transiently to
CBs before reaching their steady-state localization in nucleoli
(Narayanan et al., 1999). Hypermethylation of the 5⬘ cap of the
snoRNA, analogous to one of the cytoplasmic events in snRNP
maturation, occurs in the CB (Mouaikel et al., 2002; Verheggen
et al., 2002). Studies of U3 snoRNAs (Boulon et al., 2004;
Watkins et al., 2004) have demonstrated sequential
involvement of PHAX, the snRNA export adaptor, and CRM1,
the snRNA export factor, in their transport within the nucleus.
PHAX is proposed to recruit TMG-capped U3 precursors into
CBs, with CRM1 binding hypermethylated, TMG-capped
species in CBs and transporting them away from it. In contrast
to this, the current study suggests that the presence of CRM1
in CBs is required for the adequate retention of TMG-capped,
Sm core-containing splicing snRNPs within CBs. Previous
data have shown that new snRNPs fail to accumulate in
speckles in LMB-treated cells. Together with the data
presented here, this suggests that CRM1, or associated factors,
is required to retain new snRNPs in CBs until they are
sufficiently mature to accumulate in speckles. It is possible that
the lack of retention of Sm-containing complexes in CBs of
LMB-treated cells is an indirect, downstream consequence of
blocking snRNA export. However, the alteration in dynamics
after relatively short treatments and the interactions detected
between CRM1 and Sm-containing complexes by coimmunoprecipitation argue for a direct effect.
In summary, newly imported snRNPs are able to interact with
CBs but unable to interact with speckles, despite their ability to
access areas of the nucleus containing speckles and the presence
of complete Sm core domains. Conversely, snRNPs at steady
state are able to form interactions with speckles of sufficient
affinity for their rapid accumulation in them to be seen. They
are, however, prevented from making any detectable return to
the cytoplasm. The physical differences between snRNPs
exhibiting these two types of behaviour are yet to be elucidated,
but the presence or absence of CRM1 and snRNP-specific
proteins such as U2B⬙ and U1A are likely to be involved.
Treatment of HeLa cells with the metabolic inhibitor LMB
results in a quantifiable change in the dynamics of interaction
of snRNPs with CBs, leading to a lack of retention of snRNPs.
Inhibition of CRM1 with LMB results in a dramatic
rearrangement of CB proteins over a period of 6 hours,
Nuclear snRNP trafficking
including the depletion of snRNPs from CBs. Using dynamic
live-cell microscopy, these changes can be measured at a
molecular level at much shorter timepoints, suggesting that
these techniques are capable of detecting subtle differences in
the behaviour of protein complexes within the nucleus, and thus
will prove powerful tools in the analysis of nuclear function.
FLIP
Materials and Methods
Cell fixation, immunostaining and microscopy
Cell culture and transfection
Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal calf serum and 100 U/ml penicillin and streptomycin (Life
Technologies). For immunofluorescence assays, cells were grown on coverslips and
transfected (if necessary) using Effectene transfection reagent (Qiagen) according
to the manufacturer’s instructions. For the preparation of cell lysates, cells were
grown in 10-cm diameter dishes. For live-cell microscopy, cells were grown on 40
mm coverslips (Intracel).
Plasmid constructs and cell lines
Plasmids pEYFP-SmB, pECFP-SmB, pECFP-SmD1 and stable cell line YFP-SmBE1 have been described previously (Sleeman et al., 2003). The expression plasmid
pPA-GFP-SmB for photoactivatable-GFP-tagged SmB was generated by subcloning the SmB sequence from pEYFP-SmB as an EcoRI/SalI fragment into
plasmid pPA-GFP-C1 (a gift from J. Lippincott-Schwartz, NIH, Bethesda, MD).
CRM1-GFP was a gift from M. Fornerod.
Journal of Cell Science
Regions of interest in cells expressing YFP-SmB were defined and bleached using
a 20 mW 532 mn laser at 50% power, by automatic movement of the stage and
repeated pulsing with a diffraction-limited spot. The cells were imaged three times
before each bleach event and using a 10-second adaptive timecourse after each
bleach event. The bleach sequence was repeated 10 times. The intensity of
representative nuclear, cytoplasmic and CB regions was measured, corrected for
bleaching during imaging by subtraction of the fluorescence from a background
region within an unbleached cell, and plotted against time.
HeLa cells grown on glass coverslips were fixed for 5 minutes at room temperature
with 3.7% paraformaldehyde in PHEM buffer [60 mM PIPES, 25 mM HEPES, 10
mM EGTA, 2 mM MgCl2 (pH 6.9)]. Immunostaining was performed as described
previously (Sleeman et al., 2003). Cells were mounted in Citifluor medium (Agar
Scientific). Antibodies used were mouse monoclonal antibody (mAb) Ab1 antiTMG (Calbiochem) (dilution 1:50), mouse Y12 mAb anti-Sm (AbCam) (dilution
1:100), rabbit 5641 anti-CRM1 (a gift from M. Fornerod) (dilution 1:500), rabbit
204,10 anti-coilin (a gift from A. I. Lamond, University of Dundee, Dundee, UK)
(dilution 1:500), FITC-conjugated goat anti-mouse and TRITC-conjugated goat
anti-rabbit IgG (Jackson ImmunoResearch Laboratories) (dilution 1:500).
Immunostained specimens were examined using a Zeiss 63⫻ 1.4NA PlanApochromat objective and recorded using a Hammamatsu C4742-80-12AG camera.
Optical sections separated by 200 nm were collected using a binning of 2⫻2. Images
were restored using an iterative deconvolution algorithm using a calculated pointspread function (Volocity; Improvision, UK).
Preparation of cell lysates, immunoblotting and
immunoprecipitation
Live-cell microscopy
HeLa cells grown on glass coverslips were cotransfected with appropriate
constructs. 24 or 48 hours after transfection, cells were mounted in CO2-independent
medium (Invitrogen Life Technologies) in an open chamber (Zeiss) within an
environmental incubator (Solent Scientific) on an Olympus DeltaVision Spectris
Microscope (Applied Precision) with a Quantifiable Laser Module including a 20
mW 532 nm laser to photobleach YFP without bleaching CFP (for FRET and FLIP)
and a 20 mW 406 nm laser for photoactivation of PA-GFP. Data analyses were
performed using Volocity (Improvision, UK) and Prism 4 (GraphPad).
Acceptor-photobleaching FRET
FRET measurements were performed on live cells, basically as described previously
(Chusainow et al., 2005). Photobleaching was performed using a 200 msecond 532
nm stationary pulse at 50% laser power. The first image was acquired 1–2 mseconds
after the bleach event. Images of donor (ECFP) and acceptor (EYFP) were taken in
separate subsequent measurements, bleaching exactly the same spot before
collecting post-bleach images. A non-bleached nuclear region in the same cell was
included in the data analysis as a control. A region of background fluorescence was
defined outside the cell and subtracted from both the bleached and control regions.
Data were normalized against the mean intensity of the whole image over time to
account for normal photobleaching. FRET efficiency was calculated as:
E = (ID(post)–ID(pre)) / ID(post) ,
where ID(pre) and ID(post) are donor intensity before and after photobleaching,
respectively.
Kinetic analyses using PA-GFP
HeLa cells were grown on glass coverslips, transfected with pPA-GFP-SmB and
scanned for low levels of expression of PA-GFP-SmB using a 405/40-nm excitation
filter and a standard FITC emission filter. Selected cells were activated by a 200
msecond 406 nm pulse at 50% laser power focused to a diffraction-limited spot of
approximately 1.5 ␮m diameter. A single z-section of each cell was imaged using a
FITC filter at three timepoints before the laser pulse and using an adaptive timecourse
35 seconds after the laser pulse. For analyses of dynamics before and after LMB
treatment, cells were marked in a point list so that they could be revisited after
incubation with the drug for 1.5 hours. The fluorescence intensity of photoactivated
CBs was measured for each timepoint. A region of background fluorescence was
defined within a neighbouring non-activated cell and subtracted from these values.
The intensity values were normalized against the initial preactivation fluorescence
and plotted against time. To compare half-lives before and after LMB treatment,
single-phase exponential decay curves were fitted to each data set and the
representative half-lives obtained compared using a two-tailed t-test.
Equations used were as follows:
Y = Span1 ⫻ exp (–K1⫻X) + Plateau
and
1549
Y = Span1 ⫻ exp (–K1⫻X) + Span2 ⫻ exp (–K2⫻X) + Plateau .
For experiments requiring sequential activation of fluorescence in the same region
of interest, multiple laser pulses were used with images recorded at three timepoints
before each activation and over a 10-second adaptive timecourse after each activation.
Lysates were prepared as described previously (Sleeman et al., 2003),
electrophoresed on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose
membranes for immunoblotting. Immunoprecipitation using anti-GFP antibodies
(Roche) was performed as described previously (Trinkle-Mulcahy et al., 2001).
Primary antibodies used were anti-GFP mouse monoclonal (Roche), dilution
1:1000; 5641 rabbit anti-CRM1 (a gift from M. Fornerod), dilution 1:1000; 4G3
anti-U2B⬙ (a gift from A. I. Lamond), dilution 1:500; MANSMA1 anti-SMN (a gift
from G. Morris, RJAH Orthopaedic Hospital, Oswestry, UK), dilution 1:500; Y12
anti-Sm (AbCam), dilution 1:250; and 456 anti-U1A (a gift from A. I. Lamond),
dilution 1:500.
I would like to thank Jennifer Lippincott-Schwartz for PA-GFP-C1,
Maarten Fornerod for anti-CRM1, Laura Trinkle-Mulcahy for
pCFPYFP, Alan Prescott, Brian McStay and Sam Crouch for
comments on the manuscript, and Giles Thomas for help with curve
fitting and statistical analysis. J.E.S. is a Royal Society URF. This
work was supported by Tenovus Tayside.
References
Andersen, J. S., Lyon, C. E., Fox, A. H., Leung, A. K., Lam, Y. W., Steen, H., Mann,
M. and Lamond, A. I. (2002). Directed proteomic analysis of the human nucleolus.
Curr. Biol. 12, 1-11.
Bachellerie, J. P., Cavaille, J. and Huttenhofer, A. (2002). The expanding snoRNA
world. Biochimie 84, 775-790.
Boulon, S., Verheggen, C., Jady, B. E., Girard, C., Pescia, C., Paul, C., Ospina, J. K.,
Kiss, T., Matera, A. G., Bordonne, R. et al. (2004). PHAX and CRM1 are required
sequentially to transport U3 snoRNA to nucleoli. Mol. Cell 16, 777-787.
Carmo-Fonseca, M. (2002). The contribution of nuclear compartmentalization to gene
regulation. Cell 108, 513-521.
Carvalho, T., Almeida, F., Calapez, A., Lafarga, M., Berciano, M. T. and CarmoFonseca, M. (1999). The spinal muscular atrophy disease gene product, SMN: A link
between snRNP biogenesis and the Cajal (coiled) body. J. Cell Biol. 147, 715-728.
Chen, Y., Mills, J. D. and Periasamy, A. (2003). Protein localization in living cells and
tissues using FRET and FLIM. Differentiation 71, 528-541.
Chusainow, J., Ajuh, P. M., Trinkle-Mulcahy, L., Sleeman, J. E., Ellenberg, J. and
Lamond, A. I. (2005). FRET analyses of the U2AF complex localize the
U2AF35/U2AF65 interaction in vivo and reveal a novel self-interaction of U2AF35.
RNA 11, 1201-1214.
Darzacq, X., Jady, B. E., Verheggen, C., Kiss, A. M., Bertrand, E. and Kiss, T. (2002).
Cajal body-specific small nuclear RNAs: a novel class of 2’-O-methylation and
pseudouridylation guide RNAs. EMBO J. 21, 2746-2756.
Day, R. N., Periasamy, A. and Schaufele, F. (2001). Fluorescence resonance energy
transfer microscopy of localized protein interactions in the living cell nucleus. Methods
25, 4-18.
Dundr, M., Hebert, M. D., Karpova, T. S., Stanek, D., Xu, H., Shpargel, K. B., Meier,
U. T., Neugebauer, K. M., Matera, A. G. and Misteli, T. (2004). In vivo kinetics of
Cajal body components. J. Cell Biol. 164, 831-842.
Eggert, C., Chari, A., Laggerbauer, B. and Fischer, U. (2006). Spinal muscular atrophy:
the RNP connection. Trends Mol. Med. 12, 113-121.
Journal of Cell Science
1550
Journal of Cell Science 120 (9)
Fischer, U., Sumpter, V., Sekine, M., Satoh, T. and Luhrmann, R. (1993). Nucleocytoplasmic transport of U snRNPs: definition of a nuclear location signal in the Sm
core domain that binds a transport receptor independently of the m3G cap. EMBO J.
12, 573-583.
Fornerod, M., Ohno, M., Yoshida, M. and Mattaj, I. W. (1997). CRM1 is an export
receptor for leucine-rich nuclear export signals. Cell 90, 1051-1060.
Hamm, J., Darzynkiewicz, E., Tahara, S. M. and Mattaj, I. W. (1990). The
trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear
targeting signal. Cell 62, 569-577.
Izaurralde, E., Lewis, J., Gamberi, C., Jarmolowski, A., McGuigan, C. and Mattaj,
I. W. (1995). A cap-binding protein complex mediating U snRNA export. Nature 376,
709-712.
Izaurralde, E., Kutay, U., von Kobbe, C., Mattaj, I. W. and Gorlich, D. (1997). The
asymmetric distribution of the constituents of the Ran system is essential for transport
into and out of the nucleus. EMBO J. 16, 6535-6547.
Jady, B. E., Darzacq, X., Tucker, K. E., Matera, A. G., Bertrand, E. and Kiss, T.
(2003). Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal
body following import from the cytoplasm. EMBO J. 22, 1878-1888.
Kambach, C., Walke, S. and Nagai, K. (1999a). Structure and assembly of the
spliceosomal small nuclear ribonucleoprotein particles. Curr. Opin. Struct. Biol. 9, 222230.
Kambach, C., Walke, S., Young, R., Avis, J. M., de la Fortelle, E., Raker, V. A.,
Luhrmann, R., Li, J. and Nagai, K. (1999b). Crystal structures of two Sm protein
complexes and their implications for the assembly of the spliceosomal snRNPs. Cell
96, 375-387.
Kiss, T. (2002). Small nucleolar RNAs: an abundant group of noncoding RNAs with
diverse cellular functions. Cell 109, 145-148.
Kramer, A., Ferfoglia, F., Huang, C. J., Mulhaupt, F., Nesic, D. and Tanackovic, G.
(2005). Structure-function analysis of the U2 snRNP-associated splicing factor SF3a.
Biochem. Soc. Trans. 33, 439-442.
Kruhlak, M. J., Lever, M. A., Fischle, W., Verdin, E., Bazett-Jones, D. P. and Hendzel,
M. J. (2000). Reduced mobility of the alternate splicing factor (ASF) through the
nucleoplasm and steady state speckle compartments. J. Cell Biol. 150, 41-51.
Lam, Y. W., Lyon, C. E. and Lamond, A. I. (2002). Large-scale isolation of Cajal bodies
from HeLa cells. Mol. Biol. Cell 13, 2461-2473.
Lefebvre, S., Burglen, L., Reboullet, S., Clermont, O., Burlet, P., Viollet, L.,
Benichou, B., Cruaud, C., Millasseau, P., Zeviani, M. et al. (1995). Identification
and characterization of a spinal muscular atrophy-determining gene. Cell 80, 155165.
Lehmeier, T., Raker, V., Hermann, H. and Luhrmann, R. (1994). cDNA cloning of
the Sm proteins D2 and D3 from human small nuclear ribonucleoproteins: evidence
for a direct D1-D2 interaction. Proc. Natl. Acad. Sci. USA 91, 12317-12321.
Leung, A. K. and Lamond, A. I. (2002). In vivo analysis of NHPX reveals a novel
nucleolar localization pathway involving a transient accumulation in splicing speckles.
J. Cell Biol. 157, 615-629.
Liu, Q. and Dreyfuss, G. (1996). A novel nuclear structure containing the Survival of
Motor Neurons protein. EMBO J. 15, 3555-3565.
Lührmann, R., Kastner, B. and Bach, M. (1990). Structure of spliceosomal snRNPs
and their role in pre-mRNA splicing. Biochim. Biophys. Acta 1087, 265-292.
Massenet, S., Mougin, A. and Branlant, C. (1998). Posttranscriptional modification in
the U small nuclear RNAs. In Modification and Editing of RNA (ed. H. Grosjean and
R. Benne), pp. 201-227. Washington, DC: ASM Press.
Massenet, S., Pellizzoni, L., Paushkin, S., Mattaj, I. W. and Dreyfuss, G. (2002). The
SMN complex is associated with snRNPs throughout their cytoplasmic assembly
pathway. Mol. Cell. Biol. 22, 6533-6541.
Matera, A. G. and Shpargel, K. B. (2006). Pumping RNA: nuclear bodybuilding along
the RNP pipeline. Curr. Opin. Cell Biol. 18, 317-324.
Mattaj, I. W. (1986). Cap trimethylation of snRNA is cytoplasmic and dependent on U
snRNP protein binding. Cell 46, 905-911.
Misteli, T. (2005). Concepts in nuclear architecture. BioEssays 27, 477-487.
Misteli, T., Caceres, J. F. and Spector, D. L. (1997). The dynamics of a pre-mRNA
splicing factor in living cells. Nature 387, 523-527.
Mouaikel, J., Verheggen, C., Bertrand, E., Tazi, J. and Bordonne, R. (2002).
Hypermethylation of the cap structure of both yeast snRNAs and snoRNAs requires
a conserved methyltransferase that is localized to the nucleolus. Mol. Cell 9, 891901.
Narayanan, A., Lukowiak, A., Jady, B. E., Dragon, F., Kiss, T., Terns, R. M. and
Terns, M. P. (1999). Nucleolar localization signals of box H/ACA small nucleolar
RNAs. EMBO J. 18, 5120-5130.
Narayanan, U., Ospina, J. K., Frey, M. R., Hebert, M. D. and Matera, A. G. (2002).
SMN, the spinal muscular atrophy protein, forms a pre-import snRNP complex with
snurportin1 and importin beta. Hum. Mol. Genet. 11, 1785-1795.
Narayanan, U., Achsel, T., Luhrmann, R. and Matera, A. G. (2004). Coupled in vitro
import of U snRNPs and SMN, the spinal muscular atrophy protein. Mol. Cell 16, 223234.
Nesic, D., Tanackovic, G. and Kramer, A. (2004). A role for Cajal bodies in the final
steps of U2 snRNP biogenesis. J. Cell Sci. 117, 4423-4433.
Ohno, M., Segref, A., Bachi, A., Wilm, M. and Mattaj, I. W. (2000). PHAX, a mediator
of U snRNA nuclear export whose activity is regulated by phosphorylation. Cell 101,
187-198.
Ospina, J. K., Gonsalvez, G. B., Bednenko, J., Darzynkiewicz, E., Gerace, L. and
Matera, A. G. (2005). Cross-talk between snurportin1 subdomains. Mol. Biol. Cell 16,
4660-4671.
Pellizzoni, L., Charroux, B. and Dreyfuss, G. (1999). SMN mutants of spinal muscular
atrophy patients are defective in binding to snRNP proteins. Proc. Natl. Acad. Sci. USA
96, 11167-11172.
Phair, R. D. and Misteli, T. (2000). High mobility of proteins in the mammalian cell
nucleus. Nature 404, 604-609.
Politz, J. C. and Pederson, T. (2000). Review: movement of mRNA from transcription
site to nuclear pores. J. Struct. Biol. 129, 252-257.
Raker, V. A., Plessel, G. and Luhrmann, R. (1996). The snRNP core assembly pathway:
identification of stable core protein heteromeric complexes and an snRNP subcore
particle in vitro. EMBO J. 15, 2256-2269.
Saitoh, N., Spahr, C. S., Patterson, S. D., Bubulya, P., Neuwald, A. F. and Spector,
D. L. (2004). Proteomic analysis of interchromatin granule clusters. Mol. Biol. Cell 15,
3876-3890.
Schaffert, N., Hossbach, M., Heintzmann, R., Achsel, T. and Luhrmann, R. (2004).
RNAi knockdown of hPrp31 leads to an accumulation of U4/U6 di-snRNPs in Cajal
bodies. EMBO J. 23, 3000-3009.
Shpargel, K. B. and Matera, A. G. (2005). Gemin proteins are required for efficient
assembly of Sm-class ribonucleoproteins. Proc. Natl. Acad. Sci. USA 102, 1737217377.
Sleeman, J. E. and Lamond, A. I. (1999). Newly assembled snRNPs associate with
coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway. Curr.
Biol. 9, 1065-1074.
Sleeman, J., Lyon, C. E., Platani, M., Kreivi, J.-P. and Lamond, A. I. (1998).
Dynamic interactions between splicing snRNPs, coiled bodies and nucleoli revealed
using snRNP protein fusions to the green fluorescent protein. Exp. Cell Res. 243, 290304.
Sleeman, J. E., Ajuh, P. and Lamond, A. I. (2001). snRNP protein expression enhances
the formation of Cajal bodies containing p80-coilin and SMN. J. Cell Sci. 114, 44074419.
Sleeman, J. E., Trinkle-Mulcahy, L., Prescott, A. R., Ogg, S. C. and Lamond, A. I.
(2003). Cajal body proteins SMN and Coilin show differential dynamic behaviour in
vivo. J. Cell Sci. 116, 2039-2050.
Snaar, S., Wiesmeijer, K., Jochemsen, A. G., Tanke, H. J. and Dirks, R. W. (2000).
Mutational analysis of fibrillarin and its mobility in living human cells. J. Cell Biol.
151, 653-662.
Stanek, D., Rader, S. D., Klingauf, M. and Neugebauer, K. M. (2003). Targeting of
U4/U6 small nuclear RNP assembly factor SART3/p110 to Cajal bodies. J. Cell Biol.
160, 505-516.
Tavanez, J. P., Calado, P., Braga, J., Lafarga, M. and Carmo-Fonseca, M. (2005). In
vivo aggregation properties of the nuclear poly(A)-binding protein PABPN1. RNA 11,
752-762.
Terns, M. P. and Dahlberg, J. E. (1994). Retention and 5⬘ cap trimethylation of U3
snRNA in the nucleus. Science 264, 959-961.
Trinkle-Mulcahy, L., Sleeman, J. E. and Lamond, A. I. (2001). Dynamic targeting of
protein phosphatase 1 within the nuclei of living mammalian cells. J. Cell Sci. 114,
4219-4228.
Verheggen, C., Lafontaine, D. L., Samarsky, D., Mouaikel, J., Blanchard, J. M.,
Bordonne, R. and Bertrand, E. (2002). Mammalian and yeast U3 snoRNPs are
matured in specific and related nuclear compartments. EMBO J. 21, 2736-2745.
Watkins, N. J., Lemm, I., Ingelfinger, D., Schneider, C., Hossbach, M., Urlaub, H.
and Luhrmann, R. (2004). Assembly and maturation of the U3 snoRNP in the
nucleoplasm in a large dynamic multiprotein complex. Mol. Cell 16, 789-798.
Will, C. L. and Luhrmann, R. (2001). Spliceosomal UsnRNP biogenesis, structure and
function. Curr. Opin. Cell Biol. 13, 290-301.
Winkler, C., Eggert, C., Gradl, D., Meister, G., Giegerich, M., Wedlich, D.,
Laggerbauer, B. and Fischer, U. (2005). Reduced U snRNP assembly causes motor
axon degeneration in an animal model for spinal muscular atrophy. Genes Dev. 19,
2320-2330.
Wouters, F. S., Verveer, P. J. and Bastiaens, P. I. (2001). Imaging biochemistry inside
cells. Trends Cell Biol. 11, 203-211.
Xu, H., Pillai, R. S., Azzouz, T. N., Shpargel, K. B., Kambach, C., Hebert, M. D.,
Schumperli, D. and Matera, A. G. (2005). The C-terminal domain of coilin interacts
with Sm proteins and U snRNPs. Chromosoma 114, 155-166.