Howard Hughes Medical Institute, Children's
Hospital, Harvard Medical School, Boston, Massachusetts
Received 8 December 1998/Returned for modification 4 February
1999/Accepted 15 March 1999
The U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins
(snRNPs) form essential components of spliceosomes, the machinery that
removes introns from pre-mRNAs in eukaryotic cells. A critical initial
step in the complex process of snRNP biogenesis is the assembly of a
group of common core proteins (Sm proteins) on spliceosomal snRNA. In
this study we show by multiple independent methods that the protein
pICln associates with Sm proteins in vivo and in vitro. The binding of
pICln to Sm proteins interferes with Sm protein assembly on
spliceosomal snRNAs and inhibits import of snRNAs into the nucleus.
Furthermore, pICln prevents the interaction of Sm proteins with the
survival of motor neurons (SMN) protein, an interaction that has been
shown to be critical for snRNP biogenesis. These findings lead us to
propose a model in which pICln participates in the regulation of snRNP
biogenesis, at least in part by interfering with Sm protein interaction
with SMN protein.
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INTRODUCTION |
The function of pICln has been
debated since it was cloned by screening a Madin-Darby canine kidney
(MDCK) cell library for the induction of a novel chloride current in
Xenopus laevis oocytes. Although we initially proposed that
pICln is an integral chloride channel component, pICln lacks predicted
hydrophobic transmembrane domains and structural homology to known
channel proteins (31). Furthermore, pICln was not detected
in the plasma membrane; it is localized predominantly in the cytoplasm
and nucleus, while a small fraction is associated with the cytoskeleton
(1, 15, 26). Thus, the structure and localization of pICln
suggest that it is not itself a chloride channel. Nevertheless, a
consistent conclusion from previous studies of pICln is that its
overexpression induces the appearance of a chloride conductance
(1, 2, 28, 31). The pathway connecting pICln overexpression
to activation of the chloride current is not known. It is possible that
activation of this chloride current is a side effect of exogenous
protein expression in oocytes rather than an effect related to the
physiologic function of pICln (1, 2). In order to improve
our understanding of the physiologic role of pICln, we have identified
several proteins that associate with pICln. In this study, we
demonstrate that pICln binds several proteins which are components of
spliceosomal small nuclear ribonucleoproteins (snRNPs).
The small nuclear ribonucleoproteins U1, U2, U4, and U5 are essential
components of the pre-mRNA splicing machinery (13). Each
snRNP is made up of U RNA (U1, U2, U4, or U5), a set of core snRNP
proteins (Sm proteins) which are common to all snRNPs, and a set of
snRNP-specific proteins. There are at least eight Sm proteins, named B'
(29 kDa), B (28 kDa), D1 (16 kDa), D2 (16.5 kDa), D3 (18 kDa), E (12 kDa), F (11 kDa), and G (9 kDa) (21). While the
snRNP-specific proteins have snRNP-specific functions in the splicing
reaction, Sm proteins are only known to be involved in the biogenesis
of snRNPs (reviewed in reference 23). U1, U2, U4,
and U5 snRNAs are transcribed in the nucleus by RNA polymerase II
and acquire a monomethyl cap. The RNAs are then exported to the
cytoplasm, where they bind the Sm proteins to form the Sm core (3,
36). Subsequently, the monomethyl cap is hypermethylated to a
trimethyl cap (22), and the assembled U snRNP is actively imported into the nucleus. Nuclear import depends on the presence of at
least two import signals, the Sm core and the trimethyl cap (4,
9). SnRNP nuclear import requires importin 
and snurportin1, a
nuclear import receptor which binds to both trimethyl cap structures
and importin (12, 29).
Sm proteins form a complex with the protein SMN (survival of motor
neurons [20]), the protein which is altered in the
autosomal recessive disease spinal muscular atrophy (16).
SMN is tightly associated with the protein SIP1 (SMN-interacting
protein 1 [20]). In Xenopus oocytes,
antibodies directed against SIP1 blocked assembly of Sm proteins with U
RNA and inhibited snRNP import into the nucleus. Antibodies specific
for SMN enhanced the assembly of Sm proteins on U RNA and had no
influence on snRNP nuclear import (6). These results were
interpreted as evidence that SMN and SIP1 have a role in U snRNP
assembly and are consistent with a model in which the SMN-SIP1 complex
interacts with Sm proteins and thereby mediates their assembly onto U RNA.
In the process of identifying proteins which bind to pICln, we found
that pICln interacts with several Sm proteins. We show that Sm protein
binding to pICln inhibits their assembly on U RNA and interferes with
snRNP biogenesis. Furthermore, we show that pICln inhibits SMN
binding to Sm proteins.
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MATERIALS AND METHODS |
Constructs and yeast two-hybrid analysis.
pT7-U RNA plasmids
(9) for in vitro transcription of U RNAs were kindly
provided by I. Mattaj. U1
was generated by using PCR to delete bases
127 to 164 of Xenopus U1, which contains the Sm binding site
(24). Clones used for coupled in vitro transcription and
translation of SmB', SmD2, SmD3, and SmF were derived from full-length
human expressed sequence tagged (EST) clones (GenBank accession no.
aa084844, aa280959, aa452419, and n71826, respectively). SmD1, SmE, and
SmG coding regions were amplified from a human fetal brain cDNA library
(Stratagene) by PCR. SmE-H6, SmF-H6, and
SmG-H6 were expressed from PCR products which included a T7 promoter, the entire coding sequence, and a C-terminal hexahistidine (H6) sequence.
Plasmids expressing glutathione-S-transferase (GST) fused to
full-length rat pICln (GST-ICln), Xenopus pICln (GST-xICln), or deletion mutants of human pICln were generated by subcloning the
appropriate PCR products into pGEX-2T (Pharmacia). The deletion mutant
hICln[1-34] was generated by using a naturally occurring restriction
site, resulting in the addition of two amino acids onto the C terminus.
The plasmids expressing SMN fused to GST or to thioredoxin (Trx) were
constructed by using PCR to subclone the SMN coding region from an EST
clone (accession no. n73104) into pGEX6P-2 (Pharmacia) or pET32a
(Novagen), respectively.
The plasmid expressing LexA-pICln was constructed by subcloning the
pICln coding region into pBTM116KN (11). A library
expressing the Gal4 activation domain fused to cDNA from human brain
(Clontech) was screened (2 × 106 colonies) in the
yeast strain L40 (11). All EST clones and constructs made by
PCR were verified by DNA sequencing.
Recombinant protein purification.
GST fusion proteins were
expressed in Escherichia coli BL21, purified over
glutathione-Sepharose according to the manufacturer's protocols, and
dialyzed against intracellular buffer (IB; 5 mM HEPES, 100 mM KCl [pH
7.4]). Trx fusion proteins were expressed in E. coli
BL21(DE3)pLys and purified over an immobilized nickel resin (Qiagen)
according to the manufacturer's protocol.
Purification of pICln-bound proteins.
pICln-bound proteins
(IBPs) were purified in one step by affinity chromatography over
immobilized GST-ICln fusion protein. Purified GST or GST-ICln was
covalently immobilized on 
-aminobutyl agarose (Sigma) by using
sulfo-SMPB (Pierce) as described previously (30) at 2 mg of
protein per ml of resin. Cytosolic MDCK proteins were isolated from
1.5 × 108 cells as described previously
(15) and precleaned by batch rotation with immobilized GST
resin for 3 h. Supernatant was supplemented with NaCl to 0.4 M and
rotated overnight with 200 µl of GST-ICln agarose. After vigorous
washing with isolation buffer containing 0.4 M NaCl, bound proteins
were eluted with sodium dodecyl sulfate (SDS) loading buffer, separated
by SDS-polyacrylamide gel electrophoresis, transferred to
polyvinylidene difluoride film (Millipore), and visualized by Coomassie
staining. Excised protein bands were trypsinized, peptides were
separated by high-pressure liquid chromatography, and two to three
peptides from each protein band were microsequenced.
Immunochemistry.
Generation and purification of rabbit
polyclonal anti-pICln antibody and monoclonal anti-pICln antibody were
described previously (15). Y12 anti-Sm antibody and anti-TMG
antibody were purchased commercially (Y12 from Labvision; anti-TMG from
Calbiochem). 2B1 anti-SMN monoclonal antibody (5) was a gift
from G. Dreyfuss. Immunoprecipitation from MDCK cell cytosolic and
nuclear extracts was performed as described previously (15).
When RNA from immunoprecipitates of cytosolic and nuclear extracts was
analyzed, the immunoprecipitates were washed extensively with a buffer
containing 20 mM Tris (pH 7.6), 350 mM NaCl, and 1% Triton X-100. RNA
from immunoprecipitates was recovered by proteinase K digestion, phenol
extraction, chloroform extraction, and ethanol precipitation. The RNA
was analyzed on a 10% polyacrylamide gel containing 7 M urea,
electroblotted onto nylon, and detected with a radiolabeled U1-specific
probe. For immunoprecipitation of Sm-bound RNA from oocytes, oocytes
were homogenized in phosphate-buffered saline (PBS) by pipetting, and soluble proteins were recovered by centrifugation. RNA was recovered and analyzed as described above.
Cell culture, [35S]methionine labeling, and cell
lysis.
MDCK cells were grown in Dulbecco modified Eagle medium
(DMEM) supplemented with 10% fetal calf serum (FCS). Proteins were labeled by growing cells in DMEM minus methionine supplemented with
10% dialyzed FCS and containing 50 µCi of
[35S]methionine (Amersham)/ml. Cell extracts
were prepared as described previously (15).
In vitro translation and binding assays.
Coupled in vitro
transcription and translation were performed by using the TNT system
(Promega) and [35S]methionine. SmE, SmF, and SmG were
expressed as C-terminal hexahistidine fusions so that their correct
synthesis could be demonstrated by purification on a metal affinity
resin (data not shown). GST binding assays were performed by incubating
5 µg of purified GST fusion protein with 10 µl of in
vitro-translated protein in binding buffer (25 mM Tris, 150 mM NaCl, 1 mM MgCl2, 1% Triton X-100 [pH 7.5]) containing 20 µl
of glutathione-Sepharose beads (Pharmacia) for 1 h at room
temperature. The beads were then washed five times with binding buffer,
once with 25 mM Tris (pH 7.5), and then boiled in SDS sample buffer.
The bound proteins were analyzed by electrophoresis on an SDS-15%
polyacrylamide gel. The lower chamber buffer contained standard running
buffer supplemented with 0.1 M sodium acetate. For binding assays
involving both SMN and pICln fusion proteins, the thioredoxin fusion
protein was preincubated with in vitro-translated Sm proteins for 30 min. Glutathione-Sepharose beads coated with 1 µg of GST fusion
protein were then added, and incubation was continued for 30 more min.
The samples were then washed and analyzed as described above.
In vitro transcription of U RNA and injection of oocytes.
pT7 U RNA plasmids were linearized at restriction enzyme sites designed
to generate U RNAs of an appropriate length. In vitro transcription was
performed by incubating 40 U of T7 RNA polymerase with 1 µg of
linearized plasmid in 1× transcription buffer (Promega) containing 10 mM dithiothreitol (DTT); a 500 µM concentration (each) of ATP, CTP,
and UTP; 50 µM GTP; 30 µCi of [32P]GTP (Amersham); 1 mM m7GpppG (Pharmacia); and 20 U of RNasin (Promega). After
2 h at 37°C, the reaction was terminated by incubating with
DNase for 15 min, followed by phenol extraction, chloroform extraction, G-50 spin column chromatography (Pharmacia), and ethanol precipitation. The pellet was resuspended at 106 cpm/µl. Stage 5 to 6 Xenopus oocytes were injected (Drummond microinjector) in
the cytoplasm with 50 nl of either IB, GST (4 mg/ml), or GST fusion
proteins (4 to 5 mg/ml) in the animal pole. Four hours later, 20 nl of
radiolabeled U RNA was injected into the vegetal pole. After 16 h
at 18°C, the oocytes were fixed by boiling for 3 min. The nucleus was
manually dissected from the cytoplasm. RNA from each fraction was
purified by proteinase K digestion, phenol-chloroform extraction, and
ethanol precipitation and analyzed by electrophoresis through an 8%
polyacrylamide gel containing 7 M urea.
For bandshift analysis, five injected oocytes per sample were
homogenized by pipetting in 50 µl of ice-cold buffer (25 mM Tris, 50 mM NaCl, 250 µg of yeast tRNA/ml). Ten microliters of the soluble
fraction was incubated with 1 µl of 100 mM DTT, 40 U of RNasin
(Promega), and 2 µl of either buffer, purified anti-Sm monoclonal
antibody (2 µg), or purified anti-pICln monoclonal antibody (21 µg). After 15 min at room temperature, an 8-µl aliquot was mixed
with 1 µl of loading buffer (30% glycerol, 0.25% bromphenol blue,
0.25% xylene cyanol) and run on a 6% polyacrylamide gel buffered with
0.5× Tris-borate-EDTA (TBE). RNA was recovered from a 4-µl aliquot
of the bandshift reaction mixture as described above and analyzed on an
8% polyacrylamide gel containing 7 M urea.
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RESULTS |
pICln binds Sm proteins.
pICln was immunoprecipitated from
cytosolic extracts of metabolically labeled MDCK cells by using a
polyclonal antibody generated to pICln. Several proteins consistently
coimmunoprecipitated with pICln (IBPs), with electrophoretic mobilities
of the major proteins corresponding to molecular masses of 72, 42, 28, and 17 kDa (Fig. 1, middle panel). We
have previously reported the purification and preliminary
characterization of IBP72 (14). This novel protein has
significant homology to yeast Skb1 protein, which binds a yeast homolog
of p21Cdc42/Rac-activated protein kinase and is implicated
in the regulation of cell morphology (8).
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FIG. 1.
pICln associates with several cytosolic proteins in
vivo, including Sm proteins. (Left panel) MDCK cell cytosolic proteins
purified by binding to immobilized GST (lane 2) or GST-pICln (lane 3).
Lane 1, molecular size standards. To better visualize nonspecifically
bound proteins, lane 2 was loaded with five times more sample than lane
3. (Middle panel) Proteins coimmunoprecipitated with endogenous pICln
from the cytosolic fraction of [35S]methionine-labeled
MDCK cells. (Right panel) MDCK cell cytosolic extract was
immunoprecipitated with a monoclonal anti-Sm antibody and immunoblotted
with a polyclonal anti-pICln antibody.
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IBP42, IBP28, and IBP17 were purified from MDCK cell cytosolic extracts
by affinity chromatography on immobilized GST-ICln fusion protein. The
IBPs were significantly enriched in eluates from the pICln affinity
resin (Fig. 1, left panel). The interaction was specific, since these
proteins did not bind to immobilized GST, and their electrophoretic
mobilities matched those of the proteins which coimmunoprecipitated
with pICln. The purified IBP42, IBP28, and IBP17 proteins were digested
with trypsin, and the resulting peptides were microsequenced. Using
peptide sequences obtained from IBP42, we cloned the corresponding
cDNA. IBP42 is a novel protein which contains several WD40 motifs
(26) and is most similar to the -subunit of photoreceptor
G-protein transducin. The significance of this protein interaction is
currently under study.
Several peptide sequences identified IBP28 and IBP17 as SmB/B' and
SmD3, respectively. SmB and SmB' are products of alternative splicing
of the same precursor RNA and differ only by 11 carboxy-terminal residues (34). The peptide sequences from IBP28 correspond
to the region common to SmB and SmB'. Both SmB and SmB' may bind pICln,
given that a second protein with a molecular weight slightly higher
than that of IBP28 coimmunoprecipitated with pICln (Fig. 1).
To confirm the interaction of pICln with Sm proteins, we used several
independent approaches. First, Sm proteins were immunoprecipitated from
MDCK cell cytosolic extract by using the anti-Sm monoclonal antibody
Y12 (19), and pICln was detected in the precipitate by
immunoblotting with an anti-pICln monoclonal antibody (Fig. 1, right
panel). In control experiments, pICln was not detected in
immunoprecipitates generated with an unrelated monoclonal antibody (data not shown). Second, we used a LexA-pICln fusion protein as bait
to screen a human brain library in the yeast two-hybrid system. From
this screen, we isolated five clones that contained full-length SmD3
cDNA. Clones encoding the other Sm proteins were not isolated, although
not all positives were examined due to a high degree of background
activation by the LexA-pICln fusion protein itself. Third, we tested
the ability of recombinant, purified pICln to associate with in
vitro-translated Sm proteins. When in vitro-translated Sm proteins were
incubated with GST-ICln immobilized on glutathione-Sepharose beads,
SmB', SmD1, and SmD3 bound to GST-ICln but not to GST alone (Fig.
2). The finding that in vitro-translated SmD1 binds to GST-ICln is compatible with our coimmunoprecipitation experiments, since SmD1, SmD2, and SmD3 have similar electrophoretic mobilities (18), and there is clearly more than one protein with an electrophoretic mobility corresponding to 17 kDa which coimmunoprecipitates with pICln and which binds to pICln affinity resin
(Fig. 1). There was no detectable binding of SmF and SmG to GST-ICln.
SmD2 and SmE bound to GST-ICln, although the ratio of specific to
nonspecific binding for these two proteins was lower than for SmB',
SmD1, and SmD3. We did not investigate the possible interaction of SmD2
and SmE with pICln further, because these interactions appeared to be
the weakest, and we lacked independent data to substantiate these
interactions.
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FIG. 2.
pICln interacts with Sm proteins in vitro. (a) (Left
panel) 35S-labeled Sm protein in vitro translation products
input into binding assays. (Right panel) GST-ICln fusion proteins used
in binding assays, separated on an SDS-10% polyacrylamide gel and
stained with Coomassie blue. GST-hICln deletion mutants are labeled
with the numbers of the pICln amino acids present in the fusion
protein. (b) GST or GST-ICln immobilized on glutathione-Sepharose beads
was incubated with in vitro-translated 35S-labeled Sm.
After extensive washing, bound proteins were analyzed by polyacrylamide
gel electrophoresis and visualized by using a phosphorimager. In vitro
translation of SmD3 consistently resulted in two products, both of
which bound to GST-ICln. (b) Deletion analysis of pICln-Sm interaction.
In vitro-translated SmB', SmD1, or SmD3 was incubated with deletion
mutants of pICln fused to GST and immobilized on glutathione-Sepharose
beads. Input levels of in vitro-translated proteins are shown in panel
a, and binding to GST or GST-ICln from the same experiment is shown in
panel b. 35S-labeled proteins were visualized by using a
phosphorimager.
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To outline the region of pICln that interacts with Sm proteins, we
generated proteins consisting of various deletions of pICln fused to
GST and analyzed the ability of these proteins to bind in
vitro-translated SmB', SmD1, and SmD3 (Fig. 2c). hICln[1-199] binds
to SmB', SmD1, and SmD3 as efficiently as the wild type. However,
hICln[1-36] and hICln[175-237] do not bind Sm proteins, indicating
that the extreme N-terminal and C-terminal ends of the pICln are not
necessary for Sm protein binding. We have previously shown that the
C-terminal portion of pICln is necessary and sufficient for pICln
interaction with IBP72 (14). The failure of the truncated pICln fusion proteins to binding Sm proteins also provides additional negative controls for binding specificity in the in vitro-binding assay.
pICln is not a component of snRNPs.
We next asked whether
pICln interacts with Sm proteins assembled in snRNPs in addition to
free Sm proteins. To determine if pICln is a component of snRNPs, we
used two independent methods. First, we immunoprecipitated RNAs
containing a trimethylguanine (TMG) from MDCK cell nuclear and
cytoplasmic extracts by using a TMG antibody. Since U1, U2, U4, and U5
RNAs contained in snRNPs possess a TMG cap, while free U RNAs possess a
monomethylguanine cap, TMG immunoprecipitates contain predominantly
snRNPs. When the TMG immunoprecipitates were immunoblotted with a
pICln-specific antibody, we failed to detect pICln (Fig.
3a), suggesting that it is not a
component of snRNPs. In contrast, a pICln antibody immunoprecipitated
pICln from the same extracts, indicating that pICln was detectable by
this assay. Second, we completely immunoprecipitated pICln from MDCK
cell nuclear and cytoplasmic extracts and probed for the presence of U1
RNA. U1 RNA was not coimmunoprecipitated by the pICln-specific antibody
above the background amount precipitated by an unrelated antibody (Fig.
3b). In contrast, U1 RNA was immunoprecipitated by anti-TMG and
coimmunoprecipitated by anti-Sm antibodies. Since U1 RNA is an integral
component of U1 snRNPs, this result further demonstrates that pICln is
not a part of snRNPs.
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FIG. 3.
pICln is not a component of snRNPs. (a) SnRNPs were
immunoprecipitated from MDCK cell cytosolic (C) or nuclear (N) extracts
with TMG-specific antibody or pICln-specific antibody. The
immunoprecipitate, the supernatant (Sup), and the untreated extract
were analyzed by immunoblotting with a monoclonal pICln-specific
antibody. (b) MDCK cell cytosolic (C) and nuclear (N) extracts were
immunoprecipitated with antibodies specific for pICln, TMG, Sm, and an
unrelated antigen. Ctrl Ab, control antibody. RNA from the
immunoprecipitates and from untreated extract was deproteinized,
electrophoresed on a denaturing polyacrylamide gel, and transferred to
a nylon membrane. U1 RNA was detected by hybridization to a U1-specific
probe. U1 content in the pICln immunoprecipitate was negligible and
indistinguishable from the control immunoprecipitate, in contrast to
prominent U1 signals in TMG and Sm immunoprecipitates.
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If pICln binding to non-snRNP Sm proteins is functionally important,
then it would be reasonable to expect that a significant fraction of
cytosolic Sm proteins is bound to pICln. To measure the fraction of
cytoplasmic Sm proteins bound to pICln, we immunodepleted cytosolic
extracts of pICln with an anti-pICln-specific antibody and measured
SmB/B' content of the depleted extract by immunoblotting with an
Sm-specific antibody (Fig. 4).
Greater-than-95% pICln depletion was verified by immunoblotting with a
monoclonal anti-pICln antibody (Fig. 4a). We found that immunodepletion
of pICln reduced the amount of SmB/B' by 80% (Fig. 4b), indicating
that the majority of cytoplasmic SmB/B' was bound to pICln. These
immunodepletion experiments were performed under stringent conditions
(1% Triton X-100 and 350 mM NaCl) to ensure minimal nonspecific
binding SmB/B' to the immunoprecipitates. Under more-physiological
conditions (150 mM NaCl and no detergent), more than 95% of SmB/B' was
depleted by anti-pICln antibody (data not shown). We were unable to
perform a similar experiment with SmD1 or SmD3, since our preparation of Y12 antibody poorly recognized these proteins in cytosolic extract.
However, SmD3 has been shown to form stable RNA-free complexes with
SmB/B' (7, 33) and therefore it is likely that the majority
of SmD3 also coimmunoprecipitates with pICln.
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FIG. 4.
The majority of cytosolic SmB/B' is bound to pICln. (a)
pICln was immunoprecipitated from MDCK cell cytosolic extract with a
purified polyclonal anti-pICln antibody. More than 95% of pICln was
immunoprecipitated, as demonstrated by immunostaining the supernatant
with an anti-pICln antibody (top panel). Immunostaining the pICln
immunoprecipitate with anti-Sm protein antibody shows that SmB/B'
coimmunoprecipitated with pICln (bottom panel). An unrelated antibody
(Ctrl) did not immunoprecipitate pICln or SmB/B'. (b) Immunodepletion
of pICln results in depletion of SmB/B'. Cytosolic extract from MDCK
cells was immunodepleted of pICln as shown in panel a. SmB/B' content
in the extract prior to (No antibody) and after immunoprecipitation
with pICln or unrelated antibody was assessed by Western blotting with
Y12 antibody (insert) and quantitated by densitometry in four
independent experiments. The amount of SmB/B' in pICln-depleted samples
was normalized to the amount in control samples in each experiment.
Treatment of sample with an unrelated antibody and protein A beads did
not change SmB/B' content in the extract. Bars indicate standard errors
of the means.
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pICln inhibits Sm protein assembly on U RNA.
Our finding that
pICln binds to Sm proteins only when they are not assembled in snRNPs
raised the possibility that pICln may affect Sm protein assembly on U
RNA. Much of our understanding of snRNP biogenesis has come from
experiments in the X. laevis oocyte model. Oocytes differ
from most cell types in that the vast majority of Sm and SMN proteins
are localized in the cytoplasm (6, 36). The oocyte model has
been used successfully to study snRNP biogenesis in vivo, including Sm
assembly on U RNA, U RNA cap hypermethylation, and snRNP nuclear import
(3, 24, 36). We elected to use this model to study the
effect of pICln on snRNP biogenesis. For these experiments, we used
pICln cloned from an ovarian Xenopus cDNA library
(15). pICln is expressed in Xenopus oocytes
(15) and is associated with Sm proteins, as was demonstrated by coimmunoprecipitation experiments (data not shown).
To determine if pICln interaction with Sm proteins influences Sm core
domain formation on U RNA, we injected oocytes with Xenopus
pICln fused to GST (GST-xICln). The oocytes were then injected with
32P-labeled, in vitro-transcribed U1, U2, U4, U5, and U6
snRNAs. Subsequently, the oocytes were homogenized, and U RNA bound by Sm proteins was immunoprecipitated by using an anti-Sm antibody and
analyzed by denaturing polyacrylamide gel electrophoresis (Fig.
5a). In control oocytes injected with
buffer or GST, more than 80% of injected U1, U2, U4, and U5 RNA
coimmunoprecipated with Sm proteins. The immunoprecipitation was
specific, because U6 RNA, which does not bind Sm proteins
(10), is not significantly precipitated by the anti-Sm
antibody (Fig. 5a). Injection of GST-xICln significantly inhibited
binding of U1, U2, U4, and U5 RNAs to Sm proteins (P < 0.05; Fig. 5a and b), with U5 association with Sm protein most
affected (85% inhibition). The degrees of inhibition were different
for each U RNA (P < 0.01; Fig. 5b), except for those
for U2 and U4.
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FIG. 5.
pICln inhibits Sm binding to U RNA. (a) Oocytes were
injected with buffer, GST, or GST-xICln, followed by a mixture of
32P-labeled U1, U2, U4, U5, and U6 RNA. The oocytes were
then homogenized, and Sm-associated U RNA was immunoprecipitated with
the anti-Sm antibody Y12. The immunoprecipitate and supernatant were
deproteinized and analyzed on a denaturing polyacrylamide gel. Each
sample represents a pool of four oocytes. Ctrl indicates the mixture of
U RNAs prior to injection. The different amounts of each U RNA reflect
different amounts of U RNA produced from each template in in vitro
transcription reactions. (b) Quantitation of the results of panel a.
The amount of U RNA present in the immunoprecipitate and supernatant
was quantitated by measuring radioactivity in gels on a phosphorimager.
The percentage of U RNA found in the precipitate of pICln-injected
oocytes was plotted relative to the percentage of U RNA in the
precipitate in the buffer-injected control oocytes.  , significant
inhibition due to GST-xICln injection compared to buffer or GST
controls (P < 0.03). Each column represents the
average (error bar, standard error of the mean) for five (GST) or six
(buffer, GST-xICln) samples. (c) Bandshift analysis of Sm binding to U
RNA. Oocytes were injected with GST (G) or GST-xICln (I) and
subsequently injected with either 32P-labeled U1 (1), U5
(5), or U1 (), a U1 mutant that lacks the Sm binding site. The
oocytes were homogenized, and either buffer, anti-Sm antibody, or
anti-pICln antibody was added. An aliquot was analyzed by nondenaturing
polyacrylamide gel electrophoresis (top panel). RNA was recovered from
another aliquot of the same bandshift reaction mixture and analyzed by
denaturing gel electrophoresis (bottom panel). The square bracket
indicates U RNA with reduced mobility due to protein binding. The arrow
indicates complexes with very low mobility due to antibody binding. The
positions of free U1, U5, and U1 probes are shown.
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Although pICln inhibition of U RNA immunoprecipitation by anti-Sm
antibody strongly suggests that pICln association with Sm proteins
prevents Sm protein binding to U RNA, an alternative explanation is
that pICln binding to Sm proteins prevents them from being recognized
by the anti-Sm antibody. To exclude this possibility and confirm the
inhibitory effect of pICln by an independent method, we used the
mobility shift assay to assess Sm protein binding to U RNA. Oocytes
were injected with GST-xICln, followed by injection with
32P-labeled, in vitro-transcribed U1. Extracts from these
oocytes were then analyzed on a nondenaturing polyacrylamide gel. In
control oocytes injected with GST, several discrete complexes were
present (square bracket, Fig. 5c). The complex contained Sm proteins
specifically bound to U1 RNA, since the mobility of the complex was
reduced by incubation with anti-Sm antibody (arrow, Fig. 5c).
Furthermore, this complex does not form on U1, a U1 deletion mutant
that lacks the Sm binding site (lane 1). Although a small amount of
U1 is bound in a lower mobility complex, this complex does not
involve Sm proteins because the mobility of the complex is not further reduced by anti-Sm antibody (lane 4). When oocytes were injected with
GST-xICln, the amount of U1 RNA incorporated into the Sm-U1 RNA complex
was decreased (lane 2 versus lane 3), as was the amount which could be
supershifted by the addition of anti-Sm antibody (lane 5 versus lane
6). Similar results were found by using a U5 RNA probe. U5 RNA forms
protein complexes with reduced mobility (lane 9); these complexes
involve Sm protein since they can be supershifted by the addition of
anti-Sm antibody (lane 12), and pICln interferes with the U5-Sm protein
interaction (lane 9 versus lane 10). The inhibitory effect of pICln was
not due to degradation of the labeled U RNA probe, since the same
amount of intact U RNA probe was recovered from the GST and GST-xICln
bandshift reactions (Fig. 5c, bottom panel). Thus, in agreement with
the previous experiment, pICln inhibited Sm protein binding to U RNA.
Finally, the anti-pICln antibody had no effect on the electrophoretic
mobility of the Sm-containing complex (Fig. 5c, lanes 7, 8, and 11),
consistent with our finding that pICln was not associated with snRNPs
(Fig. 3).
pICln inhibits U RNA nuclear transport in Xenopus
oocytes.
Since snRNP biogenesis requires Sm core domain formation
on U RNA and pICln inhibits this assembly, we predicted that pICln should influence snRNP biogenesis in vivo. To test this hypothesis, we
studied the nuclear import of U RNA, the final step of snRNP biogenesis. We injected oocytes in the cytoplasm with GST-xICln and
then with 32P-labeled, in vitro-transcribed U snRNAs. We
measured U RNA nuclear import by quantitating labeled U RNA recovered
separately from nuclear and cytoplasmic fractions. In control oocytes
injected with buffer or GST, 25 to 30% of the injected RNA was
imported into the nucleus (Fig. 6a). In
contrast, following injection of GST-xICln, nuclear import of U RNA was
significantly inhibited (Fig. 6a). The effect of pICln varies for the
different U RNAs (Fig. 6b), with transport of U1 and U5 most inhibited.
U2 and U4 nuclear import was not substantially inhibited by pICln.
Thus, the degree to which pICln inhibited U RNA interaction with Sm proteins (Fig. 5b) correlated with the ability of pICln to inhibit nuclear import (Fig. 6b), suggesting that inhibition of nuclear import
by pICln is due at least in part to inhibition of Sm core domain
assembly.
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FIG. 6.
pICln inhibits nuclear import of U RNA. (a) Oocytes were
injected with buffer, GST, or GST-xICln and then with
32P-labeled, in vitro-transcribed U1, U2, U4, and U5 RNAs.
RNA was recovered from nuclear (N) and cytoplasmic (C) fractions and
analyzed by denaturing polyacrylamide gel electrophoresis. Each sample
represents a pool of five oocytes. The lane labeled control contains
the mixture of RNAs prior to injection. U RNAs imported into the
nucleus were slightly smaller than the injected U RNAs due to 3' end
trimming (27). The standard is a labeled RNA that was added
in equal amounts to the nuclear and cytoplasmic fractions following
dissection to ensure equal recovery during processing of the samples.
(b) Quantitation of the effect of GST-xICln on individual U RNAs. The
amount of each U RNA in the nuclear and cytoplasmic fractions of GST or
GST-xICln injected oocytes was determined by analyzing gels, such as
shown in panel a, on a phosphorimager. Results are displayed as the
percentage of nuclear import of each U RNA in GST-xICln injected
oocytes relative to GST injected oocytes. Each column represents the
average (error bar, standard deviation) of two independent
experiments.
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|
pICln inhibits SMN binding to Sm proteins.
We have shown that
pICln binds to Sm proteins and inhibits their assembly on U RNA. The
protein SMN also binds Sm proteins and was suggested to mediate their
assembly on U RNA (5, 20). SMN and pICln do not coexist in a
single complex with Sm proteins, since an anti-SMN antibody failed to
detect SMN in pICln immunoprecipitates (data not shown). We
hypothesized that a complex containing all three proteins was not
detected because pICln and SMN binding to Sm protein was mutually
exclusive. To test this hypothesis, we asked whether pICln binding to
Sm proteins could prevent these proteins from interacting with SMN. For
this experiment, we measured in vitro binding of in vitro-translated Sm
proteins to SMN fused to GST (GST-SMN) in the presence of pICln
competitor (a thioredoxin-ICln fusion protein, Trx-ICln). SmB', SmD1,
and SmD3 binding to SMN was significantly decreased by Trx-ICln
compared to an equal amount of Trx alone (Fig.
7). This inhibitory effect of pICln was
most pronounced for SmD1 and SmD3, which reduced binding to SMN by 82 and 99%, respectively. We also performed the reverse experiment, in
which we measured the binding of in vitro-translated Sm proteins to
pICln (GST-ICln) in the presence of SMN competitor (Trx-SMN). SMN
inhibited the binding of SmB', SmD1, and SmD3 by pICln. Since in both
experiments we used nearly saturating amounts of the inhibitory proteins but the inhibitory effect was not complete and not identical for pICln and SMN, the inhibitory effect may not reflect simple competition for Sm binding. These results, together with the absence of
SMN in pICln immunoprecipitates, lead us to conclude that pICln interferes with SMN binding to Sm proteins in vivo and in vitro.
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FIG. 7.
pICln interferes with SMN binding to Sm proteins. (a)
Fusion proteins used, separated on an SDS-10% polyacrylamide gel and
stained with Coomassie blue. (b) In vitro-transcribed and -translated,
35S-labeled SmB', SmD1, and SmD3 were preincubated with 12 µg of either Trx, Trx-ICln, or Trx-SMN. The mixture was then
incubated with 1 µg of either GST, GST-SMN, or GST-ICln bound to
glutatione beads, and the beads were vigorously washed. Bound Sm
proteins were analyzed by SDS-polyacrylamide gel electrophoresis. (c)
Quantitation of the results in panel b. A phosphorimager was used to
measure the amount of each Sm protein bound in the presence of Trx or
inhibitor protein (Trx-SMN or Trx-ICln). Relative binding was
calculated as the percent of Sm protein bound in the presence of
inhibitor compared to the amount bound in the presence of Trx (i.e.,
binding in the presence of Trx was taken as 100%). Each column
represents the average (error bar, standard deviation) of five to six
independent measurements.
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|
| |
DISCUSSION |
We have demonstrated that pICln binds to Sm proteins by several
independent methods. First, microsequencing of proteins purified by
affinity to pICln identified two pICln-binding proteins as SmB/B' and
SmD3. Second, immunoprecipitation of Sm proteins coimmunoprecipitated pICln. Third, a bait containing pICln extracted SmD3 from a cDNA library in a yeast two-hybrid screen. Fourth, purified GST-ICln bound
to in vitro-translated SmB', SmD1, and SmD3. Finally, proteins which
coimmunoprecipitated with pICln from MDCK cell cytoplasmic extracts had
electrophoretic mobilities appropriate for SmB/B' and SmD1 and SmD3.
Although these experiments do not define which Sm proteins directly
bind to pICln, they clearly demonstrate that Sm proteins and pICln form
a complex. Furthermore, this interaction is likely to be functionally
important, since immunodepletion experiments showed that in MDCK cells
the majority of cytoplasmic Sm protein (or at least SmB/B') is bound to pICln.
Binding of Sm proteins to pICln inhibited their assembly on U RNA to
form the Sm core domain, as determined by immunoprecipitation and
mobility shift assays. By inhibiting Sm core domain assembly on U RNA,
high concentrations of pICln interfered with snRNP nuclear import in
Xenopus oocytes. At the maximal GST-xICln concentration tested (5.5 µM), there was a 2.5-fold inhibition of nuclear import. Although this concentration of pICln was 25-fold higher than the normal
oocyte pICln concentration (0.2 µM [15]), this was
expected given the high concentration of Sm proteins in the oocyte
cytoplasm. In previous studies, 1.5 to 15 µM U1 RNA was required to
completely bind the endogenous cytoplasmic pool of free Sm proteins
(4). Using this as a minimal estimate of the oocyte
cytoplasmic Sm protein concentration, the concentration of pICln
necessary for effective inhibition of nuclear import was reasonable if
inhibition required stoichiometric binding of pICln to Sm proteins.
Although lack of U RNA, rather than the presence of pICln, is likely
the primary factor which regulates snRNP assembly in oocytes
(36), the nuclear import assay demonstrates clearly a
functional consequence of pICln binding to Sm proteins. In contrast to
oocytes, in the majority of mammalian cells the cytoplasmic Sm
concentration is low and pICln is relatively abundant, and the majority
of cytoplasmic Sm is bound to pICln. Under these conditions, pICln may
play an important role in the regulation of snRNP assembly.
Much has been learned recently about the assembly pathway of the Sm
core domain. The Sm proteins have been found in three stable RNA-free
complexes (D1-D2, E-F-G, and D3-B/B') (5, 7, 17, 33). The
D1-D2 and E-F-G Sm protein complexes can together form a stable complex
with U RNA. This subcore particle then binds the D3-B/B' complex to
form the complete core domain (33). Our finding that pICln
binding to complexes containing SmB/B', D1, and D3 interfered with the
ability of Sm proteins to associate with U RNA is consistent with this model.
We have also found that pICln binding to Sm proteins inhibited their
association with SMN protein. This protein forms a complex with the
protein SIP1, and the SMN-SIP1 complex binds to SmB/B', D1-3, and E
(20). Antibodies directed against SIP1 blocked assembly of
the Sm core domain on U RNA, while anti-SMN antibodies enhanced this
process. These results implicate the SMN-SIP1 complex in Sm core domain
assembly and suggest that by binding Sm proteins, the complex may
facilitate their binding to U RNA (5). In combination with
our results, these data lead us to propose a model in which pICln
inhibits Sm protein assembly on U RNA, at least in part by inhibiting
Sm protein interaction with SMN (Fig. 8).
Consistent with this model, U1 and U5 RNAs efficiently
coimmunoprecipitated with SIP1 or SMN and were most inhibited by pICln,
while U2 and U4 RNAs were weakly coimmunoprecipitated with SIP1 or SMN
(5) and were relatively insensitive to pICln (Fig. 5 and 6).
Although this model is compatible with the existing data, alternative
models are possible. For instance, we have depicted SMN as an integral component of the assembly pathway, whereas it may actually be a
regulatory element. SMN and pICln both influence snRNP biogenesis at
the level of Sm core domain assembly, but a role for these proteins in
regulating other steps in the biogenesis pathway has not been excluded.
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FIG. 8.
Model of pICln and SMN regulation of Sm protein assembly
on U RNA. pICln is depicted as inhibiting Sm core domain assembly and U
RNA nuclear import by preventing Sm interaction with SMN.
Hypermethylation and nuclear import of U RNA occur after Sm core domain
assembly, although the timing of these events relative to SMN
dissociation is not known.
|
|
In addition to promoting snRNP biogenesis, SMN may also be essential
for proper spliceosome function, since a dominant-negative SMN mutant
inhibits pre-mRNA splicing in vitro and causes dramatic nuclear snRNP
reorganization in vivo (32). The inhibitory effect of the
dominant-negative mutant on in vitro splicing was observed only when
the SMN mutant was preincubated with splicing extract prior to addition
of pre-mRNA (32). One interpretation of these results is
that splicing results in snRNP rearrangement to an inactive form and
that SMN is necessary for the regeneration of functional snRNPs. Since
pICln is also present in the nucleus, pICln could potentially regulate
snRNP recycling by modulating the interaction of SMN with snRNPs. In
keeping with the participation of pICln in a critical cellular
function, loss of pICln in mice results in embryonic lethality between
3.5 and 7.5 days postcoitus, and embryonic stem cells lacking both
pICln alleles cannot be generated in tissue culture (35).
In summary, we have described a novel interaction between pICln and
several Sm proteins, and we have demonstrated that this interaction
inhibits association of Sm proteins with SMN, Sm protein binding
to U RNA, and snRNP biogenesis. We have presented a model that accounts
for these observations. Experiments are in progress to test this
model and to determine the mechanism by which loss of pICln leads to
embryonic lethality.
W.T.P. and G.B.K. contributed equally to this work.
We thank John Leszyk for his peptide microsequencing expertise and
Kevin Wickman for helpful discussions. I. Mattaj kindly provided T7-U
RNA in vitro transcription plasmids. G. Dreyfuss generously provided us
with anti-SMN antibody.
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Abstract
Introduction
