1 Medical Research Council Protein Phosphorylation Unit, Division of Cell
Signalling, University of Dundee, Dundee DD1 5EH, Scotland, UK
2 Division of Gene Regulation and Expression, School of Life Sciences,
University of Dundee, Dundee DD1 5EH, Scotland, UK
* Author for correspondence (e-mail: p.t.w.cohen{at}dundee.ac.uk)
Accepted 3 February 2003
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Summary |
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Key words: Gemin4, Gemin3, Spinal muscular atrophy, Spliceosome, Cajal bodies (coiled bodies), snRNP
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Introduction |
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SMN is expressed in all tissues and localises both in the nucleus and the
cytoplasm. In the nucleus of most cultured cells and primary neurons, SMN and
Gemin2 localise in the Cajal (also termed coiled) bodies
(Carvalho et al., 1999;
Matera, 1999
;
Sleeman and Lamond, 1999
).
These bodies are discrete nuclear structures that are known to contain
components such as small nuclear ribonucleoproteins (snRNPs), small nucleolar
ribonucleoproteins (snoRNPs) and transcription factors, which suggest that
Cajal bodies have a role in snRNP and snoRNP biogenesis and metabolism. In a
small proportion of rapidly proliferating cells in some cell lines in culture,
SMN localises in discrete foci, often near the Cajal bodies called gems
(gemini of coiled bodies) (Liu and
Dreyfuss, 1996
; Liu et al.,
1997
).
Several studies implicate SMN complexes in spliceosome assembly and
regeneration of splicesomal components. In the cytoplasm, the SMN complex
appears to be involved in the binding of small nuclear uridine-rich (U) RNAs
to Sm proteins for the assembly of spliceosomal snRNPs
(Bühler et al., 1999;
Fischer et al., 1997
;
Meister et al., 2000
). The SMN
complex is then believed to accompany the assembled snRNPs to the Cajal bodies
in the nucleus, where it might be involved in the generation of active
spliceosomes and the recycling of snRNPs after each round of pre-mRNA splicing
(Matera, 1999
;
Pellizzoni et al., 1998
). An
SMN complex has also been implicated in the assembly and metabolism of snoRNPs
required for pre-rRNA splicing (Pellizzoni
et al., 2001a
). A recent study indicates that an SMN complex may
play a central role in the assembly of transcriptosomes
(Pellizzoni et al., 2001b
),
large complexes containing RNA polymerase I, II or III, transcription factors
and spliceosomes for the coordinated synthesis and processing of mRNA and
snRNA (Gall et al., 1999
). The
interaction between the SMN protein and RNA polymerase II was shown to be
mediated by RNA helicase A, which although not a `core' component of the SMN
complex, may be present in a subset of SMN complexes and is suggested to be a
substrate of the SMN complex (Pellizzoni
et al., 2001b
). Interestingly, hnRNP-R, a protein that is involved
in RNA processing, is predominantly expressed in the axons of motor neurons
and interacts with SMN (Rossoll et al.,
2002
). To date there is no information on how the functions of the
SMN complex are regulated or coordinated with other cellular functions.
Reversible protein phosphorylation is a key mechanism for the control of
cellular processes in eukaryotes. PPP4 (originally termed PPX) is a ubiquitous
protein phosphatase that dephosphorylates serine and threonine residues
(Cohen, 1997). The catalytic
subunit, PPP4c, is very highly conserved from mammals to Drosophila
(91% amino acid identity) (Brewis and
Cohen, 1992
) and production of a D. melanogaster mutant
deficient in PPP4c has demonstrated that this phosphatase is essential for the
nucleation, growth and/or organisation of microtubules at centrosomes
(Helps et al., 1998
). Analysis
of the homologue in Caenorhabditis elegans by RNA-mediated
interference showed that PPP4c is also essential for formation of the mitotic
spindle in mitosis and is required for sperm meiosis
(Sumiyoshi et al., 2002
). In
accordance with these data, PPP4c exhibits a prominent localisation at
centrosomes in cultured mammalian cells
(Brewis et al., 1993
). However,
its high expression in the nucleus with weak expression in the cytoplasm
suggests that PPP4c regulates additional cellular functions.
PPP4c is a member of the PPP family of protein phosphatases and is most
closely related to PP2Ac (65% amino acid identity) and PPP6c (
60%
identity), with less similarity to PP1 (
45% identity). The PP2A
holoenzyme comprises a heterodimeric `core' of PP2Ac complexed to an A (PR65)
regulatory subunit, and this dimer may then bind to one of a number of
different B regulatory subunits (Janssens
and Goris, 2001
). However, PP1c forms heterodimeric holoenzymes
complexes in which the catalytic subunit is bound to one of more than 40
distinct regulatory subunits (Cohen,
2002
). Interaction occurs via a short conserved motif that is
present in many of the regulatory subunits, and the different regulatory
subunits may target PP1c to different subcellular locations. Sit4p, the S.
cerevisiae homologue of mammalian PPP6c, also exists as heterodimeric
complexes containing one variable subunit
(Luke et al., 1996
).
PPP4 exists as high molecular mass complexes of 450-600 kDa, and two
putative regulatory subunits have been identified, R1
(Kloeker and Wadzinski, 1999)
and R2 (Hastie et al., 2000
).
However, the subunit composition of higher molecular mass complexes of PPP4 is
unclear, and the structures of R1 and R2 regulatory subunits do not provide
any additional information on the location or function of these PPP4
complexes. Here, we examine the PPP4 complexes that contain R2 and identify as
novel `variable' regulatory subunit(s) Gemin3 and/or Gemin4, which are
components of the SMN complex. We also present data suggesting that R2-PPP4c
enhances the maturation of snRNPs, a function in which the SMN complex is
implicated.
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Materials and Methods |
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Fluorescence in situ hybridisation
The Vysis Nick Translation Kit was used to directly label purified R2 cDNA
or BAC DNA with the fluorochrome Spectrum Red as described in the
manufacturer's protocol. The labelled probe was used for hybridisation of
metaphase chromosome spreads.
Screening of LLNL human single chromosome libraries
Chromosome 3 and 5 libraries, constructed in the cosmid Lawrist 16 at
Lawrence Livermore National Laboratory, USA, were kindly provided by the UK
HGMP Resource Centre in the form of high-density gridded filters. The
libraries had an average insert size of 30-50 kb and contained approximately
20,000 clones providing a three- to four-fold coverage of each chromosome. The
filters were screened by Southern blotting, using the R2 coding region cDNA
(0.8 kb) as a probe. Positive clones, kindly provided by the HGMP, were
analysed using PCR with R2 specific primers or Topo shotgun cloning
(Invitrogen, Groningen, Netherlands). Three chromosome 5 R2 positive clones
were sequenced in both directions.
RNA analyses
Northern blots (Clontech, Palo Alto, CA) contained approximately 2 µg
poly (A)+ RNA from different tissues. The blots were hybridised
with R2 and PPP4c probes according to the manufacturer's instructions, with
the last wash in 15 mM NaCl/1.5 mM sodium citrate/0.1% SDS at 55°C.
Following autoradiography, blots were stripped by washing the membrane in 0.5%
SDS at 100°C for 5 minutes and subsequently reprobed with a control
ß-actin probe.
Cell culture, transfection and preparation of lysates
Human embryonic kidney (HEK) 293 cells were cultured, transfected and lysed
as described previously (Hastie et al.,
2000). HeLa cells expressing cyan fluorescent protein tagged SMN
protein (Sleeman et al., 2003
)
were cultured and lysed as described previously
(Hastie et al., 2000
).
Purification of Flag-R2PPP4c complexes
20-100 10 cm3 dishes of 293 cells were transfected with
pCMV5-Flag-R2 and cultured for 44 hours prior to lysis in buffer A (50 mM
Tris/HCl pH 7.5, 0.03% Brij-35, 2 mM EDTA, 0.1 mM EGTA) plus 5% glycerol, 0.15
M NaCl and `Complete' protease inhibitor cocktail (Roche Diagnostics Ltd,
Lewes, UK). The lysate was mixed with 5 ml of anti-Flag agarose (Sigma, Poole,
UK) in a tube by end over end rotation for1 hour at 4°C. The agarose beads
were separated by centrifugation and washed in buffer A containing 5% glycerol
and 500 mM NaCl several times for 1 hour at 4°C. Bound material was eluted
from the anti-Flag agarose column by the addition of 20 ml Flag peptide (100
µg/ml) and collected in 20x1 ml fractions. These fractions were
analysed for co-elution of Flag-R2 and PPP4c. Peak fractions were then pooled,
concentrated and desalted in buffer A using a Vivaspin column (Vivascience,
Lincoln, UK) prior to further study. Gel filtration analysis of the purified
Flag-R2PPP4c material on Superose 6 columns was performed as described
previously (Hastie et al.,
2000). Molecular mass marker proteins used were thyroglobulin (670
kDa), ferritin (450 kDa),
-globulin (158 kDa), ovalbumin (44 kDa),
myoglobin (17 kDa) and vitamin B12 (1.35 kDa).
Identification of proteins co-eluting with Flag-R2PPP4c
Purified Flag-R2PPP4c material was fractionated by SDS-PAGE and
stained with Coomassie Blue. Proteins that consistently co-eluted with Flag-R2
and PPP4c were excised from the gel and digested in situ with trypsin as
described previously (Jensen et al.,
1997). Tryptic peptide masses were analysed using a thin film
matrix of 4-hydroxy-
-cyanocinnamic acid/nitrocellulose (2:1) in an
Elite STR mass spectrometer (PerSeptive Biosystems, Foster City, CA, USA) in
reflectron mode. Spectra were internally calibrated with matrix and trypsin
autolysis ions and the peptide masses were used to search databases within the
UCSF Protein Prospector program using MS-FIT analysis software with a mass
error set at 50 ppm. Flag-R2 and PPP4c were initially identified by
immunoblotting but were subsequently verified by identification using mass
spectrometry.
Immunological techniques
Immunoblotting was performed following fractionation of proteins by
SDS/PAGE, using NuPage 4-12% Bis-Tris gels (Invitrogen, Groningen,
Netherlands), and transfer to nitrocellulose membranes (Shliecher and Schull,
Dassel, Germany). The blots were probed with affinity-purified antibodies, and
antibody binding was detected using anti-sheep IgG antibodies conjugated to
horseradish peroxidase, followed by enhanced chemiluminescence (Amersham
International, Little Chalfont, UK). Anti-PPP4c antibodies were raised against
the N-terminal 57 amino acids of human PPP4c
(Brewis et al., 1993).
Anti-Gemin4 antibodies were made against the peptide AEGIGPEERRQTLLQKMSSF
(corresponding to amino acids 1039-1059 of Gemin4) and coupled to keyhole
limpet haemocyanin. The above antibodies were raised in sheep at the Scottish
Antibody Production Unit, Carluke, Penicuik, Midlothian, UK and affinity
purified. Murine anti-Gemin3 antibodies were purchased from BD Transduction
Laboratories (Lexington, KY, USA) and murine anti-Flag antibodies were
purchased from Sigma. Anti-GFP and anti-Xpress antibodies were purchased from
Invitrogen (De Schelp, Netherlands). Anti-sheep and anti-mouse secondary
antibodies were from Pierce (Chester, UK).
Immunoprecipitation of tagged proteins expressed in mammalian cells was
carried out using lysates containing 1 mg of total cell protein for each
immunoprecipitation. The lysates were precleared by incubation at 4°C for
50 minutes on a shaking platform with sheep preimmune IgG covalently coupled
to protein G-Sepharose using dimethylpimelimidate
(Harlow and Lane, 1988).
Following centrifugation for 1 minute at 16,000 g, the
supernatant was removed and incubated for 1 hour as above, with 5 µg
antibody coupled to 10 µl of protein G-Sepharose. The immunoprecipitates
were washed five times with 1 ml of buffer A containing 0.35 M NaCl and
resuspended in SDS loading buffer for analysis by SDS-PAGE and immunoblotting.
All controls for the immunoprecipitation experiments were carried out in
parallel as described above using cell lysates containing another protein
expressed with the same epitope-tag.
Micoinjection of expression plasmids into HeLa cells
Plasmid DNAs were diluted to 15 µg/ml with 100 mM glutamic acid
(titrated to pH 7.2 with citric acid), 140 mM KOH, 1 mM MgSO4 and 1
mM DTT and injected into HeLa cells using an Eppendorf 5242 microinjector.
Cells were cultured at 37°C in Dulbecco's modified Eagles' medium. Two
hours after injection, cells were washed in phosphate-buffered saline, fixed
for 5 minutes at 37°C in 3.7% (w/v) paraformaldehyde in 60 mM PIPES, 27 mM
HEPES, 10 mM EGTA, 4 mM MgSO4 titrated to pH 7.0 with 10 M KOH.
Permeabilisation was performed with 1% Triton in PBS for 15 minutes at room
temperature. Mouse anti-HA monoclonal antibodies (clone 12CA5) were from Roche
Diagnostics Ltd (Lewes, UK). Secondary anti-mouse IgG antibodies conjugated to
Cy3 were from Jackson Immuno research Labs. Inc, (West Grove, PA).
Immunofluorescence was detected using a Zeiss DeltaVision Restoration
microscope (Applied Precision Inc.) equipped with a 3D motorised stage and a
100x NA 1.4 Plan-Apochromat objective. Optical sections separated by 200
nm were recorded and images were restored using an iterative deconvolution
algorithm. For EGFP, EYFP, Cy3 and DAPI excitation wavelengths were 460 nm
(bandwidth 20 nm), 523 nm (20 nm) 555 nm (28 nm), 360 nm (40 nm) and emission
wavelengths 500 nm (bandwidth 22 nm), 568 nm (50 nm), 617 nm (73 nm), 457 nm
(50 nm), respectively.
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Results |
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Five genes encoding R2, located on chromosomes 3 and 5, are found in the
NCBI Homo sapiens genomic contig sequences database. The R2 gene
located at 3p14-2 is probably the functional gene, as its sequence (NT_005561
/Hs3_5683) is virtually identical to that of the cDNA (Accession No. AJ
271448) (Hastie et al., 2000)
and other R2 partial cDNA sequences in the human databases. In addition, this
R2 gene contains nine introns and is spread over more than 40 kb. Two other R2
genes (NT_022440.4/Hs3_22596 and NT_015983.5/Hs3_16139) with
>98% DNA coding sequence identity, each containing only the most 3'
intron are located at 3q28. Two more R2 genes
(NT_029228.1/Hs5_29447 and
NT_006611.5/Hs5_6768) with >96% DNA coding sequence
identity, each containing only the most 3' intron are located at 5p15.5.
Each pair of genes at 3q28 and 5p15.5 have very similar 5' and 3'
UTR sequences, suggesting that they are likely to have arisen by gene
duplication. Isolation of a BAC clone carrying R2 genes located on chromosome
5 allowed mapping of R2 genes to 3(q27-qter) and 5p15.5 (kindly performed by
Mark Sales and Norman Pratt, Ninewells Hospital Medical School, Dundee, UK),
in accordance with the data in the genome database. No genomic clones encoding
R2 were identified in the LLNL human single chromosome 3 library, and the high
identity (>90%) of the 3' UTR sequences of all the R2 genes prevented
ascertainment of whether the two R2 mRNAs
(Fig. 1) are derived from the
gene at 3p14-21 as predicted.
Identification of proteins interacting with PPP4c and PPP4R2
Following transfection of cDNA encoding the Flag-tagged regulatory subunit
R2 of PPP4c into HEK 293 cells, proteins co-sedimenting with anti-Flag agarose
beads in the cell lysate were examined.
Fig. 2 shows the co-elution of
Flag-R2 and PPP4c from an anti-Flag agarose column. Use of the Flag peptide
for elution minimises the detachment of proteins bound non-specifically to
agarose. Analysis of the eluted Flag-R2-PPP4c material by gel filtration on a
Superose 6 column indicated that the purified Flag-R2-PPP4c material comprises
high molecular mass complexes, ranging from 450 kDa to >670 kDa
(Fig. 3). A large proportion of
this material eluted at 450 kDa, a size identical to that of the complex
formed with purified free PPP4c and baculovirus-expressed R2 in previous
experiments (Hastie et al.,
2000). Therefore the pool of protein eluting at 450 kDa is likely
to be a complex of Flag-R2 and PPP4c. There is also further material eluting
at a much higher molecular mass (approximately 800 kDa-450 kDa), which is
likely to represent Flag-R2PPP4c complexed with other proteins.
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Fig. 4 shows a
Coomassie-Blue-stained gel of material eluted from the anti-Flag agarose
column. Following excision of protein bands and tryptic digestion, the
proteins were identified by mass spectrometry and comparison of the peptide
ions generated with sequences of previously characterised proteins in the UCSF
Protein Prospector database. Along with Flag-R2 (with 21 peptides matched,
giving 60% sequence coverage) and PPP4c (19 peptides matched, giving 65%
sequence coverage) other proteins that were identified are Gemin4 (with 35
peptides matched, giving 40% sequence coverage) and Gemin3 (with 20 peptides
matched, giving 33% sequence coverage). Both and ß-tubulins were
also identified, which may be relevant to the function of R2-PP4c at the
centrosomes (Helps et al.,
1998
; Sumiyoshi et al.,
2002
).
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Gemin3 and Gemin4 have recently been identified in a number of studies
analysing the SMN protein complex (Campbell
et al., 2000; Charroux et al.,
2000
; Charroux et al.,
1999
). On the basis of amino-acid sequence alignments, Gemin3 is
thought to be a member of the DEAD box family of putative ATP-dependent RNA
helicases and is also termed DEAD-box protein103 kDa (DP103). The name `DEAD'
is derived from the single amino code for a conserved motif, Asp-Glu-Ala-Asp,
which confers ATPase activity. Gemin3 (824 amino acids) has been detected in
cellular lysates by SDS-PAGE as a 103 kDa protein, although the calculated
molecular mass from the cDNA is 92.2 kDa, in agreement with the size of 92.5
kDa determined on SDS-PAGE after in vitro transcription/translation
(Charroux et al., 1999
;
Grundhoff et al., 1999
).
Gemin4, also termed Gemin3-interacting protein 1 (GIP1) comprises 1058 amino
acids and has a calculated molecular mass of 119.9 kDa but has an apparent
molecular mass on SDS-PAGE of 97 kDa
(Charroux et al., 2000
). No
functional or conserved domains of Gemin4 have been identified, but it has
been shown to interact directly with Gemin3 and is thought to be a component
of the SMN complex via this interaction.
Co-elution of Gemin3 and Gemin4 from the anti-Flag agarose column with
Flag-R2-PPP4c was readily reproducible; therefore peptide antibodies were
raised against these two proteins. The antibodies recognised a major band of
Gemin3 at 94 kDa and a major band of Gemin4 at
97 kDa on immunoblots
of Flag-R2PPP4c-interacting proteins (data not shown) and HEK 293 cell
lysates (Fig. 5), which
validates the identification of these proteins by mass spectrometry
(Fig. 4). Although the protein
methylase JBP1 was also found co-eluting with Flag-R2PPP4c and
methylation of proteins is known to be important in assembly of SMN complexes
(Friesen et al., 2001
;
Meister et al., 2001
), it is
presently unclear whether this is a specific interaction, because JBP1 has
also been found in the eluates with other Flag-tagged proteins (G.K.C., N.M.
and P.T.W.C., unpublished).
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The anti-Gemin peptide antibodies did not immunoprecipitate endogenous Gemin3 or Gemin4. Therefore cDNA encoding Gemin4 was obtained by screening a human multi-tissue cDNA panel using PCR. Following ligation of the Gemin4 cDNA into an expression vector and transfection into HEK 293 cells, a reciprocal immunoprecipitation experiment was carried out using lysate from HEK 293 cells expressing epitope-tagged Gemin4. Fig. 6 shows the co-immunoprecipitation of Gemin4 with endogenous Gemin3 (a positive control), R2 and PPP4c. This suggests that the interaction between R2-PPP4c and Gemin4 and/or Gemin3 may be a specific interaction that occurs in vivo.
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Co-immunoprecipitation of PPP4c with the SMN complex
Since Gemin3 and Gemin4 have both been recently identified as components of
the SMN protein complex, it was important to determine whether R2-PPP4c
interacted with these proteins when they were part of the SMN protein complex.
Therefore lysates from a HeLa cell line expressing the SMN protein with a cyan
fluorescent protein (CFP)-epitope tag
(Sleeman et al., 2003) were
subjected to immunoadsorption using anti-GFP antibodies (which readily cross
react with CFP) and analysed for co-sedimentation of known components of the
SMN complex and PPP4c. The results show that the SMN protein fused to the 27
kDa CFP tag forms a complex with Gemin2, Gemin3, Gemin4, as previously
described, and in addition with PPP4c (Fig.
7). Control immunoprecipitates from lysates of HeLa cells
transfected with GFP showed no Gemin2, Gemin3, Gemin4 or PPP4c. These results
indicate that PPP4 interacts with the SMN protein complex and points to a
novel role for this phosphatase associated with the function of the SMN
complex.
|
Analysis of snRNP localisation in the presence of the R2-PPP4c
complex
Newly formed snRNPs can be detected by injecting plasmids into cultured
cells capable of expressing Sm proteins tagged with a fluorescent label, and
the maturation of the snRNPs can be monitored by observing temporal changes in
the fluorescent pattern (Sleeman and
Lamond, 1999). To determine whether the R2-PPP4c complex has an
effect on the pathway of entry of new snRNPs into the nucleus and/or their
subsequent movements, plasmid DNAs capable of expressing YFP-SmB, GFP-R2
and/or HA-PPP4c were injected into HeLa cells. Of the 200-250 cells injected
for each condition approximately one third could be visualised and scored
clearly. Two hours after injection, cells expressing the plasmid encoding
YFP-SmB alone showed a YFP-SmB signal in Cajal bodies in 92% of cells
(Fig. 8E) and a signal in the
nucleoli as well as the Cajal bodies in only 8% of cells, in accordance with
previous studies (Sleeman and Lamond,
1999
). In contrast, cells expressing all three tagged proteins
showed marked accumulation of YFP-SmB-labelled snRNPs within the nucleoli and
Cajal bodies in 89% of cells (Fig.
8B) and a YFP-SmB signal in Cajal bodies but not the nucleoli in
11% of cells. Nucleolar localisation is not usually seen in the majority of
cells on expression of YFP-SmB alone until later time points (3 to 7 hours).
Cells expressing YFP-SmB and GFP-R2 in the absence of HA-PPP4c
(Fig. 8H) or YFP-SmB and
HA-PPP4c in the absence of GFP-R2 (Fig.
8K) showed the normal localisation of YFP-SmB to Cajal bodies at
this time point. The experiments gave the same results on three separate
occasions. The signal intensities for YFP-SmB and GFP-R2 were similar and the
filters used eliminated cross-talk between the YFP and GFP channels. These
studies demonstrate that the R2-PPP4c complex modifies the localisation of
newly formed snRNPs. Furthermore, HA-PPP4c and GFP-R2 colocalised with YFP-SmB
in the nucleolus [Fig. 8A-C and
merged images (data not shown)]. Since it is possible snRNPs are continuously
cycling through the Cajal bodies and nucleoli and R2-PPP4c blocks their exit
from a nucleolar localisation, a late time point was examined, which showed
that YFP-SmB exhibited a `mature' localisation, being present in Cajal bodies
and interchromatin granule clusters (speckles) within the nucleoplasm in
80% of cells expressing YFP-SmB, GFP-R2 and HA-PPP4c as seen for those
expressing YFP-SmB alone. Thus the R2-PPP4c complex does not block the
progress of YFP-SmB and the overall results indicate that R2-PPP4c enhances
the movement of newly formed snRNPs through the first stages of their normal
maturation pathway.
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Discussion |
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RNA helicases have been implicated in nearly all processes that are linked
to RNA metabolism, such as translation initiation, pre-mRNA splicing, ribosome
assembly and mRNA stabilisation and transport
(Linder, 2000). Gemin3 was
identified as a nuclear phosphoprotein interacting with Epstein-Barr virus
encoded antigens EBNA2 and EBNA3C, both of which form a complex with the
cellular transcription factor RBP-J
and thereby modulate the expression
of target genes (Grundhoff et al.,
1999
). Independent studies detected Gemin3 as a component of the
SMN protein complex interacting directly with the SMN protein
(Campbell et al., 2000
;
Charroux et al., 1999
). Since
the Sm proteins bind to the SMN complex, the helicase activity of Gemin3 may
be required for the U RNAs to bind to the Sm proteins for the assembly of
snRNPs. Gemin4 does not interact directly with the SMN protein and its
presence in the SMN complex is probably the result of its direct and stable
interaction with Gemin3 (Charroux et al.,
2000
). Gemin4, the SMN protein and Gemin3 associate with U1 and U5
RNAs in the cytoplasm of Xenopus oocytes, but not after these RNAs
have been assembled into snRNPs and imported into the nucleus. Therefore it is
thought that the SMN complex containing Gemin4 dissociates from the
spliceosomal snRNPs either immediately before or shortly after nuclear entry.
Nevertheless Gemin4 is found in the nucleus, colocalising with SMN in Cajal
bodies and is also detected in nucleoli
(Carvalho et al., 1999
;
Charroux et al., 2000
;
Sleeman et al., 2001
). Our
studies show that R2 and PPP4c are found predominantly in the nucleus and are
also detected in nucleolar accumulations
(Fig. 8), localisations that
are consistent with interaction of this phosphatase with Gemin4 and
association with the SMN complex.
The R2-PPP4c complex influences the temporal localisation of newly
formed snRNPs in the nucleus
SnRNAs are transcribed in the nucleus and transported to the cytoplasm
where Sm proteins (a group of seven polypeptides termed B/B', D1, D2,
D3, E, F and G) bind, and the 5' end of the snRNA is modified to form
the characteristic trimethylguanosine cap
(Fischer et al., 1993;
Hamm et al., 1990
;
Lührmann et al., 1990
;
Mattaj, 1986
;
Nagai and Mattaj, 1994
). The
newly formed snRNPs are then imported into the nucleus. The fully mature
snRNPs also contain snRNP-specific proteins and numerous base and sugar
modifications of the snRNA but the location(s) where these modifications occur
is unclear. Newly assembled snRNPs show a characteristic temporal sequence of
localisation patterns on their initial import into the nucleus, which can be
followed using fluorescently labelled Sm proteins
(Sleeman et al., 2001
;
Sleeman and Lamond, 1999
). At
early time points following injection (1-2 hours) of expression plasmids
encoding GFP- or YFP-tagged Sm proteins, new snRNPs are detected as a diffuse
pool throughout the cell, with accumulation also seen in nuclear Cajal bodies
containing SMN. Later (between 3 and 7 hours following injection), new snRNPs
show nucleolar accumulation in addition to accumulation in Cajal bodies. Only
at later time points (7 hours onwards) do the majority of cells show the
mature pattern of snRNP localisation to Cajal bodies and interchromatin
granule clusters (speckles). The R2-PPP4c complex alters this temporal
sequence of localisation, with the 3-7 hour pattern being observed 2 hours
after injection, results that are consistent with the R2-PPP4c complex playing
a role in enhancing the movement of snRNPs through the first stages of their
normal maturation pathway. This result agrees with the data indicating that R2
interacts with Gemin3 and/or Gemin4 and that PPP4c is present in some SMN
complexes.
There are many points at which PPP4 might regulate the snRNP maturation
pathway (Gall, 2000). Since
PPP4c is present at low levels in the cytoplasm, it is possible that PPP4 may
be required for the binding of U RNAs to the Sm proteins and/or for the entry
of snRNPs into the nucleus. PPP4c may dephosphorylate one or more component in
the SMN complex. Gemin3 is reported to be a phosphoprotein and therefore PPP4
could regulate its helicase activity by dephosphorylation, which may modulate
the assembly of snRNPs. Alternatively, dephosphorylation of Gemin3 or another
SMN component may be a necessary prerequisite for the binding of the snRNPs to
nuclear importers. The movement and/or processing of snRNPs as they flow
through the Cajal bodies and enter the nucleolus may be subject to regulation
by PPP4. It is also possible that PPP4 may participate in the nuclear SMN
function(s) of recruiting components to the spliceosomes and transcriptosomes
and regenerating snRNPs after pre-mRNA splicing. It will be important to
ascertain whether PPP4 is associated with a particular SMN complex found
either in the cytoplasm and/or the nucleus or, like many protein phosphatases,
participates in the regulation of several cellular processes by interacting
transiently with several SMN complexes. Although we consistently found Gemin3
and Gemin4 in the R2-PP4c immunoprecipitates, we did not find SMN or Gemin 2.
This data suggest that that Gemin3-Gemin4-R2-PPP4c may be only loosely
associated with the `core' SMN complex and therefore easily lost from it,
which would explain the low level of PPP4c that we coprecipitate with CFP-SMN
(Fig. 7). Distinct pools of
Gemin3-Gemin4 may exist within the cell and therefore it is also possible that
an independent complex of Gemin3-Gemin4-R2-PPP4c may possess different
functions to that interacting with the SMN complex. In this respect it will be
interesting to know whether the viral EBNA2 and EBNA3C proteins modulate
transcription by binding to Gemin3 within the SMN complex or to a different
pool of Gemin3. The nuclear transcriptional activator E2 of papillomavirus has
been shown to modulate transcription through an interaction with the SMN
complex (Strasswimmer et al.,
1999
). Further experiments are being undertaken to delineate the
precise role(s) of Gemin3-Gemin4-R2-PPP4c complexes in relation to SMN and the
assembly of complexes involved in pre-mRNA splicing, pre-rRNA splicing and
transcription.
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Acknowledgments |
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References |
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