From the Department of Biological Sciences, State University of New
York, Buffalo, New York 14260
We report the identification and cloning of a
nuclear matrix protein termed matrin cyclophilin or matrin CYP.
The derived sequence of matrin cyp encodes a protein of 752 amino acids with a predicted mass of 88 kDa. A 172-residue stretch at
the amino terminus shows high identity with the ubiquitous family of
cyclophilins. Clustered throughout the carboxyl half of the protein are
a series of serine-arginine (SR) repeats that are a characteristic
feature of many RNA splicing factors. Antibodies raised against matrin CYP recognize a 106-kDa antigen that is detected in isolated nuclei and
quantitatively subfractionates in the nuclear matrix. Laser scanning
confocal microscopy localizes most of the anti-matrin CYP-specific
antigen within the nucleus in a pattern of large bright speckles that
co-localize with splicing factors and diffuse nucleoplasmic staining. A
strikingly similar pattern of staining is observed in cells extracted
for in situ nuclear matrices. A fusion protein containing
the cyclophilin domain of matrin CYP exhibits cyclosporin A
(CsA)-sensitive, peptidylprolyl cis-trans-isomerase activity that is characteristic of native cyclophilins. Although total
rat liver nuclei contains predominantly CsA-resistant PPIase activity,
the corresponding activity in the nuclear matrix is largely
CsA-sensitive.
 |
INTRODUCTION |
The many functions associated with the cell nucleus are temporally
and spatially regulated. Nuclear proteins and nucleic acids are often
partitioned into functional domains (1). Examples include the nucleoli,
heterochromatin, DNA replication sites (2, 3), and transcription
domains (4, 5). In addition, a number of other domains have been
identified that may also perform specific functions, such as the
nuclear speckles (1, 6) and coiled (7) and promyelocytic leukemia
(8-10) bodies. Determining which molecules comprise these structures
and how these molecules interact will lead to a greater understanding
of the mechanisms responsible for their diverse functions and the role
of nuclear architecture in these processes.
The proteins and nucleic acids in the cell nucleus that resist
solubilization by high salt extraction, nuclease digestion, and
detergent solubilization constitute a structure termed the nuclear
matrix (11, 12). The isolated nuclear matrix has been shown to maintain
a structure similar to and containing many of the functional properties
associated with the nucleus (13). Several major proteins of the
internal nuclear matrix have been identified by immunological methods
and termed the nuclear matrins (14). The nuclear matrins include
previously characterized proteins such as human
RNP1 A, human nRNP B (15),
the nucleolar protein, B23/numatrin (14, 16), the hyperphosphorylated
form of RNA pol II LS (17, 18), numerous SR-related proteins (19), and
a 125-kDa acidic protein, termed matrin 3 (20). A recent study has
confirmed the RNP nature of many of the major nuclear matrix proteins
(21).
Here we present results on the isolation of a cDNA that encodes an
88-kDa protein with a cyclophilin domain at the amino terminus and a
series of SR repeats throughout the carboxyl half of the protein. This
protein, termed matrin cyclophilin or matrin CYP, is enriched in the
nuclear matrix, co-localizes with splicing factors at nuclear speckles,
and undergoes dynamic rearrangement during mitosis. Matrin CYP fusion
protein expresses a cyclosporin A (CsA)-sensitive peptidylprolyl
cis-trans-isomerase (PPIase) activity that is characteristic
of cyclophilins. We further measure, for the first time, PPIase
activity in isolated cell nuclei and demonstrate the quantitative
recovery of total nuclear CsA-sensitive activity in the nuclear
matrix.
 |
EXPERIMENTAL PROCEDURES |
Screening of a Rat Insuloma Library--
A random primer
digoxigenin-11 dUTP-labeled rat liver partial length cDNA probe was
used to screen a pCD-X rat insuloma library (22) via colony
hybridization. The probe was detected with the Genius chemiluminescence
kit (Boehringer Mannheim). 250,000 colonies were screened and two
positives obtained from the quaternary screening. Both positives
contained similar sized inserts, and their restriction maps were
indistinguishable.
Sequence Analysis of Matrin cyp cDNA--
Twelve fragments
encompassing the entire matrin CYP full-length cDNA were subcloned
into pGEM-3Z (Promega) and sequenced. Sequencing was performed by the
dideoxy method using [35S]dATP and the Sequenase 2.0 kit
(U. S. Biochemical Corp.) (23). Additional sequencing was performed
over ambiguous regions using synthesized oligo primers (Integrated DNA
Technologies). Each fragment was sequenced in the forward and reverse
directions, and overlapping sequence was obtained for parts of the
cDNA. The cDNA sequence was assembled and analyzed using the
GCG computer package. The EMBL, Swiss-protein, and National Institutes
of Health nucleotide and amino acid data bases were searched for
homology to the matrin cyp cDNA using FASTA, BLAST, and
BLITZ programs.
Construction, Expression, and Purification of GST/Matrin CYP-CT
and GST/Matrin CYP: NT Fusion Proteins--
For GST/matrin CYP-CT a
389-bp region from the matrin CYP cDNA corresponding to nucleotides
1930-2319 was amplified (GeneAmp, Perkin-Elmer) using the following
primers with engineered restriction sites:
5'-GATGGATCCAGCCAAGACAGTAAGAGTTCACACAG and
3'-GCAGAATTCGAGGGAGGCTCTTGAAAAGTAGAC (Integrated DNA
Technologies). A 536-bp region from the matrin cyp cDNA
corresponding to nucleotides 1-536 was amplified using the following
primers: 5'-CAATCGGATCCATGGGAATAAAGGTTCAGCGTCC and 3'-GACGAATTCAACCAGCTCTCCACAACTGAGTATC for GST/matrin CYP-NT. These amplified fragments were digested with EcoRI and
BamHI and subcloned into pGEX-kt (24). The open reading
frame and sequence of the pGEX-kt subclones were verified by DNA
sequencing. Each plasmid was transformed into JM101 and a single
recombinant colony transferred to 1 liter of LB media. The fusion
protein was purified by a previously described procedure (25) except
that the cells were induced with 0.1 mM isopropyl
-D-thiogalactopyranoside, lysed in a French press
(p.s.i. = 18,000), and the fusion proteins were purified over a 1-ml
glutathione-agarose bead column (Sigma). The GST/matrin CYP-CT fusion
protein was cleaved with thrombin (Sigma) while still associated with
the column and the eluate recovered as matrin CYP-CT. Conversely, the
GST/matrin CYP-NT was not cleaved with thrombin but eluted intact from
the glutathione-agarose bead column. The GST/NK-TR (26) and GST alone
were expressed and purified identically to GST/matrin CYP-NT. The
various fusion protein fractions were run on a 10% SDS-polyacrylamide
gel using standard procedures and stained with Coomassie Blue or
processed for immunoblot analysis (described below). The fusion
proteins were quantified using the BCA protein detection kit
(Pierce).
Preparation and Purification of Polyclonal Antibodies against
Matrin CYP-CT--
1 mg of the protein was emulsified in 300 µl of
Freund's complete adjuvant and then injected intramuscularly into a
28-week-old leghorn hen. After 3 weeks a boost of 500 µg of matrin
CYP-CT in incomplete Freund's adjuvant was given, and the eggs
produced by this chicken were immediately retrieved. A total of three
booster shots were separated by 4-week intervals. Isolation of IgY from egg yolks was performed as described previously (27). Antibodies were
further purified by passage over a 1-ml affinity column of matrin
CYP-CT linked to Sepharose 4B (Amersham Pharmacia Biotech). The
antibody preparation showed no detectable cross-reactivity with the GST
portion of the fusion protein.
Subcellular Fractionation and Nuclear Matrix Isolation from Rat
Liver Cells--
Livers were extracted from adult male Sprague-Dawley
rats and processed for rat liver matrices using exogenously added DNase I (28). The supernatants of the washes from the extraction procedures were saved for later use. Additionally, the first post-nuclear supernatant was retained for separation into various cytoplasmic subfractions. A 10,000 × g·h centrifugation
separated a crude mitochondrial pellet and post-mitochondrial
supernatant. Next, centifugation of the post-mitochondrial supernatant
at 100,000 × g·h resulted in a microsomal pellet and
cytosol supernatant. Protein fractions were quantified by the BCA
protein detection kit (Pierce) and separated by 7.5%
SDS-polyacrylamide gel electrophoresis.
Phosphatase Treatment--
Rat liver nuclear matrices were
pelleted to the bottom of a microcentrifuge tube and treated with
potato acid phosphatase (Sigma) at a concentration of 0.4 units of
phosphatase per µg of nuclear matrix protein for 14 h at
20 °C (29). Samples were resolved on a 7.5% SDS-polyacrylamide gel
and immunoblotted as described below.
Immunoblot Analysis--
Protein samples run on an
SDS-polyacrylamide gel were transferred overnight by wet transfer to a
nitrocellulose filter (Schleicher & Schuell). The filter was incubated
for at least 3 h in blocking buffer (10 mM Tris-HCl
(pH 7.4), 150 mM NaCl, 0.5% Tween 20, 6% nonfat dry milk)
at room temperature. The filter was incubated for 2 h with
anti-matrin CYP-CT (1:500 dilution) in blocking buffer. The filter was
washed 3 times with blocking buffer (minus the nonfat dry milk) for 5 min per wash. Goat anti-chicken IgG linked to alkaline phosphatase
(1:5000 dilution) (Kirkegaard & Perry Laboratories) was incubated with
the filter for 1 h at room temperature. The blot was washed four
times as above and developed in 1 M Tris (pH 9.5), 10 mg/ml
nitro blue tetrazolium, and 100 mg/ml bromochloroindolyl phosphate.
Some bands were quantified by scanning densitometry.
Indirect Immunofluorescence Confocal Microscopy--
Rat
parenchymal cells were prepared from rat liver tissue (30). These cells
were grown on coverslips in Dulbecco's modified Eagle's medium and
10% fetal calf serum for 1 or 2 days. Mouse 3T3 fibroblasts were grown
on coverslips as described previously (3). For mitotic studies, cells
were first collected into G0 by incubating in 0.5% calf
serum and Dulbecco's modified Eagle's medium for 3 days and released
from the G0/G1 block by the addition of 10%
calf serum in Dulbecco's modified Eagle's medium. After 22 h the
maximum number of cells were in mitosis and used for analysis. For
immunofluorescence, cells were washed quickly in three changes of PBS
and typically fixed with 3% paraformaldehyde for 4 min on ice. Other
fixation conditions included 3% paraformaldehyde for 10 min at
20 °C, 100% methanol for 2 min at
20 °C (31), acetone/ethanol
(1:1) for 1 min at
20 °C (32), or treatment with CSK buffer
followed by paraformaldehyde (33). Fixed cells were incubated with
blocking buffer (10% goat serum, 0.5% Tween 20, and 3 mM
MgCl2) in PBS for 30 min followed by reaction with anti-matrin CYP-CT (1:20 dilution) in the above blocking buffer at
37 °C for 1 h. Following three washes with 0.5% Tween 20 in PBS, the cells were incubated in goat anti-chicken IgG-fluorescein isothiocyanate (Kirkegaard & Perry Laboratories) at 37 °C for 30 min, washed 3 times with 0.5% Tween 20 in PBS, incubated with the
second primary antibody, and washed as above. The red signal was
enhanced by incubation with a biotinylated secondary antibody and
streptavidin conjugated to Texas Red (Life Technologies, Inc.). Finally, the coverslips were washed 4 times in PBS and then mounted in
SlowFade (Molecular Probes). Cells were observed by epifluorescence and
laser scanning confocal microscopy with an Olympus GB200 laser scanning
confocal microscope equipped with a 25-milliwatt argon laser operating
at 488 and 514 nm. Images were corrected for inter-channel leakage
using a leakage factor computed from a calibration image. The
calibration image was obtained from a sample that is single labeled
with fluorescein isothiocyanate-conjugated antibodies, and information
was acquired on both channels. In situ nuclear matrices were
prepared from rat parenchymal cells as previously reported (20).
Peptidylprolyl cis-trans-Isomerase (PPIase) Assay--
PPIase
activity was determined as described previously (26, 34). Briefly, the
cis-proline containing tetrapeptide substrate, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide
(Sigma), was converted to the trans-proline isomer and made
accessible to protease cleavage by chymotrypsin resulting in an
increase in absorbance of the product at 405 nm. Approximately 65% or
65 µM substrate was in the cis-isomer at the
start of the assay measurements. Absorbance readings were collected
every second using the double beam Hitachi U2000 spectrophotometer. To
simplify analysis, the assay was performed under conditions in which
[s]
Km. These reaction conditions enabled
calculation of the first-order kinetic rate constant
(kcat/Km) using the PSI-Plot
program. The IC50 of CsA was determined with 100 nM GST/matrin CYP-NT and 65 µM
cis-substrate as described (26).
Measuring PPIase Activity in Isolated Rat Liver Nuclear and
Nuclear Matrix Proteins--
Nuclei and nuclear matrices were
concentrated by centrifugation for 5000 × g·h at
4 °C. The pellets were solubilized in 9 M urea, 50 mM DTT, 2 M NaCl, 5 mM EDTA, and 20 mM Tris (pH 7.4) for 10 min on ice with mixing and then
centrifuged for 5000 × g·h to remove insoluble
material. The solubilized total nuclear or nuclear matrix proteins were
dialyzed 2 times against 100 volumes of 200 mM NaCl and 20 mM Tris (pH 7.4) at 4 °C for 8 h and used in the
PPIase assays following centrifugation for 5000 × g·h. All buffers contained the protease inhibitors 1 mM benzamidine, 1 mM phenylmethylsulfonyl
fluoride, 5 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. Typically 25 µg of solubilized rat liver nuclear or nuclear matrix
proteins were incubated with the cis-proline containing substrate, N-succinyl-AAPF-p-nitroanilide, and
the activities of total proline isomerization was measured in
micromoles of cis-substrate converted to
trans-substrate (34). Incubating solubilized fractions with
10 µM CsA allowed determination of cyclophilin-catalyzed PPIase activity. Thermal nonenzymatic proline isomerization values were
subtracted from observed protein induced activities to obtain actual
values of PPIase activity. For each sample n = 2 or
3.
 |
RESULTS |
Molecular Cloning and Sequence Analysis of the Matrin CYP
cDNA--
Chicken polyclonal antibodies raised against a nuclear
matrix protein of approximately 106 kDa were used to isolate a rat liver cDNA from a lambda gt11 expression library. The expressed fusion protein from this clone reacted with the antisera, and when
sequenced appeared to constitute only the 3' portion of a full-length
transcript (data not shown).
By using the partial length cDNA as a probe, a 3971-bp full-length
cDNA was isolated from a rat insuloma cDNA library. Sequencing of this cDNA, referred to as matrin CYP, revealed that it contains the partial length cDNA sequence as well as a continuous open reading frame of 2256 bp (Fig.
1B). The ATG at position +1 is a likely candidate for the start codon since the nucleotides around this ATG show near identity to the "Kozak" ribosome binding
consensus sequence, and five codons upstream of this ATG is a
termination codon (35). The 3'-untranslated region contains two
consensus polyadenylation signals (e.g. AAUAAA). The
consensus polyadenylation signal located at nucleotide 3557 appears to
be utilized by the full-length rat insuloma cDNA.

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 1.
The nucleotide and derived amino acid
sequence of matrin CYP. A, a schematic representation of the
derived amino acid sequence of matrin CYP indicating the cyclophilin
domain (open box), the acidic serine domain (solid
box), and the SR repeat regions (stippled boxes). The
predicted molecular mass of matrin CYP is 88 kDa, and the predicted
isoelectric point is 11.05. The boxes labeled GST/matrin
CYP-NT and GST/matrin CYP-CT represent the regions corresponding to the
amino-terminal and carboxyl-terminal fusion protein, respectively.
B, the nucleotide sequence of the full-length matrin
cyp cDNA with the predicted amino acid sequence below.
The putative polyadenylation signals are single underlined
in the 3'-untranslated region. The cyclophilin domain is single
underlined, the acidic serine region is dashed
underlined, and the SR repeats are double
underlined.
|
|
The open reading frame of matrin cyp encodes a polypeptide
of 752 amino acids. The derived amino acid sequence has a predicted molecular mass of 88,072 daltons and an isoelectric point of 11.05. One
intriguing aspect of this amino acid sequence is the preponderance of
charged and serine residues as follows: arginine and lysine residues
account for 29%, aspartate and glutamate for 16%, and serines for
15% of the total number of residues of matrin CYP (Fig.
1B). Southern and Northern blot analyses indicates that matrin CYP in rat liver is coded for by one gene and contains one
transcript whose size is consistent with the cloned cDNA fragments (data not shown).
Searching computer data bases for similarities to the nucleotide and
predicted amino acid sequences identified two regions within the amino
acid sequence that show striking similarity to several previously
cloned and characterized proteins (Fig. 1A). The first of
these domains is located between residues 6 and 178 (Fig.
1B). This region shows a high level of identity with several distinct cyclophilin proteins. Cyclophilin domains have been
demonstrated to function as both a proline isomerase and a molecular
chaperone (36, 37).
The second domain is composed of a series of SR repeats (Fig.
1B, 29 repeats are marked by bold underline) that
cover much of the carboxyl half of the protein. SR-rich regions have
been found in several protein factors that are associated with
pre-mRNA splicing (38, 39).
Adjacent to the cyclophilin domain (residue 193) is a stretch of 30 amino acids containing 19 serines and 8 aspartate or glutamate residues
(Fig. 1B, dashed underline). This acidic serine region is
immediately followed by another stretch of 30 amino acids containing 77% basic residues. Various nuclear localization signal (NLS) binding proteins contain similar domains (40, 41).
Consistent with a nuclear localization of matrin CYP, there are
numerous NLS motifs in the amino acid sequence. Three putative NLS
motifs are particularly rich in SR repeats (amino acids 342-354, 456-489, and 512-667) and resemble the SR repeat regions of the SuWA
and Tra Drosophila splicing proteins that have been
demonstrated to target cytoplasmic proteins to the nucleus (42). In
addition to the SR-rich regions, matrin CYP contains nine classical NLS motifs as follows: six different bipartite-type NLS (starting at
positions 226, 233, 338, 437, 452, 608) and three different SV40 large
T antigen-type NLS at positions 226, 243, and 619 (43).
Immunological Analysis of Matrin CYP--
A fusion protein
comprising the carboxyl-terminal residues (644-752) of the full-length
matrin cyp cDNA (GST/matrin CYP-CT) was expressed, and
polyclonal antibodies were raised against this peptide in chickens (see
"Experimental Procedures"). This region of matrin CYP was chosen
because it did not contain clusters of SR repeats nor significant
similarity with other reported proteins.
Rat liver whole cell extracts were subfractionated into cytoplasmic and
nuclear fractions and then probed with the purified anti-matrin CYP-CT
antibody on immunoblots. The antibody did not stain protein bands in
the cytoplasmic fractions but recognized a diffuse band at about 106 kDa in the purified nuclear fraction (Fig.
2, lane 7). Over 90% of the
106-kDa protein was recovered in the nuclear matrix fraction based on a
total nuclear protein recovery of 29% in the nuclear matrix and an
approximately 3-fold increase in signal from the purified nuclei to the
nuclear matrix fractions (Fig. 2, compare lanes 7 and
11). Correspondingly, no anti-matrin CYP-CT signal was
detected in the soluble protein fractions obtained from the nuclease
digestion, high salt, or detergent extractions used to prepare the
nuclear matrices (Fig. 2, lanes 8-10). Similar protein
blots stained with anti-lamin A/C and anti-matrin 3 antibodies, both
nuclear matrix-associated proteins, resulted in levels of nuclear
matrix enrichment and recovery similar to those observed for matrin CYP
(data not shown).

View larger version (96K):
[in this window]
[in a new window]
|
Fig. 2.
Immunoblot analysis of subcellular fractions
derived from rat liver cells. The lanes were loaded with 5 µg of
various rat liver cell fractions as follows: lane 1, whole
cell lysate; lane 2, postnuclear supernatant; lane
3, mitochondrial pellet; lane 4, postmitochondrial
supernatant; lane 5, microsomal pellet; lane 6,
cytosol; lane 7, purified nuclei; lane 8,
supernatant from nuclease and high salt wash; lane 9,
supernatant from second high salt wash; lane 10, supernatant
of detergent extract; lane 11, nuclear matrix. Lanes
12 and 13 are 5 µg of rat liver nuclear matrix
proteins probed with anti-matrin CYP-CT (lane 12) or
anti-matrin CYP-CT that was preincubated with an equal molar amount of
matrin CYP-CT as competitor (lane 13). The signals in
lanes 7 and 11 were quantified by densitometry.
Molecular mass markers are indicated in kDa.
|
|
The specificity of the anti-matrin CYP-CT antibody for the 106-kDa
antigen was demonstrated by preincubating the antibody with an equal
molar concentration of the matrin CYP-CT fusion protein and then
probing rat liver nuclear matrix proteins with this mixture. This
resulted in the complete inhibition of binding of the anti-matrin
CYP-CT antibody to the 106-kDa antigen (Fig. 2, lane 13),
whereas a corresponding mock treatment (Fig. 2, lane 12) had
no effect.
The difference observed between the apparent molecular mass of 106 kDa
and the predicted molecular mass of 88 kDa based on cDNA sequencing
is consistent with the highly charged nature of matrin CYP and previous
observations that numerous SR proteins exhibit apparent molecular
masses on SDS-polyacrylamide gel electrophoresis that are significantly
higher than predicted from amino acid sequences (38, 44-46). Moreover,
sequence analysis revealed 46 known phosphorylation motifs in matrin
CYP including 19 casein kinase II and 14 protein kinase C sites.
Indeed, a phosphatase-mediated shift in the migration of matrin CYP
from 106 to 98 kDa (Fig. 3, lane
1) indicates that a significant portion of the higher apparent
molecular weight exhibited by matrin CYP is due to a phosphorylated
state of this protein in the cell nucleus.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 3.
Phosphatase treatment of rat liver nuclear
matrix proteins shift the mobility of matrin CYP. 20 µg of rat
liver nuclear matrix protein was incubated with phosphatase inhibitors
and potato acid phosphatase (lane 2) or only potato acid
phosphatase (lane 1). Lane 3 is a mocked treated
sample. Following the treatments, the proteins were separated on a 6%
SDS-polyacrylamide gel. Immunoblot analysis was performed with the
anti-matrin CYP-CT. Size markers are indicated in kDa.
|
|
Matrin CYP Is Localized to Nuclear Speckles during
Interphase--
Immunofluoresence analyses using anti-matrin CYP-CT
confirmed the immunoblot results indicating that this antibody is
highly specific for a nuclear antigen. Double staining
immunofluorescence of primary cultured rat parenchymal cells with
anti-lamin A/C and anti-matrin CYP-CT was imaged by laser scanning
confocal microscopy and revealed that anti-matrin CYP-CT decorated
intranuclear structures consisting predominantly of large irregularly
shaped foci that do not correspond to the nucleoli (Fig.
4, A-C). Similar
structures were stained by anti-matrin CYP-CT using a wide variety of
fixation protocols (see "Experimental Procedures") or in other cell
lines such as mouse 3T3 fibroblasts or human HeLa cells (data not
shown). In addition to the bright foci, a less intense, diffuse
staining was detected throughout the nucleoplasm (Fig. 4A).
A very low intensity background staining was also consistently observed
throughout the cytoplasm.

View larger version (137K):
[in this window]
[in a new window]
|
Fig. 4.
Confocal immunofluorescence microscopy of
primary cultures of rat parenchymal cells. A-F,
cells were fixed in 3% paraformaldehyde and stained with anti-matrin
CYP-CT (A), anti-lamin A/C (B), and merged images
(C) or stained with anti-matrin CYP-CT (D),
anti-U1-70-kDa (E), and merged images (F).
G-I, following extraction of cells for in
situ nuclear matrix and stained with anti-matrin CYP-CT
(G), anti-lamin A/C (H), and merged images
(I).
|
|
The specificity of anti-matrin CYP for these various structures was
tested by preincubating this antibody with different levels of matrin
CYP-CT fusion protein and performing immunofluorescence with these
mixtures. By using this competitor peptide approach, the very intense
staining of the speckled sites was virtually abolished at an
antibody:matrin CYP fusion protein molar ratio of 0.3, whereas the
diffuse nucleoplasmic and cytoplasmic staining was not affected
(Fig. 5). We conclude that the matrin CYP
antibody is at least predominantly decorating matrin CYP at the
speckled sites. These findings are consistent with the immunoblot
results (Fig. 2) that did not detect matrin CYP antigens in the
cytoplasmic fractions from rat liver cells.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 5.
Confocal images of mouse 3T3 fibroblasts
stained with anti-matrin CYP-CT following preincubation with a matrin
CYP fusion protein. Increasing levels of matrin CYP-CT fusion
protein were preincubated with anti-matrin CYP-CT, and the molar ratios
of antibody to fusion protein are indicated above a representative
cell. The control cells were stained only with the secondary antibody.
Cells were fixed with 3% paraformaldehyde.
|
|
Rat parenchymal cells in primary culture were extracted for in
situ nuclear matrices (see "Experimental Procedures") and then double-stained as above. The results show that, like those observed with immunoblot analysis, the anti-matrin CYP-CT-specific antigens are
tightly associated with the nuclear matrix (Fig. 4,
G-I). Moreover, this association is maintained
in a similar distribution of bright foci and less intense nucleoplasmic
staining characteristically observed for fixed cells (Fig. 4,
G-I).
Since the predicted amino acid sequence of matrin CYP contains clusters
of SR repeats and the nucleoplasmic foci stained by anti-matrin CYP-CT
were reminiscent of splicing factor rich nuclear "speckles"
observed in other studies (32, 33, 47-49), double labeling
immunofluorescence microscopy was performed with anti-splicing factor
specific antibodies and anti-matrin CYP-CT. Indeed, antibodies specific
for the 70-kDa subunit of the U1 snRNP co-localizes with anti-matrin
CYP-CT in rat parenchymal cells (Fig. 4, D-F).
Similar results were obtained using other splicing factors
(e.g. Y12) or different mammalian cell lines,
e.g. mouse 3T3 fibroblasts or human HeLa cells (data not
shown).
Matrin CYP Redistributes during Mitosis and Associates with Other
Non-snRNP SR Proteins--
Mouse 3T3 cells were synchronized by serum
deprivation and examined at times when mitotic figures are maximal (see
"Experimental Procedures"). At the onset of mitosis, matrin CYP is
remodeled from its characteristic speckle pattern to a more diffuse
pattern that is distributed between the condensing chromosomes (Fig.
6A). In late prophase (as the
nuclear envelope breaks down) the diffuse staining of matrin CYP
extends into the cytoplasm (Fig. 6C). At prometaphase,
metaphase, and anaphase (when the chromosomes are fully condensed) the
diffuse staining of anti-matrin CYP-CT distributes uniformly throughout
the mitotic cytoplasm along with a limited number of small punctate
structures (Fig. 6, E, G, and I). The characteristic exclusion of matrin CYP from the chromosomes is maintained throughout mitosis. During telophase, matrin CYP undergoes a
significant rearrangement as the diffuse staining becomes less pronounced and many round bright foci are observed scattered throughout the cytoplasm (Fig. 6K).

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 6.
Reorganization of matrin CYP during mitosis
in mouse 3T3 fibroblasts. Mouse 3T3 fibroblasts were synchronized
by serum deprivation and examined 22 h after serum addition to
enrich the population of mitotic cells. These cells were fixed in 3%
paraformaldehyde and stained with anti-matrin CYP-CT and DAPI.
A and B, early prophase; C and
D, late prophase; E and F,
prometaphase; G and H, metaphase; I
and J, anaphase; K and L, telophase.
A, C, E, G, I, and K show anti-matrin CYP-CT
staining. B, D, F, H, J, and L show DAPI staining
of the same cell.
|
|
Similar nuclear redistributions during mitosis have been previously
reported for several antibodies specific for non-snRNP SR splicing
factors (80). Consistent with these results, we found in late telophase
cells a nearly identical localization of anti-matrin CYP-CT and the
monoclonal antibody B1C8 (Fig. 7, A and B), which recognizes an SR protein of 160 kDa (33). An antibody specific for a small constellation of non-snRNP,
SR proteins, NM-4 (19), also showed a very similar distribution as
anti-matrin CYP-CT in late telophase cells (data not shown). In
contrast, snRNP proteins, as detected by Y12, localized predominantly
to the interior of the nucleus during late telophase and showed no enrichment in the cytoplasmic foci stained by anti-matrin CYP-CT (Fig.
7, D and E). Identical results were obtained
using the U1 70-kDa specific antibodies (data not shown).

View larger version (65K):
[in this window]
[in a new window]
|
Fig. 7.
Mouse 3T3 fibroblasts in late telophase show
markedly different staining patterns for SR proteins compared with
snRNP proteins. A-C shows a cell in late telophase stained
with anti-matrin CYP-CT (A), the monoclonal antibody
specific for a 180-kDa SR protein termed B1C8 (B), and DAPI
(C). D-F shows late telophase stained
with anti-matrin CYP-CT (D), the anti-snRNP monoclonal
antibody Y12 (E), and DAPI (F).
|
|
By using two alternative procedures, we next examined whether matrin
CYP fractionates with other SR proteins. Matrin CYP was enriched from
5- to 10-fold on a protein basis in the SR protein fraction of Blencowe
et al. (19) compared with the initial HeLa nuclear extract
(Fig. 8, lanes 1 and
2). In contrast, matrin CYP was not detected in the
classical SR protein preparation of Zahler et al. (50) and
was, therefore, highly depleted in this fraction compared with the
nuclear extract (Fig. 8, lanes 1 and 3). These results support previous observations (19) that numerous SR-related proteins (including several nuclear matrix proteins) are not present in
the final preparations of Zahler et al. (50).

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 8.
Fractionation of matrin CYP with SR
proteins. Protein samples were separated on an SDS-polyacrylamide
gel and transferred to nitrocellulose membrane using a semi-dry
electrotransfer apparatus. Immunoblot analysis was then performed as
described under "Experimental Procedures." Anti-matrin CYP-CT
recognizes a protein of approximately 106 kDa in a splicing competent
HeLa nuclear extract (lane 1, 100 µg of protein). This
protein is enriched in a high salt extract from a
magnesium-precipitated pellet of this nuclear extract (lane
2, 20 µg of protein) that was previously shown (19) to contain
numerous SR-related proteins. Anti-matrin CYP-CT does not recognize an
antigen in the fraction enriched for the six soluble SR proteins
(lane 3, 100 µg of protein) defined by Zahler et
al. (50). Note that anti-matrin CYP-CT shows no cross-reactions
with other SR proteins. Protein size markers are indicated in
kDa.
|
|
Matrin CYP Is a Functional PPIase--
A fusion protein containing
the cyclophilin domain of matrin CYP linked to the carboxyl tail of
glutathione S-transferase (GST/matrin CYP-NT, see Fig. 1)
was expressed in Escherichia coli and purified to over 90%
purity from a glutathione bead column (Fig.
9A). GST/matrin CYP-NT was
capable of catalyzing the conversion of the cis-proline
tetrapeptide substrate, AAPF, to the trans-form in an
in vitro assay (Fig. 9B, line a). This activity
was well above the spontaneous background observed when no exogenous
proteins were added to the assay (Fig. 9B, line g). The
isomerase activity is a function of the matrin CYP-NT domain since the
GST portion was not capable of acting as a PPIase alone (Fig. 9B,
line f). The homologous NK-TR cyclophilin (26) also exhibited
PPIase activity, albeit at a slightly lower level than matrin CYP (Fig. 9B, line b). The calculated
kcat/Km for GST/matrin CYP-NT is 16-fold lower than those reported for human CYP A, but within the
range reported for other cyclophilins using similar tetrapeptide substrates (Table I).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 9.
Peptidylprolyl
cis-trans-isomerase activity and inhibition by cyclosporin
A in the cyclophilin domain of matrin CYP. A, both
GST/matrin CYP-NT and GST/NK-TR cyclophilin were expressed in E. coli and purified to near homogeneity. Cell lysates or purified
fusion proteins were run on a 10% SDS-polyacrylamide gel and stained
with Coomassie Blue. Lane 1, GST/matrin CYP-NT uninduced;
lane 2, GST/matrin CYP-NT induced; lane 3,
GST/matrin CYP-NT purified; lane 4, GST/NK-TR uninduced;
lane 5, GST/NK-TR induced; lane 6, GST/NK-TR
purified. B, GST/matrin CYP-NT, GST/NK-TR, and GST alone
were tested for in vitro PPIase activity in a
chymotrypsin-coupled assay using the tetrapeptide substrate
N-succinyl-AAPF-p-nitroanilide (34). The
graph indicates PPIase activity as an increase in
absorbance. Line a, 145 nM GST/matrin CYP-NT;
line b, 145 nM GST/NK-TR; line c, 36 nM GST/matrin CYP-NT; line d, 100 nM
GST/NK-TR and 10 µM CsA; line e, 100 nM GST/matrin CYP-NT and 10 µM CsA;
line f, 145 nM GST; line g, no
protein added.
|
|
The PPIase activity of this fusion protein was completely abolished by
preincubation with CsA (Fig. 9B, line e) as was its GST/NK-TR counterpart ((Fig. 9B, line d). Matrin CYP had an
inhibitory sensitivity to CsA (IC50) of 220 nM
that is within the range of IC50 values reported for other
cyclophilins (Table I). Obtaining the direct binding constants for CsA
and GST/matrin CYP-NT was not feasible for the same reasons as
previously discussed for the NK-TR cyclophilin (26).
Functional Nuclear Cyclophilins Associate with the Nuclear
Matrix--
Since matrin CYP is quantitatively recovered in the
nuclear matrix, we next investigated whether CsA-inhibited PPIase
activity is present within endogenous rat liver nuclear matrix
proteins. Rat liver nuclear and nuclear matrix proteins were incubated
in 9 M urea and 50 mM DTT that resulted in the
nearly complete (>90%) solubilization of proteins from these
fractions (Fig. 10A,
lanes 1 and 3). Most of the protein, including
matrin CYP, remained in solution after the urea and DTT were diluted
10,000-fold by dialysis (Fig. 10, A and B, lane
5). The relatively harsh procedure of urea and DTT treatment used
to solubilize rat liver nuclear and nuclear matrix proteins had little
inhibitory effect on the PPIase activity of similarly treated
GST/matrin CYP-NT (>90% recovery of total enzyme activity).

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 10.
Solubilization of total nuclear matrix
proteins includes matrin CYP. The majority (>80%) of nuclear
matrix proteins, including matrin CYP, remain in the supernatant after
solubilization and dialysis. A, rat liver nuclear matrix
proteins were solubilized in 9 M urea, 50 mM
DTT and dialyzed as described under "Experimental Procedures."
Equal volumes were loaded in lanes 1-5 as follows:
lane 1, untreated rat liver nuclear matrix (10 µg);
lane 2, pellet from urea/DTT-solubilized rat liver nuclear
matrix; lane 3, supernatant from urea/DTT-solubilized
nuclear matrix; lane 4, pellet from dialyzed and solubilized
nuclear matrix; lane 5, supernatant from dialyzed and
solubilized nuclear matrix. The gel was stained with Coomassie Blue.
B, immunoblot stained with anti-matrin CYP-CT of a gel
similar to A. Total nuclear proteins were solubilized
similarly to nuclear matrix proteins (data not shown). Size markers are
indicated in kDa.
|
|
The in vitro PPIase revealed a 2.2-fold higher specific
activity in the nuclear matrix proteins compared with the nuclear proteins (Table II). This corresponded to
a recovery of 63% of the total nuclear PPIase activity in the nuclear
matrix fraction. Moreover, the CsA-sensitive PPIase activity was
quantitatively associated with the nuclear matrix fraction (Table II)
with a 4.4-fold increase in specific activity compared with the
corresponding total nuclear CsA-sensitive activity. In contrast,
CsA-resistant activity was not significantly enriched (1.1-fold) on the
nuclear matrix compared with the total nuclear activity (Table II).
 |
DISCUSSION |
The Cyclophilin Domain of Matrin CYP--
Matrin CYP belongs to a
highly conserved and large class of proteins termed cyclophilins that
function as peptidylprolyl-isomerases (PPIases) to catalyze the
conversion of cis-proline to trans-proline in a
polypeptide chain. Cyclophilins bind the immunosuppressive drug
cyclosporin A that inhibits the associated PPIase activity (51, 52).
They are ubiquitous throughout the phylogenetic scale (53) and are
located in a wide variety of subcellular compartments (37, 54-63)
where they have been proposed to function in protein folding as well as
a chaperone for protein targeting and macromolecular assembly (26, 36,
37, 52, 55, 57, 59, 64-69). Initial studies indicate a wide range of
functional roles for cyclophilins such as involvement in T-cell
activation (70), natural (tumor) killer cell function (71), protein
secretion (59, 69), photoreceptor cell function via formation of active forms of rhodopsin 1 and 2 proteins (64, 67), steroid receptor function
(68), myeloid cell differentiation (72), tumor cell growth (73), and
formation of infectious human immunodeficiency virus-I virions through
interactions with the Gag polyprotein (74, 75).
Although matrin CYP is the first cyclophilin demonstrated to be
predominantly located within the nucleus, an FK506-binding protein
PPIase was previously found in the nucleolus (76), and a parvulin
PPIase has been localized to the nuclear speckles (77). Neither of
these PPIase activities exhibit sensitivity to CsA. Nestel et
al. (78) and Bourquin et al. (79), using the yeast two-hybrid approach, independently identified and sequenced the human
homolog of the rat matrin cyp (GenBankTM
accession numbers U40763 and X99717, respectively) that is 93%
identical in amino acid sequence. Complementing our findings, transient
transfection experiments revealed that the overexpressed human matrin
CYP protein co-localized at splicing factor-rich nuclear speckles and
exhibited a possible nuclear matrix association (79). Matrin CYP also
shows significant homology to the cyclophilin containing protein termed
NK-TR (GenBankTM accession numbers LO4288 and L04289, Ref.
71). Although originally described as a plasma membrane component,
NK-TR may also be a nuclear component involved in myeloid cell
maturation (72).
Consistent with the localization of matrin CYP in the cell nucleus and
its enrichment in the nuclear matrix, we measured significant levels of
PPIase activity associated with isolated rat liver nuclei (Table II).
The nearly complete recovery of matrin CYP and its characteristic
CsA-sensitive PPIase activity in the nuclear matrix fraction (Table II)
indicates that a part (if not all) of the CsA-sensitive PPIase activity
in the nucleus and nuclear matrix is contributed by the matrin CYP
protein. This suggests an involvement of higher order structure in the
important but poorly understood events of protein folding, targeting,
and macromolecular assembly at discrete sites of nuclear function
(e.g. DNA replication, transcription, and/or RNA
splicing).
Possible Functions of Matrin CYP--
Further studies are needed
to determine the properties of matrin CYP compared with other SR
proteins (39). The SR repeats of matrin CYP, for example, may
potentiate specific interactions with other SR proteins (81-83).
Enrichment of matrin CYP in a nuclear matrix-associated SR protein
fraction (Fig. 8 and Ref. 14) suggests a possible role of matrin CYP in
the higher order assembly of SR proteins in the cell nucleus. One
property associated with SR proteins is the specific phosphorylation of
serine residues present in the serine-arginine dipeptide repeats (39).
Misteli and Spector (84) have recently presented a model outlining a central role of protein phosphorylation/dephosphorylation in the spatial and temporal coordination of transcription and pre-mRNA splicing. For example, phosphorylation of SC-35 and other splicing factors by the cyclin-independent mitotic kinase SRPK-1 has been linked
to the disruption of nuclear speckles observed at the onset of mitosis
(85). Similarly, overexpression of the clk kinase resulted in the
redistribution of splicing factors from speckles to a diffuse pattern
(86). Clk kinase binds to a number of SR proteins and directs (at least
in part) their phosphorylation states (86). Likewise, matrin CYP has
recently been identified as a specific clk kinase binding protein via
the yeast two-hybrid approach (78). Clk kinase is, therefore, a prime
candidate for being involved in matrin CYP phosphorylation and the
regulation of matrin CYP function such as its associated PPIase
activity.
Evidence suggests that splicing factors undergo a dynamic
redistribution after participating in pre-mRNA splicing. This has been demonstrated by the cycling of snRNP and non-snRNP splicing factors to and from nuclear speckles (89) and, more recently, by direct
observations in living cells (88). We propose that matrin CYP may act
as a molecular chaperone that is involved in the dynamic regulation of
the nuclear speckle domains. Through its cyclophilin domain and
associated PPIase activity, matrin CYP may hold SR proteins in a state
from which they cannot associate with each other. This could prevent
undesirable aggregation of splicing factors and would be an efficient
means to keep a warehouse of splicing factors ready for the variable
expression needs of the cell. In addition, the proline isomerase of
matrin CYP may assist proteins in nuclear speckles to properly fold and
associate into appropriate macromolecular assemblies.
The dynamic movement of speckle-associated proteins is also closely
coupled to RNA polymerase II transcription (88). In this regard, a
hyperphosphorylated form of the large subunit of pol II (pol lIo) is
associated with pre-mRNA splicing, and a subset of SR proteins may
mediate this association through interactions with the highly
phosphorylated carboxyl-terminal domain (CTD) of pol IIo (17, 18,
90-92). Recently, a yeast two-hybrid screen has identified the human
homolog of matrin CYP as strongly interacting with the CTD of pol II
and, thus, a member of this CTD-binding protein family (79, 92). This
interaction implicates matrin CYP as a possible regulator in the
coordination of transcription and pre-mRNA splicing perhaps through
its cyclophilin domain. Indeed, the extraordinarily high proline
content of the CTD (2 prolines per heptapeptide repeat or 30% of total
amino acids) makes it an obvious target for the matrin CYP proline
isomerase.
Interaction of matrin CYP with the CTD of pol II and/or other
CTD-binding proteins may, in turn, be regulated by specific phosphorylation events mediated by clk kinase and other protein kinases
such as SAPK-1. In this regard, Bourquin et al. (79) demonstrated that human matrin CYP requires the SR repeat motifs (known
phosphorylation target sites of the clk kinase, see Ref. 86) to
interact with the CTD in vitro. In contrast, all other characterized CTD-binding proteins interact with the CTD at other regions on the proteins (90, 92, 93). The nuclear matrix association of
pol IIo (17, 18), matrin CYP (Ref. 79 and this study), and a recently
characterized CTD-binding protein termed SCAF8 (94) together imply an
important role of nuclear architecture for coupling transcription and
RNA splicing in the cell nucleus (87).
We thank Dr. Tom Maniatis for supplying the
anti-U1-70-kDa and Y12 antibodies; Drs. Nilabh Chaudray and Gunter
Blobel for supplying the anti-lamin A/C antibodies; Dr. David
Brownstein for the pC-DX cDNA library; Dr. Stephen Anderson for
supplying the NK-TR CYP fusion protein construct in pGEX; Dr. Aline
Rinfret and Dr. Roydon Price for suggestions concerning the PPIase
assay; Drs. David Hakes and Jack Dixon for the pGEX-kt plasmid; Dr.
James Sawyer for advice and assistance in preparing the rat parenchymal cells; and Dr. Ping-Chin Cheng for use and maintenance of the confocal
microscope. We are particularly grateful to Dr. Phillip Belgrader for
purifying the partial length cDNA; Dr. Jagath Samarabandu for
computer imaging programming and training; Dr. Ben Blencowe who
provided the SR protein preparations, several of the anti-SR protein
antibodies, and stimulating discussion; Leigh Leonard who helped
prepare the antibodies from chicken eggs; and Jim Stamos for preparing
the figures.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF043642.