From the Max-Planck Institute of Neurobiology, Am Klopferspitz 18a,
D-82152 Martinsried, Germany and the Departments of
Pathology and Cell Biology, Baylor College of Medicine,
Houston, Texas 77030
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ABSTRACT |
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We report on the molecular cloning of a novel
human cDNA by its interaction with the splicing factor SRp30c in a
yeast two-hybrid screen. This cDNA is predominantly expressed in
muscle and encodes a protein that is present in the nucleoplasm and
concentrated in nucleoli. It was therefore termed Nop30 (nucleolar
protein of 30 kDa). We have also identified a related cDNA with a
different carboxyl terminus. Sequencing of the NOP gene
demonstrated that both cDNAs are generated by alternative 5' splice
site usage from a single gene that consists of four exons, spans at
least 1800 nucleotides, and is located on chromosome 16q21-q23. The
alternative 5' splice site usage introduces a frameshift creating two
different carboxyl termini. The carboxyl terminus of Nop30 is rich in
serines and arginines and has been found to target the protein into the nucleus, whereas its isoform is characterized by proline/glutamic acid
dipeptides in its carboxyl terminus and is predominantly found in the
cytosol. Interaction studies in yeast, in vitro protein interaction assays, and co-immunoprecipitations demonstrated that Nop30
multimerizes and binds to the RS domain of SRp30c but not to other
splicing factors tested. Overexpression of Nop30 changes alternative
exon usage in preprotachykinin and SRp20 reporter genes, suggesting
that Nop30 influences alternative splice site selection in
vivo.
In eukaryotes, gene expression is controlled at several levels.
Due to the presence of cell-specific factors, genes can be transcribed
in a cell type or developmentally regulated manner. Moreover, primary
transcripts undergo maturation processes such as pre-mRNA splicing
(1) and capping. A growing number of proteins containing an N-terminal
RNA recognition motif and C-terminal clusters of serines and arginines
(SR proteins)1 have been
shown to be involved in constitutive and alternative splicing (2, 3).
Using the family member SRp30c (4) as an interactor in a two-hybrid
screen, we have previously shown that the matrix attachment region
element-binding protein SAF-B has the potential to link matrix
attachment region elements, RNA polymerase II, and SR proteins (5).
This supports growing evidence that gene expression achieved through
RNA biosynthesis, RNA processing, and RNA transport is a highly
coordinated process mediated by a network of proteins that has been
termed the RNA "factory" (6) or transcriptosomal complex (7). For
example, it has been shown that the carboxyl-terminal domain of
RNA polymerase II binds to proteins involved in pre-mRNA processing
(8, 9) and that 3' processing of pre-mRNA is coupled to components
of the splicing machinery (6).
RNA processing is not just confined to polymerase II transcripts, since
rRNA generated by polymerase I undergoes maturation that removes
externally and internally transcribed spacers in various steps to yield
28, 18, and 5.8 S ribosomal RNA in mammalian cells (10). This process
is confined to nucleoli, the sites of ribosome biosynthesis. Three
morphologically distinct areas of the nucleolus can be distinguished:
the nucleolar fibrillar center, the dense fibrillar region, and the
granular region. Functional studies have indicated that the fibrillar
center is the region where ribosomal RNA genes, RNA polymerase I, and
transcription factors are localized. The dense fibrillar region
consists of unprocessed nascent ribosomal RNA and associated proteins.
Mature 18 and 28 S rRNA together with intermediates in ribosome
assembly are found in the granular region. Thus, the morphological and functional compartmentalization of the nucleolus reflects specialized areas involved in rRNA synthesis, processing, and export (11, 12).
We are currently using yeast two-hybrid screens to identify novel
proteins involved in RNA processing. Employing the splicing factor
SRp30c (4) as a bait to screen a human library, we identified a
cDNA coding for a nuclear protein that is enriched in nucleoli and
migrates around 30 kDa on SDS-polyacrylamide gel electrophoresis. We
therefore termed this protein Nop30. We identified an isoform of Nop30,
which we call Myp. Myp has been previously isolated from rats in a
screen aimed to find genes induced by nerve growth factor (13).
Analysis of the NOP gene revealed that both isoforms are
generated by alternative splicing. In contrast to the nuclear localization of Nop30, its isoform Myp is found in the cytosol. Overexpression of Nop30 with a cotransfected SRp20 or preprotachykinin minigene (14) affects splicing of the corresponding alternative exons
in the reporter genes, suggesting a potential role of Nop30 in
pre-mRNA splicing. Since Nop30 binds specifically to SRp30c and its
mRNA is highly expressed in muscle, we suggest that this protein
acts as a regulator of SR protein function in muscle.
Two-hybrid Screening--
A yeast two-hybrid screen using SRp30c
as a bait in pGBT9 and a HeLa library was performed as described by
Fields and Song (15). DNA from the autotrophic bacteria was sequenced
using an ABI sequencer and analyzed with the GCG Wisconsin package
(16). To test the interaction between proteins, 1 µg of a prey
construct fused to the Gal4 activation domain in pADGal4 and 1 µg of
bait-construct fused to the Gal4 binding domain were simultaneously
co-transformed and plated onto double dropout plates lacking leucine
and tryptophan. Surviving colonies were restreaked onto triple dropout
plates lacking leucine, tryptophan, and histidine and onto triple
dropout plates supplemented with 5 and 10 mM
3-aminotriazole (Sigma).
Genomic Cloning--
Using the cDNA of Nop30 as a probe, a
human BAC library was screened (kindly performed by the German Human
Genome Project). Two PstI fragments containing the complete
clone were subcloned in pBluescript-SK(+) (Stratagene), and positive
clones were identified by colony hybridization (17).
Computer Analysis--
Calculation of splice site scores was as
described by Stamm et al.
(18).2
Chromosomal Localization--
Chromosomal localization was
performed with the Stanford radiation hybrid panel 4.0 (Research
Genetics Inc.). For PCR, the following primers were used: PEintrof1
(5'-GGTCCGGGTGAGCGCGCGGG-3'; located in intron B) and PEint1
(5'-GAGATGACGGGAACAGTGGTCAAAG-3'; located in intron C). For data
processing and mapping, World Wide Web servers were
used.3
Northern Blot--
Nop30 full-length cDNA, SRp30c cDNA,
and a In Situ Hybridization--
0.2 ng of the cloned Nop30 probe was
PCR-amplified using primers specific for Nop30 anchored by either the
T7 or Sp6 RNA polymerase recognition sequence and purified using
QiaQuick (Qiagen). Transcription using either T7 or Sp6 RNA polymerase
was done according to the manufacturer's protocol (Boehringer
Mannheim) using 250 ng of the respective PCR product as template.
In situ hybridization histochemistry and the following
washing steps were performed as described earlier (19). 20-µm-thick
dried cryostat sections were incubated with 2 × 106
cpm of the [35S]UTP (Amersham Pharmacia Biotech)-labeled
probe. After the washing steps, sections were dehydrated through graded
ethanols containing 0.3 M ammonium acetate, air-dried,
covered with a thin sheet of film emulsion (NBT-2, Eastman Kodak Co.),
and exposed at 4 °C for 7 days. They were developed in Kodak D19
developer and fixed in 24% sodium thiosulfate. Sections were
counterstained with Cresyl Violet, dehydrated, and mounted in DePeX (Gurr).
Cloning of Deletion Variants--
The following constructs were
generated with the primers indicated using PCR: EGFP-Nop30 Immunocytochemistry/DiQ Staining--
HEK293, BHK, or HeLa cells
were grown on glass coverslips and transfected with 0.5 µg of an EGFP
fusion construct (in pEGFP-C2; CLONTECH), using the
method of Chen and Okayama (20). The following EGFP fusion products
were used: Nop30, Nop30 Pull Down--
Nop30 was cloned in pCR3.1 (Invitrogen) and used
for an in vitro reticulocyte lysate
transcription/translation (TNT, coupled reticulocyte lysate system;
Promega) to obtain 35S-labeled Nop30 protein. For the
binding experiments, glutathione-Sepharose 4B (Amersham Pharmacia
Biotech) was washed three times with HNTG buffer (50 mM
HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10%
glycerol, and 0.1% Triton X-100), (23), followed by
incubation with 1 µg of GST-SRp30c, GST-ASF/SF2, or GST; 10 µl of
glutathione-Sepharose 4B; 20 µl of HNTG buffer; and 23,000 cpm of
35S-labeled Nop30 with agitation overnight at 4 °C.
After centrifugation, the supernatant was removed, and the pellet was
washed three times with 500 µl of HNTG buffer supplemented with 0.1%
Triton X-100 and loaded onto a 12% SDS-polyacrylamide gel. Exposure of
dried gels was made overnight on FujiX BAS 1000 phosphor imager plates.
Immunoprecipitation and Western Blot--
For
immunoprecipitation, human embryonic kidney (HEK) 293 cells were grown
to 50% confluency and transfected with 0.1-µg DNA constructs as
indicated. Immunoprecipitation of the EGFP constructs was performed
using an anti-GFP antibody (Boehringer Mannheim) (5). The
co-precipitating FLAG constructs were analyzed after 10-15%
SDS-polyacrylamide electrophoresis and Western blotting using an
anti-FLAG antibody (Santa Cruz Biotechnology). SDS-polyacrylamide gel
electrophoresis was performed as described by Laemmli (24), and protein
was transferred onto ECL membranes (Amersham Pharmacia Biotech).
Blocking of the membranes and protein detection was performed as
described by Nayler et al. (23).
Splicing Assays--
Transient transfection and RT-PCR was
performed as described previously (5). The indicated amounts of
pNop30-C2, pEGFP-C2 (CLONTECH) and pCG35 were
co-transfected with 2 µg of either the SRp20 (14) or PPT minigene in
HEK293 cells or in fibroblasts, respectively. The next day, the
transfection rate was assessed by fluorescence microscopy, and RNA was
isolated from equally transfected cultures using the RNeasy Kit
(Qiagen). For the SRp20 minigene, 30 cycles were used with annealing
and extension temperatures of 55 and 72 °C with the primer pair T7
(5'-TAATACGACTCACTATAGGG-3') and X16R
(5'-CCTGGTCGACACTCTAGATTTCCTTTCATTTGACC-3'). PCR products were resolved
on a 1.5% agarose gel, stained with ethidium bromide, and quantified
using the Herolab EASY system. For the PPT minigene, reverse
transcription followed by radioactive PCR using the primers T7PRO
(5'-TAATACGACTCACTATA-3') and PPTE5rev (5'-GTGAGAGATCTGACCATGCC-3') with 18 cycles was performed using annealing and extension temperatures of 70 and 72 °C, respectively. PCR products were resolved on a 5%
nondenaturing polyacrylamide gel. Bands were quantified directly from the gel using a phosphor imager. The percentage of inclusion is
the percentage of spliced RNA that contains the exon (cpm of inclusion
band/(cpm of inclusion band + cpm of exclusion band) × 100.
Sequence--
In order to find new proteins involved in RNA
processing, we performed yeast two-hybrid screens with SRp30c (4) and
identified a clone from a HeLa library, which we named Nop30. Its
sequence is shown in Fig. 1A.
The cDNA of Nop30 encodes a protein with a calculated molecular
mass of 24.3 kDa. The protein contains a highly acidic N terminus
(amino acids 1-95) with an isoelectric point of 4.9 and a basic C
terminus, enriched with arginines, serines, and prolines (amino acids
96-124) (pI = 12.7). The total protein is highly basic (pI 11.7)
(Fig. 1B). Probably due to the bipolar charge distribution,
it has an apparent molecular mass of 30 kDa upon SDS gel
electrophoresis. Inspection of the sequence shows that it contains a
potential cAMP-dependent protein kinase phosphorylation
site (amino acids 197-200), several potential protein kinase C
phosphorylation sites, and a potential casein kinase II phosphorylation
site (amino acids 41-44). The casein kinase II site may be functional,
since recombinant protein can be phosphorylated by casein kinase II
in vitro (data not shown). Due to the presence of arginine
and serine residues (Fig. 1D), the C terminus of Nop30 shows
homology to some SR and SR-related proteins (human protein kinase CLK3
(23) and splicing factors 9G8, SRp55, SRp75, and SC35 (2)).
Data base searches also showed that the first 95 amino acids of Nop30
share 94% homology to a rat protein with an as yet unknown function
that was previously found in a screen aimed to identify genes induced
with nerve growth factor (13). However, due to a frameshift, the
carboxyl domains encoded by the two cDNAs are completely different.
We searched the expressed sequence tag data base (25) and identified a
human clone (accession number AA085275) corresponding to the rat
cDNA. This expressed sequence tag clone was entirely sequenced and
found to encode the human homologue of the rat cDNA clone described
by Geertman et al. (13). The carboxyl terminus of the
encoded protein is characterized by clusters of prolines and glutamic
acids (Fig. 1, A and B). We named this protein
Myp (muscle-enriched cytosolic protein) because of its expression
pattern and intracellular localization (see below). In addition to the
different C terminus caused by the frameshift, this clone differs from
our two-hybrid isolate by the use of a different polyadenylation site
(PS-2, Fig. 1A).
Since both human sequences differ by only ten nucleotides in their
coding region, they could derive from alternative splicing. We
therefore screened a human BAC library and obtained a genomic NOP clone. Comparison of its sequence with Nop30 and its
variant revealed that the gene is composed of four exons and three
introns and generates the two isoforms by using an alternative 5'
splice site in exon 2 (Fig. 1, A and C). The size
of the gene is at least 1800 nucleotides. The same first start codon
present in all expressed sequence tags, our two-hybrid isolate, and the
rat cDNA is located in exon II. In contrast to rat, the start codon
in humans deviates slightly from the Kozak consensus sequence (26). A
remarkable feature of the gene is the small size of its introns (115 nucleotides for intron A, 155/165 nucleotides for intron B, and 102 nucleotides for intron C). This is significantly smaller than mammalian
internal introns, which average around 1127 nucleotides (27). All
intron/exon boundaries are in agreement with the mammalian consensus
sequence. However, calculation of the splice site scores revealed a
difference in the alternatively used 5' splice site of intron B. The
splice site generating Nop30 (score 5.3) deviates from the average
score of constitutive exons (score 8.1), which is typical for a subset of alternatively spliced exons (18). In contrast, the score of the
variant splice site (score 7.9) is close to the average score of
constitutive exons (score 8.1).
Using the Stanford radiation hybrid panel with the primers PEint1 and
PEintrof1, we mapped the NOP gene to human chromosome 16. The NOP gene was found 3.15 cR from WI-5594, between markers WI-5594 and WI-9392 (Fig. 2). This most
likely corresponds to the region between 81 and 84 cM on chromosome 16 (q21 to q23). In summary, we have shown that the NOP gene is
located on chromosome 16q and generates two isoforms via an alternative
splicing mechanism.
Expression--
We next determined the expression of
NOP and performed Northern blot analysis with various human
tissues using the full-length cDNA fragment of Nop30 as a probe.
Nop30 mRNA shows the highest expression in heart and skeletal
muscle (Fig. 3A). However, a faint band is visible in other tissues examined. The two bands of 1.8 and 1.3 kb most likely correspond to the two polyadenylation sites
found in the NOP gene (Fig. 1A). These expression
data are in agreement with the tissue distribution found for the rat
cDNA (13). The 1.8-kb band corresponds to the size of the longest cDNA clone, indicating that we have obtained the full-length Nop30 transcripts. By probing mouse tissue with a human cDNA probe, it
was shown that SRp30c is predominantly expressed in kidney, lung, and
spleen, whereas only low levels were detected in muscle (4). This
expression pattern would make an in vivo interaction between
SRp30c and Nop30 questionable. We therefore reprobed our filter with
human SRp30c and found considerable SRp30c expression in human striated
muscle as well as in other tissues (Fig. 3B), suggesting
that SRp30c is a possible interactor for Nop30 in muscle. The same blot
was reprobed with a
We noticed a faint signal present in nonmuscle tissues using Nop30 as a
probe (Fig. 3A) and compared Nop30 mRNA expression in
heart and brain using in situ hybridization. We used a rat Nop30 cDNA fragment containing the whole coding region, except for
the C-terminal repetitive sequence, as a probe. The Nop30 antisense
probe gave a strong signal after in situ hybridization in
heart (Fig. 4A). In brain, a
specific signal was only seen in the pia mater (Fig. 4C).
The pia mater is of mesenchymal origin, surrounds the brain (Fig.
4D), and contains blood vessels lined with smooth muscle
cells (29). In contrast, the signal derived from neuronal
or glial cells did not exceed the background. No signal could be
detected using a sense control probe (data not shown). In summary,
Northern blot analysis and in situ hybridization demonstrate
that Nop30 shows the highest expression in muscle cells. Moreover,
using RT-PCR, we did not detect a change in expression during C2C12
cell differentiation into myoblasts or during embryonic development,
suggesting that Nop30 mRNA and its isoform are constitutive parts
of muscle cells in various stages of differentiation and development
(data not shown).
Interaction with Other Proteins--
In order to obtain
information on the possible function of Nop30 and its isoform, we
tested its interaction with other proteins in the yeast two-hybrid
system. Of all proteins involved in pre-mRNA metabolism tested
(SRp20, SF2/ASF, SC35 (2), SmN (30), htra-beta1 (21), SRp30c, SRp75,
SRp55, (4) SAF-B (5), U2AF35 (31), and the carboxyl-terminal domain of
RNA polymerase II (8)), only SRp30c and Nop30 itself showed interaction
in yeast (Fig. 5A).
Interestingly, its splice variant Myp did not interact with SRp30c and
reacted only weakly with Nop30. This indicated that the different
carboxyl termini of the two splice variants are responsible for
differences in protein-protein interactions. We therefore tested them
separately. The deletion constructs used are shown schematically in
Fig. 5B. We found that deletion of the N terminus of Nop30
abolishes binding to Nop30, whereas a clone with a deleted C terminus
was still able to interact. In addition, the two C-terminal Nop30
deletion variants (Nop30
In order to verify the two-hybrid interactions biochemically, Nop30 was
translated and incubated in vitro with
glutathione-Sepharose, GST, GST-SRp30c, and GST-ASF/SF2 (Fig.
6). In this assay, Nop30 bound only to
SRp30c, but not to SF2/ASF. This confirmed our two-hybrid analysis and
indicated that Nop30 can discriminate between the related RS domains of
SF2/ASF and SRp30c.
Next, we tested Nop30 interaction under more physiological conditions
in immunoprecipitation assays. We fused Nop30, Myp, SRp30c, and their
deletion variants previously analyzed in yeast (Fig. 5B) to
EGFP and co-transfected these constructs with FLAG-tagged Nop30,
SRp30c, and ASF/SF2 in HEK293 cells. Complexes were precipitated with a
monoclonal anti-EGFP antibody, and interacting proteins were detected
with an anti-FLAG antibody. As shown in Fig.
7A, Nop30 bound to itself,
more weakly to its amino terminus (Nop30
Taken together, our binding experiments indicate that Nop30 binds to
itself and to SRp30c. Binding to SRp30c is RS
domain-dependent, and Nop30 is able to discriminate between
the RS domain of SRp30c and other SR proteins, which implies that the
binding is due to protein-protein interaction and is not RNA-mediated.
In addition, we were unable to detect binding of EGFP-Nop30 to RNA or
DNA in pull down experiments (data not shown).
Intracellular Localization--
Since the two splice variants of
NOP had different affinities toward nuclear proteins, we
determined their intracellular localization. First, we tagged Nop30
with EGFP and expressed the fusion proteins in BHK and HEK293 cells. As
shown in Fig. 8, EGFP-Nop30 is expressed in both cell lines in the nucleus, where it is concentrated in the
nucleoli, but is also present in the nucleoplasm (Fig. 8, A-C, BHK cells; Fig. 8, D-F, HEK 293 cells).
The staining in the nucleoli is not uniform, but it shows several small
foci (Fig. 8A, arrow) or one large one (Fig.
8G, arrow). Untagged EGFP is located throughout
BHK or HEK293 cells (Fig. 8, H and I). To verify that the structures identified in phase contrast were nucleoli, we
performed costaining with an anti-nucleolus antibody. These experiments
confirmed the predominant nucleolar localization of Nop30 (Fig.
9, A-C).
Three morphologically distinct areas of the nucleolus were defined on
the ultrastructural level. It is possible to distinguish them by
immunocytochemistry and confocal microscopy, using markers that have
been shown to be restricted to certain nucleolar regions by correlating
light and electron microscopy (32-34). We therefore performed
localization experiments with the nucleolar protein B23, which is a
marker for the granular component of nucleoli (34). As shown in Fig. 9,
D-F, Nop30 and B23 colocalize in the granular component of
nucleoli. It is also evident that the major part of Nop30 localizes in
the area between the foci and the granular component, which is likely
to be the fibrillar component according to the classical organization
of the nucleolus (35).
To determine whether an interaction between Nop30 and SRp30c can take
place in vivo, we analyzed the localization of EGFP-Nop30 and FLAG-SRp30c (Fig. 9, G-I) using an anti-FLAG antibody.
SRp30c does not localize in nucleoli, but both proteins were detected in the nucleoplasm, indicating that the interaction between both proteins could occur in vivo.
We next tested Myp, the splice variant of Nop30. Transfection of the
EGFP-tagged variant revealed that it is predominantly located in the
cytoplasm. Only faint staining could be detected in the nucleus, and no
staining was detected in nucleoli (Fig. 10A). Therefore, we named
this protein Myp (muscle-enriched cytoplasmic protein). Since the
intracellular localization of Myp and Nop30 is different, we tested
their common N terminus and different C termini separately. The common
N terminus gave a predominant cytoplasmic staining pattern resembling
Myp (Fig. 10C). Transfection of cells with the C terminus of
Nop30 resulted in nuclear staining, with predominant staining in the
nucleoli similar to that of full-length Nop30 (Fig. 10D). In
contrast, when we tested the C terminus of Myp, we observed staining
throughout the cell, excluding the nucleoli (Fig. 10B). The
integrity of various EGFP constructs used for fluorescence was tested
by Western blot (Fig. 10E), demonstrating that all
constructs expressed protein of the expected size.
We conclude from these experiments that the two splice variants
generated by the NOP gene have a different intracellular
localization; Nop30 is a nuclear protein concentrated in the nucleoli,
and the variant Myp is predominantly localized in the cytoplasm. The
localization to nucleoli is an intrinsic property of the basic
arginine-rich C terminus of Nop30, whereas the acidic N terminus seems
to exclude the protein from the nucleus.
In Vivo Splicing--
We next asked whether the binding properties
of Nop30 to SRp30c are relevant in vivo. Since concentration
changes of SR proteins modulate alternative splicing patterns in
vivo (3, 36, 37), we reasoned that overexpression of Nop30 would
change the SR protein composition through interaction with SRp30c,
which would change splicing patterns of a reporter gene. First, we
employed the SRp20 XB minigene. Using this construct, it has been shown
that SRp20 can promote inclusion of the alternative exon 4 in its own
mRNA, whereas SF2/ASF has the opposite effect (14). We examined the effect of Nop30 on exon 4 usage (Fig.
11A) and cotransfected the SRp20 XB minigene with an increasing ratio of pNop30-C2 to the parental
vector pEGFP-C2 (Fig. 11, A-C, top). With
increasing amounts of pNop30-C2, we detected a decrease of exon 4 inclusion from ~34 to 10% (Fig. 11A). In order to compare
the effect of SRp30c on this minigene, we performed a similar
experiment and found that EGFP-SRp30c also decreases exon 4 usage (Fig.
11B). However, less EGFP-SRp30c cDNA needs to be
transfected to reduce exon 4 usage. To examine the combined effect of
SRp30c and Nop30 on exon 4 splicing, we performed cotransfection
experiments in which increasing amounts of pNop30c were added to a
constant amount of SRp20 XB minigene and SRp30c (Fig. 11C).
We found that, in the presence of EGFP-SRp30c, less pNop30-C2 was
necessary to induce a visible decrease in exon 4 usage. Transfection of
5 µg of pNop30-C2 abolished inclusion of the alternative exon (Fig.
11C), whereas this amount of pNop30-C2 did not change exon 4 usage significantly in the absence of SRp30c (Fig. 11A).
Overexpression of EGFP alone (first lane in each
experiment) did not induce exon skipping. To test the effect of Nop30
in another system, we used a PPT minigene (38). As shown in Fig.
11D, cotransfection of 3 and 10 µg of pNop30-C2 promotes
alternative exon inclusion in the PPT system, again indicating that
concentration changes of Nop30 changes alternative exon usage in
vivo. In contrast, overexpression of the splicing factor SC35 had
no significant effect on exon 4 inclusion. We conclude from these
experiments that Nop30 can influence alternative splicing in
vivo.
We report on the molecular cloning and characterization of the
NOP gene and its two products, which are generated by
alternative splicing. Northern blot analysis indicates that the gene is
predominantly expressed in muscle cells. In order to investigate a weak
signal present in all tissues examined, we performed in situ
hybridization with rat brain sections and found that the NOP
gene is expressed in the pia mater, indicating a specific expression
pattern. Therefore, we assume that the faint signal detected in
nonmuscle tissues with Northern blot analysis might result from
muscle-derived cells that line blood vessels in all tissues. Therefore,
it is possible that Nop30 expression is muscle-specific. However, from
our data we cannot exclude a weak basal NOP expression in
other tissues. The NOP gene is located on chromosome 16 between 16q21 and 16q23. Although only a few genes are identified in
this region, it is interesting that a 16q23.1 deletion leads to
disorders in musculoskeletal systems (39). Further analysis will be
necessary to investigate whether Nop30 is involved in one of these disorders.
The Nop30 cDNA was obtained in a two-hybrid screen for proteins
that interact with SRp30c. Using the three independent methods of yeast
two-hybrid assays, GST pull down experiments and immunoprecipitations, we were able to demonstrate that Nop30 protein can bind to itself and
to SRp30c. The multimerization of Nop30 protein depends on its N
terminus. The interaction with SRp30c is dependent on the RS domain of
SRp30c and both N- and C-terminal parts of Nop30. Our results
demonstrate that protein-protein interactions mediated by RS domains
can be extremely specific. In order to test whether an interaction of
Nop30 with SRp30c is possible in vivo, we determined the
distribution of SRp30c in human tissues. We found that human SRp30c is
found in all tissues, including muscle. Furthermore, we show that both
SRp30c and Nop30 colocalized in the nucleoplasm (Fig. 9,
G-I). Taking the expression and localization experiments together, it is likely that Nop30 interacts with SRp30c in the nucleoplasm of muscle cells.
Alternative splicing is a commonly used mechanism to create protein
isoforms (1, 40). A usual feature of alternatively used exons is the
introduction of stop codons or frameshifts (18) that can either switch
off genes (41) or create proteins with different C termini, which can
result in a different intracellular localization. For example, the
nuclear localization of the estrogen receptor (42) and the
transcription factors E2F (43), p105 NF- Nucleoli vary in size and shape, reflecting the cellular activity (52,
53). In addition, nucleoli of transformed cells have been reported to
be extremely irregular in number, shape, and size (54, 55), as can also
be seen in our Figs. 8 and 9. No significant difference between
nucleoli of transfected and untransfected cells was observed by bright
field and fluorescence microscopy using the B23 antibody (Fig.
9F), indicating that Nop30 overexpression does not change
the nucleolar shape. Nop30 protein expression is restricted to
specified areas in the nucleolus. Colocalization with B23 revealed that
Nop30 is present in the granular component, where the later stages of
ribosome biosynthesis occur. In addition, it is detected in the more
central region that could be the dense fibrillar component.
Furthermore, we noticed that, based on their size, number, and shape
(56, 57), Nop30 is absent from discrete foci that are most likely the
fibrillar centers.
The function of Nop30 proteins has yet to be demonstrated directly.
Colocalization and binding to SRp30c suggest a role of Nop30 in
pre-mRNA processing. Indeed, we found an effect of Nop30 on
splicing of a PPT and a SRp20 reporter gene in vivo. As with all mammalian factors involved in pre-mRNA processing, the specific target gene is unknown (3). Nop30 has a different effect on the two
minigenes tested; it promotes exon inclusion in the PPT minigene but
causes exon skipping when tested with the SRp20 construct. This is
reminiscent of opposing effects of splicing factors on natural
minigenes in vivo. For example, SF2/ASF promotes exon EN
inclusion when tested with the clathrin light chain B minigene (36) but
causes exon 4 skipping with the SRp20 minigene (14).
The predominant nucleolar localization of Nop30 also points to a
potential role in RNA processing in muscle nucleoli. There are several
examples of intimate connections between nuclear compartments. For
example, in S. cerevisiae rRNA processing and pre-mRNA
splicing are coupled via the ribosomal protein L32, which influences
both splicing of its own transcript and the processing of rRNA (58). Another link between splicing and nucleolus comes from the observation that the transcript of the mouse ribosomal protein S24 gene is alternatively spliced (59). Therefore, it seems likely that Nop30 has a
dual role in splicing as well as in the nucleolus.
Several studies in vivo (3, 36, 37, 60) and in
vitro (3, 4, 48, 61, 62) have shown that SR protein concentration can influence alternative splice site selection. Specifically, an
increase in the nuclear concentration of a transiently overexpressed SR
protein changes alternative exon usage. Tissue-specific differences in
the ratio of SR proteins and hnRNP A1 suggest a role in tissue-specific splicing (63), and alterations in the levels of individual SR proteins
have been associated with changes in CD44 alternative splicing during T
cell activation (4). Therefore, SR proteins are likely to be involved
in tissue-specific regulation of alternative splicing. Due to its
binding properties to SRp30c, its ability to influence splice site
decisions, and its high expression in muscle, it is possible that Nop30
changes the free concentration of SRp30c by specifically
sequestering this SR protein in muscle cells. This muscle-specific
sequestration would change the SR protein composition in a cell
type-specific way and might indirectly lead to muscle-specific
alternative splicing decisions. Alternatively, similar to results
obtained with SRm160 (64), it is possible that Nop30 enhances effects
of SR proteins on certain genes, which would be consistent with the
synergistic effect of Nop30 and SRp30c on exon 4 of the SRp20
minigene (Fig. 11C). It remains to be seen whether other
tissue-specific proteins exist that can discriminate between different
SR proteins and might influence alternative splicing.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin probe (CLONTECH) were labeled with
radioactive [
-32P]dCTP using the megaprime DNA
labeling system from Amersham Pharmacia Biotech. A human multiple
tissue Northern blot (CLONTECH) was probed
according to the manufacturer's instructions.
N with
PEC (5'-GGCCATGAATTCACCATGGCTACCGGGACCGCAGCTATG-3') and PEOBam
(5'-GGCCATGGATCCCTATCCAGCATGGGCGGGGCAC-3'); Nop30
C with PEOEco
(5'-GGCCATGAATTCATGGGCAACGCGCAGGAGCGG-3') and PEN (5'-GGCCATGGATCCTCAGTGCTGCCAGTCCCAAGCGGG-3'); Myp
N with est1b (5'-GGCCATGAATTCGTGGGTCCGGGCTACCGGGAC-3') and est1c
(5'-GGCCATGGATCCCTATCCAGCATGGGCGGGGCAC-3'); 30c
RS with 30cdelr
(5'-GGCCATGGATCCCTCAGGATAAACTCGGATGTAGG-3') and 30cdelf
(5'-GGCCATGAATTCATGTCGGGCTGGGCGGACGAG-3').
N, Nop30
C, Myp, Myp
N, SRp30c, and
SRp30c
RS. Cells were cultured in Dulbecco's modified Eagle medium
(Life Technologies, Inc.) supplied with 10% fetal calf serum (Life
Technologies) for 18-24 h in 5% CO2. To
counterstain cells, DiQ
(4-(4-(dihexadecylamino)styryl)-N-methylquinolinium iodide; Molecular Probes, Inc., Eugene, OR) was added, followed by
another 2 h of incubation. DiQ is a long-chain dialkylcarbocyanine that stains membranes and components of the cytoplasm, excluding the
nucleus. Immunocytochemistry was performed as described (21). The
following antibodies in phosphate-buffered saline, 0.1% Triton were
used: a monoclonal anti-nucleolus antibody (1:300; Calbiochem), a
polyclonal rabbit anti-FLAG antibody (1:150; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and a monoclonal B23 antibody (1:100; Ref. 22).
For B23 staining, cells were permeabilized for 1 min with 100% acetone
at
20 °C. After washing three times, the cells were incubated for
1 h with the appropriate antibody linked to Cy3 (1:500 in
phosphate-buffered saline, 1% Triton). The cells were mounted using
Gelmount (Biomeda) and analyzed by confocal microscopy (Leica).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence and structure of the NOP
gene and its derived cDNAs Nop30 and Myp. A,
sequence of the NOP gene. Exons are indicated by
capital letters, introns by small
letters. The two starting points of cDNA clones are
indicated by arrows (S1 for Nop30, S2
for Myp). The translation of the protein corresponding to Nop30 is
shown between the DNA sequence and the protein sequence of Myp. The
start codon, the alternatively spliced region, and the stop codons of
the two cDNAs are shown in boldface type. The
polyadenylation signals are boxed (open
box). The glutamic acid/proline-rich repeats of Myp are
indicated by shaded boxes. The ends of the two
cDNA clones are indicated by triangles (PS-1
for Nop30, PS-2 for Myp). The numbering starts at the S2
site. B, domain structure of Nop30 and its isoform Myp. The
common N terminus is indicated by gray shading,
and the divergent C termini are black and
striped, respectively. Isoelectric points are shown
above these domains. C, schematic representation
of the NOP gene organization. Exons are shown as
boxes, introns as lines. Exons generating Nop30
are shown above the line, and those forming Myp
are shown below the line. Coding regions are
indicated by shading. Different
shading indicates differences resulting from the frameshift
introduced by the alternative 5' splice site. D, change of
the amino acid composition in the C terminus caused by alternative 5'
splice site usage. aa, amino acids.
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Fig. 2.
Chromosomal localization of the
NOP gene. Cytogenetic (left) and
radiation hybrid map (right) of chromosome 16. NOP maps 3.15 cR from WI-5594, which corresponds to a region
between 16q21 and 16q23. The most likely location on the radiation map
of chromosome 16 is indicated by an arrow.
-actin control (CLONTECH) to verify that equivalent amounts of RNA were loaded. In heart and skeletal muscle, the additional muscle-specific actin isoforms at 1.8 kb were detected (28).
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Fig. 3.
Northern blot analysis of Nop30
expression. A, Northern blot analysis of NOP
transcripts on various human tissues (CLONTECH)
using a full-length Nop30 probe. The signals at 1.8 and 1.3 kb in heart
and skeletal muscle correspond to NOP transcripts processed
at the two polyadenylation sites. The faint band detected in nonmuscle
tissues is indicated by a triangle. B, the same
filer was rehybridized with a human SRp30c full-length probe. The
highest expression was detected in pancreas, followed by kidney,
placenta, and heart. C, the same filer was rehybridized with
a -actin control probe. The cytoskeletal
-actin isoform is
detected at 2.0 kb. The probe also hybridizes to the muscle-specific
and
isoforms (1.8 kb) in heart and muscle.
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Fig. 4.
In situ analysis of Nop30
expression in heart and brain. A, dark field picture of
rat heart tissue probed with a rat Nop30 antisense cRNA. B,
cresyl violet counterstain of the same area as in A). The
blood vessel present in this area is indicated. C, dark
field picture of rat brain probed with Nop30 antisense cRNA. The signal
is located in the pia mater. No tissue is present between the top of
this section and the pia mater. The signal there represents the
backgound. D, cresyl violet counterstain of the same area as
in C.
C) were also found to interact. Thus, Nop30
dimerizes or multimerizes via its N terminus. In contrast, deleting
either the C or N-terminal part of Nop30 resulted in a lack of
interaction with SRp30c. In addition, deletion of the RS domain of
SRp30c prevented binding of SRp30c to Nop30 (Fig. 5A). We
conclude from this analysis that Nop30 interacts with the RS domain of
SRp30c, using parts of both its N- and C-terminal domain.
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Fig. 5.
Binding of Nop30 and Myp to other proteins in
the yeast two-hybrid assay. A, interaction of Nop30,
Myp, SRp30c, and deletion constructs. The interacting partners are
indicated. The first interactor was fused to the Gal4 binding domain of
pGBT9 (bait constructs); the second partner, indicated in
boldface type, was fused to the Gal4 activation
domain (prey constructs). Positive clones were restreaked on
His plates containing 10 mM 3-aminotriazole.
B, schematic diagram of SRp30c, Nop30, Myp, and their
deletion constructs used in the two-hybrid analysis. RRM,
RNA recognition motif; aa, amino acids.
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Fig. 6.
Nop30 interaction in vitro. Nop30
was transcribed and translated in vitro in the presence of
[35S]Met. 23,000 cpm of radiolabeled Nop30 was incubated
with 1 µg of GST, GST-SRp30c, or GST-SF2/ASF bound to
glutathione-Sepharose 4B. , no protein bound. 30% of Nop30 were
specifically retained by GST-SRp30c.
C), and to SRp30c. As in
yeast, there was no detectable interaction between EGFP-Nop30 and
FLAG-SF2/ASF. The variant EGFP-Myp did not bind to FLAG-SRp30c, and
EGFP alone did not interact with FLAG-Nop30 and FLAG-SRp30c (Fig.
7A, lanes 9 and 10).
Despite inclusion of protease inhibitors, a nonspecific background was repeatedly observed in our experiments (Fig. 7A,
lanes 3, 5, 6, and
7), which might be due to proteolytic fragmentation of the antibody used in the immunoprecipitation. Fig. 7B
demonstrates that all EGFP-tagged constructs were immunoprecipitated
with the anti-GFP antibody and migrated with the expected size.
However, there is a faint band below some GFP signals, which again
might be due to degradation. Similarly, all FLAG constructs were
expressed with the predicted size (Fig. 7C).
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Fig. 7.
Detection of Nop30 interactions by
immunoprecipitation. 0.1 µg of plasmid DNA encoding the EGFP and
FLAG fusion proteins indicated in A (top) was
transfected into HEK293 cells. A, The EGFP constructs were
immunoprecipitated with a monoclonal anti-GFP antibody, and the
co-immunoprecipitating FLAG-constructs were detected with an anti-FLAG
antibody. Immunoprecipitated binding partners are indicated in
boldface type. The location of FLAG-Nop30 that
migrates at 30 kDa is indicated by an arrow on the
left. B, Western blot with anti-GFP antibody to
detect the precipitated EGFP fusion protein. The fusion proteins
migrate according to their molecular masses indicated: EGFP-Nop30, 51 kDa; EGFP-Nop30 N, 38 kDa; EGFP-Nop30
C, 41 kDa; EGFP-Myp, 50 kDa;
EGFP-SRp30c, 52 kDa; EGFP-SRp30c
SR, 48 kDa; EGFP, 27 kDa.
C, Western blot with an anti-FLAG antibody of the cell
lysates prior to immunoprecipitation. FLAG-SRp30c and FLAG-Nop30 fusion
proteins migrate at 30 kDa; FLAG ASF/SF2 (lane
11) migrates slighty more slowly.
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Fig. 8.
Intracellular localization of Nop30.
A, pNop30-C2 expressing EGFP-Nop30 was transfected in BHK
cells. The protein is present in the nucleoplasm and is enriched in
nucleoli excluding discrete foci (arrows). B, the
same cell stained with DiQ. C, overlay of A and
B. D, expression of EGFP-Nop30 in HEK293 cells.
The pattern is similar to that of BHK cells. E, the cells of
D were counterstained with DiQ. F, overlay of
D and E. G, BHK cell transfected with
pNop30-C2. Instead of multiple foci as in A, some cells have
a large region inside the nucleoli from which Nop30 is absent.
H, BHK cell expressing EGFP. I, HEK293 cells
expressing EGFP.
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Fig. 9.
Colocalization experiments with nucleolar
antigens or SRp30c. A, HEK293 cells expressing
EGFP-Nop30. The unstained foci are indicated by an arrow.
B, the same cell was stained with an anti-nucleolus antibody
(Calbiochem). C, overlay of A and B.
Colocalization is indicated by yellow color.
D, HEK293 cells transfected with pNop30-C2. E,
the same cells as in D were stained with a monoclonal
anti-B23 antibody (22), which marks the granular component of the
nucleoli. F, colocalization of EGFP-Nop30 and B23 is
indicated by yellow color. G, HEK293
cells expressing EGFP-Nop30. H, the same cells as in
G expressing FLAG-SRp30c. I, colocalization of
EGFP-Nop30 and FLAG-SRp30c is observed in the nucleoplasm but not in
the nucleoli.
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Fig. 10.
Intracellular localization of Myp and Nop30
deletion variants. BHK cells were transfected with the EGFP-tagged
constructs indicated. A, EGFP-Myp localizes predominantly in
the cytoplasm with a weak nuclear staining. B, the C
terminus of Myp (EGFP-Myp N; amino acids 99-208) is distributed
throughout the whole cell excluding the nucleoli. C, N
terminus of EGFP-Nop30 (EGFP-Nop30
C; amino acids 1-95). The
localization is primarily cytoplasmic. D, the C terminus of
EGFP-Nop30 (EGFP-Nop30
N; amino acids 96-219) localizes, similar to
full-length Nop30, in nucleoli and is also present in the nucleoplasm
without a cytoplasmic staining. E, Western blot of EGFP
constructs used in the transient transfection assays.
Immunoprecipitation was performed with a monoclonal anti-GFP antibody
(Boehringer Mannheim); detection was performed with a polyclonal rabbit
anti-GFP antibody (CLONTECH). The fusion proteins
have the following molecular masses: EGFP, 27 kDa; EGFP-Nop30, 51 kDa;
EGFP-Nop30
C, 41 kDa; EGFP-Nop30
N, 38 kDa; EGFP-Myp, 50 kDa;
EGFP-Myp
N, 39 kDa.
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Fig. 11.
In vivo splicing assays.
A, a total amount of 10 µg of an increasing ratio of
pNop30-C2 (encoding EGFP-Nop30) to empty vector pEGFP-C2 was
cotransfected with 2 µg of the SRp20 XB minigene in HEK293 cells and
analyzed by RT-PCR. The amount of transfected EGFP constructs is
indicated at the top of the gel. PCR products were stained
with ethidium bromide and quantified. The size difference between the
upper and the lower band corresponds to the size of alternative exon 4 (435 nucleotides). The structure of the PCR products is indicated
schematically on the right. Primers used are indicated as
arrows. Exon 4 inclusion was calculated as follows: (amount
of inclusion band/(amount of inclusion band + amount of exclusion band) × 100. The percentage of exon inclusion is shown under each
gel. Error bars represent the S.D. from three
independent experiments. M, pBR322 MspI digest.
B, a similar experiment as in A was performed
with pSRp30c-C2 instead of pNop30-C2. C, in vivo
splicing was investigated using a total amount of 10 µg of a mixture
of three different EGFP constructs (pNop30-C2, pSRp30c-C2, and
pEGFP-C2) and 2 µg of the SRp20 minigene. The amount of transfected
pSRp30c-C2 was 5 µg in every case, whereas 5 µg of an increasing
ratio of pNop30-C2 (encoding EGFP-Nop30) to empty vector (pEGFP-C2)
were cotransfected. D, chicken fibroblast cells were
cotransfected with 2 µg of PPT minigene and 0, 3, and 10 µg of
either pNop30-C2 or pCG35 (expressing SC35). Control, only
the PPT minigene was transfected. RNA was isolated, and splicing of the
minigene was analyzed by radioactive RT-PCR with the primer pair T7PRO
and PPTE5rev. The RT-PCR products containing exon 4 were separated from
those lacking exon 4 on a 5% polyacrylamide gel. The quantification of
three independent experiments is shown. Exon inclusion was calculated
as follows: (cpm of inclusion band/(cpm of inclusion band + cpm of
exclusion band) × 100. White bar, control; shaded
bars, pNop30-C2; black bars, pCG35.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (44), and a
protein-tyrosine phosphatase (45) as well as the intracellular
localization of the NADH-cytochrome b5 reductase (46) are controlled by alternative splicing. In the NOP
gene, the intracellular localization of the gene products is dependent on alternative 5' splice site usage, causing a frameshift that creates
two different carboxyl termini. Nop30 is found in the nucleoplasm and
is concentrated in the nucleoli, whereas its variant Myp is distributed
throughout the cytosol. Myp did not interact with SRp30c (Fig.
5A), indicating that alternative splicing determines the
binding specificity of the NOP gene products. Therefore, in muscle, the amount of the interactor of the splicing factor SRp30c could be regulated by alternative splicing. This is reminiscent of
other proteins involved in pre-mRNA splicing that are regulated by
alternative splicing. For example, in mammals, splice variants of
SRp55, SRp40 (4, 47), and ASF/SF-2 (48), and the mammalian homologue of
SWAP (49) and transformer-2 (21) have been described. In the case of
transformer-2 (50), SWAP (51), and X16/SRp20 (14), the alternative
splicing event seems to be autoregulated.
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ACKNOWLEDGEMENTS |
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We thank Clair Lo for excellent technical assistance, Claudia Cap for sequencing, and Peter Nielsen for providing the SRp20 minigenes. We thank Robert Ochs for sending us the B23 antibody and Petra Kioschis from the German Genome Center for performing the genomic screen.
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FOOTNOTES |
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* This work was supported by the Max Planck Society, Human Frontier Science Program Grant RG562/96 (to S. S.), and National Institutes of Health Grant HL45565 (to T. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF064598, AF064599, and AF064600.
§ To whom correspondence should be addressed. Tel.: 49 89 8578 3625; Fax: 49 89 8578 3749; E-mail: stamm{at}pop1.biochem.mpg.de.
2 This calculation can be performed on the World Wide Web at http://cookie.imcb.osaka-u.ac.jp/stamm/.
3 The servers can be found on the World Wide Web at http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl and http://www.ncbi.nlm.nih.gov/cgi-bin/SCIENCE96/msrch2?CHR=16.
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ABBREVIATIONS |
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The abbreviations used are: SR proteins, serine/arginine-rich proteins; PPT, preprotachykinin; DiQ, 4-(4-(dihexadecylamino)styryl)-N-methylquinolinium iodide; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; GST, glutathione S-transferase; kb, kilobase pair(s); BHK, baby hamster kidney; EGFP, enhanced green fluorescent protein.
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