1
Laboratory of Cell Regulation, Imperial Cancer Research Fund, PO Box 123, 44
Lincoln's Inn Fields, London WC2A 3PX, UK
2
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of
Matter, Hiroshima University, and `Unit Process and Combined Circuit', PRESTO,
JST, Higashi-Hiroshima 739-8526, Japan
3
Department of Cellular and Molecular Pharmacology, Finch University of Health
Science, The Chicago Medical School, North Chicago, IL 60064, USA
4
Department of Microbiology and Immunology, Finch University of Health Science,
The Chicago Medical School, North Chicago, IL 60064, USA
5
Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt
University, Nashville, TN 37232, USA
*
These authors contributed equally to this work
Present address: Nomura Research & Advisory Co. Ltd., Urbannet Otemachi
Building 2-2-2, Otemachi, Chiyoda-ku, Tokyo 100-8130, Japan
Present address: Fundacion Inbiomed. Paseo Mikeletegi 61, bajo, 20009 San
Sebastian, Gipuzkoa, Spain
¶
Author for correspondence (e-mail:
toda{at}icrf.icnet.uk
)
Accepted May 1, 2001
![]() |
SUMMARY |
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Key words: Fission yeast, Genome integrity, Spindle, Splicing, Transcription, WD repeats
![]() |
INTRODUCTION |
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Schizosaccharomyces pombe is an ideal organism in which to study
genetic control of sister chromatid segregation (Umesono et al.,
1983; Yanagida,
2000
). The cell usually
proliferates as a haploid with ploidy being maintained faithfully. In fission
yeast, aneuploidy is usually deleterious (Niwa and Yanagida,
1985
; Niwa et al.,
1989
); however, genome
instability can be detected by chromosome loss of nonessential artificial
mini-chromosomes or diploidisation phenotypes (Broek et al.,
1991
; Takahashi et al.,
1994
; Kominami and Toda,
1997
). Using these phenotypic
markers, previous studies have identified molecules and the genetic network,
which ensure genome stability. These include periodic activation and
inactivation of Cdc2/cyclins (Broek et al.,
1991
; Hayles et al.,
1994
; Moreno and Nurse,
1994
; Kominami and Toda,
1997
), temporal expression of
S-phase regulators (Nishitani and Nurse,
1995
; Nishitani et al.,
2000
), centromere and
kinetochore integrity (Allshire et al.,
1995
; Ekwall et al.,
1995
; Freeman-Cook et al.,
1999
; Goshima et al.,
1999
; Saitoh et al.,
1997
; Takahashi et al.,
2000
), sister chromatid
cohesion (Furuya et al., 1998
;
Tomonaga et al., 2000
), and
chromosome architecture (Yanagida,
1998
).
Maturation of pre-mRNA not only plays a housekeeping role in cell division,
but also plays an important part in cell cycle progression. For example,
several fission yeast mutants defective in splicing reactions show cell-cycle
specific phenotypes (Lundgren et al.,
1996; Burns et al.,
1999
; McDonald et al.,
1999
; Ohi et al.,
1994
; Ohi et al.,
1998
; Potashkin et al.,
1998
; Urushiyama et al.,
1996
; Urushiyama et al.,
1997
; Beales et al.,
2000
), although the reason for
this remains unknown. The spliceosome consists of a large complex comprising
some 80 components. One of the first splicing factors to interact with the
pre-mRNA is U2 auxiliary factor (U2AF). U2AF consists of a large (Prp2 in
fission yeast) and a small subunit, and it is the large subunit that is
required for binding of the U2 snRNP to the branch point sequence (Parker et
al., 1987
; Ruskin et al.,
1988
; Wu and Manley,
1989
; Zhuang and Weiner,
1989
). Unlike other splicing
mutations that produce cell-cycle arrest at G2 phase, temperature-sensitive
prp2 mutants show different phenotypes, such as mitotic defects and
failure in cell elongation (Takahashi et al.,
1994
; Beales et al.,
2000
), suggesting that Prp2
might play a role in progression of the cell cycle that is distinct from other
splicing components.
We have performed a large-scale screen to identify mutants with defects in
the maintenance of genome ploidy (Kominami and Toda,
1997). We have previously
identified Pop1 and Pop2, two F-box proteins consisting of the SCF ubiquitin
ligase (Skp1-Cullin-1-F-box) (Kominami and Toda,
1997
; Kominami et al.,
1998a
; Patton et al.,
1998
; Deshaies,
1999
). Loss of either Pop1 or
Pop2 results in diploidisation, which is attributable to the failure in the
ubiquitin-proteasome-dependent degradation of the CDK inhibitor Rum1. High
levels of Rum1 in these mutants lead to the bypass of M phase and successive
occurrence of S phase, which results in doubling of genome ploidy.
In this study, we have characterised pop3-5235, a diploidising
mutant that was isolated in the same screen as the pop1 mutant. Pop3
is a conserved protein composed of WD repeats (Neer et al.,
1994) and is the same as Wat1,
which has been identified as the protein required for proper cell morphology
and F-actin localisation (Kemp et al.,
1997
). Pop3/Wat1 has also been
isolated as a partner of Prp2 by two-hybrid screening. We will show that,
unlike pop1 or pop2 mutants, the reason wat1
mutants diploidise is marked reduction of
-tubulin levels, which
results in unequal chromosome separation. Our study highlights a novel
mechanism for the maintenance of genome stability involving microtubule
integrity via transcriptional regulation.
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MATERIALS AND METHODS |
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Cloning of the pop3+ gene
An S. pombe genomic library (Barbet et al.,
1992) was used for the
isolation of genes which complemented the ts pop3-5235 mutant. Three
ts+ transformants showed haploid-sized cells, and became fertile.
This suppression was plasmid dependent. Plasmid DNAs were recovered from these
transformants and restriction enzyme mapping showed that they contained
overlapping inserts. Identity of the cloned gene as
pop3+/wat1+ was confirmed by genetic crosses
between a tagged strain and the original mutant. Random spore analysis between
a Pop3/Wat1-HA strain (kanamycin-resistance as a marker) and a ts
pop3/wat1-5235 strain showed that the gene that was isolated was
derived from the pop3+/wat11+ locus (no
recombinants were obtained from more than 103 haploid
segregants).
Yeast two-hybrid screening
The entire coding region of prp2+ was cloned into the
pGBT9 vector and used as bait. An S. pombe cDNA library constructed
in pGADGH (Clonetech, Palo Alto, CA) was used for screening and 30 positive
clones were obtained (Wentz-Hunter and Potashkin,
1996; McKinney et al.,
1997
). Restriction mapping
analysis and nucleotide sequencing of recovered cDNA showed that one of these
clones (clone G) encoded the wat1+ gene (previously
designated uap3+,
U2AF59-associated-protein).
Nucleic acid preparation and manipulation
Enzymes were used as recommended by the suppliers (New England Biolabs and
Roche Diagnostics). Total RNA was prepared and Northern analysis was performed
as described previously (Suda et al.,
2000). Nucleotide sequence
data reported in this paper are in the DDBJ/EMBL/GenBank databases under
Accession Number AB016895
(pop3+/wat1+).
Gene disruption
The entire ORF of the wat1+ gene was deleted using
PCR-generated fragments (Bähler et al.,
1998). Tetrad dissection was
performed using a standard method (Moreno et al.,
1991
).
C-terminal epitope tagging of Wat1
Epitope tagging of Wat1 with HA or Myc peptide was performed using
PCR-generated fragments (Bähler et al.,
1998). Wild-type Wat1 was
tagged with 3HA, while mutant Wat1 protein derived from wat1-5235 was
tagged with 13Myc.
Immunological methods
Total cell extracts were prepared after disruption of cells with glass
beads in the lysis buffer as described previously (Kominami and Toda,
1997). For
immunoprecipitation, cell extracts were prepared without boiling. Mouse
monoclonal anti-Cig2 (3A11), anti-Cdc2 (Y100), anti-
-tubulin (Sigma),
anti-ß-tubulin (KMX-1), anti-actin (N350, Amersham), anti-HA (16B12,
BAbCO), anti-Myc antibodies (9E10, BAbCO), and rabbit polyclonal anti-Cdc13,
anti-Cig2, anti-Rum1 and anti-Cdc18 antibodies were used. Antibodies to Prp2
were raised in rabbits against a synthetic peptide corresponding to the amino
acid sequence LSSGSSRIPKRHRDYRDEE (amino acids 7-25 of the protein product;
Multiple Peptide Systems, San Diego, CA). The peptide was coupled to keyhole
limpet hemocyanin (KLH) via a C-terminal cysteine residue not present in Prp2.
Each inoculation contained 0.5 mg of the synthetic peptide. Horseradish
peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse IgG (BioRad
Laboratories) and a chemiluminescence system (ECL, Amersham) were used to
detect bound antibody.
Indirect immunofluorescence microscopy
Cells were fixed with methanol and primary antibodies (TAT-1, 1/50)
applied, followed by Cy3-conjugated goat anti-mouse IgG (Sigma).
Immunofluorescence images were viewed with a chilled video-rated CCD camera
(model C5985, Hamamatsu) connected to a computer (Apple Power Macintosh
G3/400) and processed by use of Adobe® Photoshop (version 5). A confocal
microscope LSM510 (Zeiss Co.) was also used.
Gel filtration chromatography
Soluble protein extracts were prepared in buffer A (20 mM Tris-HCl pH 7.5,
20% glycerol, 0.1 mM EDTA, 1 mM mercaptoethanol, 5 mM ATP plus a cocktail of
inhibitors, Sigma) as described previously (Vardy and Toda,
2000). Gel filtration
chromatography was performed on a Superose-6 column by FPLC (Pharmacia
Biotech). To determine molecular weight, a parallel column was run with
standards consisting of dextran (2000 kDa), thyroglobulin (669 kDa) and
ovalbulin (43 kDa).
Purification of Wat1-interacting proteins
The Wat1-containing complex was immunopurified according to the method that
was used for purification of a Cdc5-containing multiprotein complex (McDonald
et al., 1999). Wat1-HA tagged
strains were metabolically labelled with Tran[35S]-label and used
for extract preparation.
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RESULTS |
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|
|
The wat1+ gene is not essential, but is required
for genome stability
Gene disruption showed that the wat1+ gene is
non-essential for cell growth (Fig.
1C). Dissection of asci from heterozygous diploid cells showed
that four viable spores were obtained from 20 tetrads dissected and uracil
auxotrophic phenotype segregated 2:2. wat1-deleted
(wat1) cells, however, grew very poorly at 27°C (doubling
times of
wat1 are 180% and 120% longer than wild type and
wat1-5235 mutants, respectively) and showed, like wat1-5235,
cs and ts growth defects.
wat1 is sterile, which is caused by
defects in G1 arrest under nitrogen starvation conditions (Kominami and Toda,
1997
). We realise that sterile
phenotypes are not stable in the wat1 mutant and that phenotypic
reversion occurs spontaneously, which could explain why sterility was not
described in the previous study (Kemp et al.,
1997
). Furthermore,
wat1 haploid cells tended to diploidise
(Fig. 1D). These results
indicate that Wat1 is a conserved WD-repeat protein that is required for
genome stability.
Isolation of the wat1+ gene as a U2AF-Prp2
interacting partner
In a separate set of experiments, we have performed yeast two-hybrid
screening using the prp2+ gene as bait (Fields and Song,
1989; McKinney et al.,
1997
). Prp2 is the large
subunit of U2AF and forms a complex with the small subunit p23 (Potashkin et
al., 1993
; Wentz-Hunter and
Potashkin, 1996
). In order to
identify additional proteins that interact with Prp2, we screened a fission
yeast cDNA library. One of the plasmids we identified from this screen, clone
G, encodes Wat1. Interaction between Prp2 and Wat1 was specific, as either
protein alone without a partner failed to activate the GAL promoters
(Table 2). Moreover Wat1
interacted with neither p23 nor Uap2, which was identified as a Prp2-binding
protein from the same screening (McKinney et al.,
1997
).
|
To confirm an in vivo interaction between Prp2 and Wat1, immunoprecipitation was performed. For this purpose, a strain containing C-terminally tagged Wat1-HA was constructed. Cell extracts were prepared from this strain and immunoprecipitation was performed using anti-HA antibody; the filter was probed with an anti-Prp2 antibody. As shown in Fig. 3A, Prp2 co-immunoprecipitated with Wat1-HA (lane 2). The interaction was specific, as a non-tagged control did not precipitate Prp2 (lane 1). Reciprocal immunoprecipitation was also performed using an anti-Prp2 antibody and preimmune serum, and the interaction was examined with an anti-HA antibody. As shown in Fig. 3B, Wat1-HA was precipitated with this antibody, although a small amount of Wat1-HA was also precipitated with preimmune serum (lanes 1 and 2). Thus, Wat1 forms a complex with the large subunit of U2AF, possibly independently of its small subunit.
|
-tubulin levels are reduced in the wat1 mutant
We addressed the mechanism(s) that underlie diploidisation in the
wat1 mutant. Unlike pop1 or pop2 mutants, which
accumulate the CDK inhibitor Rum1 to high levels, Rum1 levels were not
increased in wat1 mutants (data not shown). Consistent with this,
again unlike pop1 or pop2 (in which polyploid phenotypes are
dependent upon the presence of Rum1; Kominami and Toda,
1997; Kominami et al.,
1998a
), the
rum1wat1-5235 double mutant still diploidised (data not shown). This
result indicated that, although Wat1 was identified from the same screen as
Pop1, the reason that wat1 mutants diploidise is distinct from that
for pop1 and pop2.
During a series of immunoblotting experiments to find proteins abnormally
produced in wat1 mutants, we noticed that -tubulin levels were
substantially decreased in this mutant, even under the permissive condition
(Fig. 4A). This decrease of
-tubulin levels appeared to be specific, as the amount of other
proteins examined, such as ß-tubulin, actin, Cdc13 (B-type cyclin) and
Cdc2, was not altered significantly (Fig.
4A). Temperature-shift experiments of wat1 mutants showed
that
-tubulin levels became further reduced upon an upwards shift to
36°C (Fig. 4B). In
wat1-deleted cells, more drastic reduction of
-tubulin levels
was observed (lanes 3, 6 and 9). This result suggests that Wat1 plays an
important role in the homeostasis of
-tubulin levels.
|
Microtubule structure and function are compromised in the
wat1 mutant
Given the inability to maintain -tubulin levels, immunofluorescence
microscopy with anti-
-tubulin antibody was performed in the
wat1 mutant using confocal microscopy. As shown in
Fig. 5A, compared with
wild-type cells (left panel), wat1 mutant cells grown at 36°C (2
hours, right panel) and even at 26°C (middle panel) contained shorter and
fewer cytoplasmic microtubules. Nuclear staining with DAPI showed that, in a
significant fraction of wat1 mutants, in addition to positional
displacement, pairs of chromosomes separating to opposite poles at anaphase
were not equal and that these segregation defects became especially obvious at
36°C (20% of mitotic cells Fig.
5B). This indicated that unequal sister chromatid segregation
occurred in wat1mutants, which resulted in spontaneous
diploidisation. Consistent with abnormal and compromised microtubule
structures, wat1 mutants were hypersensitive to
microtubule-destabilising drugs (Fig.
5C). Taking these results together, Wat1 regulates microtubule
integrity by maintaining
-tubulin levels, and lack of Wat1 results in
unequal chromosome separation and failure in the maintenance of genome
ploidy.
|
Wat1 may play a crucial role in the maintenance of mature mRNA
levels
We sought to examine the involvement of Wat1 in mRNA metabolism. It is
known that ts prp2 mutants are defective in splicing reactions when
incubated at 36°C, which could be detected with Northern analysis using
intron sequence as probes (Potashkin et al.,
1989; Potashkin et al.,
1993
; Urushiyama et al.,
1996
). Total RNA was prepared
from wild type,
wat1 or ts prp2 mutants incubated at
26°C or 36°C, and Northern analysis was performed. Two probes were
used, which corresponded to intron and exon sequences of the
tbp1+ gene (encoding the TATA-binding protein; Hoffman et
al., 1990
). While unspliced
premature RNA species were accumulated in ts prp2 mutants to high
levels upon upwards temperature shift (Fig.
6A, lanes 4 to 6 in top panel), no equivalent bands were observed
in wat1 mutants (lanes 7 to 9) or in wild type (lanes 1 to 3),
suggesting that Wat1 may not be involved in the splicing reaction per se.
Instead, what was clearly defective in this mutant was that the total amount
of tbp1+ mRNA was significantly reduced, especially when
incubated at 36°C (lanes 7 to 9 in bottom panel).
|
In order to examine the specificity of the reduction of mRNA levels,
filters were rehybridised with probes corresponding to other genes. Genes
examined included nda2+ (encoding 1-tubulin; Toda
et al., 1984
),
cdc2+, cig2+ (encoding S-phase cyclin;
Bueno and Russell, 1993
;
Connolly and Beach, 1994
;
Obara-Ishihara and Okayama,
1994
) and
act1+ (encoding actin) (Mertins and Gallwitz,
1987
), among which
nda2+ and cdc2+ contain introns, while
cig2+ and act1+ do not. As shown in
Fig. 6B, irrespective of the
presence or absence of introns, in all cases, total levels of mRNA were
substantially reduced in wat1 mutants. Therefore, although it binds
Prp2 in vivo, unlike Prp2, Wat1 appears to play a role in either synthesis or
stability of mRNA, but not splicing.
Wild type Wat1, but not its mutant protein, forms a large complex in
the cell
It has been generally believed that WD repeats are hallmarks for
protein-protein interactions (Wall et al.,
1995; Lambright et al.,
1996
). As shown before, Wat1
binds at least Prp2 in vivo. In order to examine the native size of the
Wat1-containing complex, gel filtration chromatography was performed using a
tagged strain in both wild-type and wat1-5235 mutant strains. In
wild-type cells, the cellular Wat1 protein was found to be almost exclusively
included in a large complex (>2000 kDa,
Fig. 7A). In clear contrast, in
wat1-5235 mutants, the majority of the Wat1 protein was eluted in
much smaller fractions, most likely monomers or dimers
(Fig. 7B). This result
indicates that Wat1 forms a large complex in the cell, which is crucial for
its protein function.
|
We next attempted to purify directly proteins that interact with Wat1. Large-scale immunoprecipitation using anti-HA antibody was performed with cell extracts from a Wat1-HA strain labelled with [35S]. Fig. 7C shows the [35S]-labelling coprecipitated proteins. At least five protein species (marked by arrows), possibly more, specifically co-purified with Wat1; one protein, p33 (smaller than Wat1), notable appeared to bind Wat1 in a stoichiometric manner. Identification of these co-purified proteins is in progress. This analysis has firmly established that, consistent with the protein-protein-interacting WD-repeat structure, Wat1 forms a stable complex with other proteins in the cell.
![]() |
DISCUSSION |
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Selective reduction of -tubulin protein levels in the
wat1 mutant
Finding low -tubulin levels in wat1 mutants has led us to
explore the molecular function of Wat1 in microtubule integrity. We show that
Wat1 is involved in the maintenance of mRNA levels of not only
-tubulin
but also other genes, suggesting that the primary function of Wat1 is for
general transcription. Our results then raised further questions. For example,
while transcript levels of all the genes examined are reduced in the
wat1 mutant, why are only
-tubulin protein levels
significantly decreased? How is the translation rate of
-tubulin mRNA
regulated in fission yeast? It is possible that the efficiency or kinetics of
translation from each transcript is varied among individual mRNA species and
this may be the reason that only
-tubulin protein levels appear to be
low in wat1 mutants. Perhaps in
-tubulin synthesis, efficiency
of translation is dependent upon the levels of its transcripts or requires
stable mRNA or the half-life of
-tubulin is much shorter than that of
other proteins examined in this study. It should be noted that, although we
have shown only
-tubulin levels are decreased, circumstantial evidence
suggests that
-tubulin is not alone and other protein levels are also
dependent upon Wat1.
Previous work identified Wat1 as a protein that is required for proper
localisation of F-actin (Kemp et al.,
1997). Thus, Wat1 plays a
crucial role in establishment of both the actin and microtubule cytoskeleton.
Unlike
-tubulin, actin levels are not noticeably reduced in
wat1 mutants. This suggests that some factor(s) that is required for
F-actin localisation is defective in wat1 mutants. In addition, the
wat1 mutant is sterile, which is attributable to a failure in G1
arrest upon nutrient starvation (Kemp et al.,
1997
; Kominami and Toda,
1997
) a prerequisite
for conjugation in fission yeast (Kumada et al.,
1995
; Kominami et al.,
1998b
). We predict that the
levels of protein(s) required for G1 arrest are also reduced in this
mutant.
Wat1 is an evolutionarily conserved WD repeat protein
In budding yeast the Wat1 homologue, Lst8 was identified as a factor that
is required for transport of amino acid permeases from the Golgi to the plasma
membrane (Roberg et al.,
1997). Given the role of Wat1
in mRNA metabolism, it is possible that in this organism, protein levels
responsible for transport of amino acid permeases are tightly controlled by
Lst8-mediated mRNA maturation. In mouse, expression of this homologue in
adipocytes is regulated by insulin, while in human the homologue has been
isolated as a gene induced by DNA damage and overexpressed in promyelocytic
leukemia cell lines (HL-60; database search from DDBJ and NCBI). This suggests
that Wat1 also plays a role in response to internal and external cues.
Consistent with this, fission yeast wat1 mutants are hypersensitive
to various chemicals and drugs (Kemp et al.,
1997
; I. O. and T. T.,
unpublished).
In contrast to the previous report (Kemp et al.,
1997), we do not see abnormal
cell shape in either wat1/pop3-5235 or
wat1/
pop3-deletion strain that we have isolated and
characterised in this study. It is possible that this phenotype is
allele-specific; in fact, a previously isolated deletion allele of
wat1+ (wat1.d) only removed part of the protein,
while the entire ORF was deleted in
wat1.
Link between mRNA splicing and other maturation processes
Wat1 binds Prp2, the large subunit of U2AF. Although Prp2 is required for
pre-mRNA splicing (Parker et al.,
1987; Ruskin et al.,
1988
; Wu and Manley,
1989
; Zhuang and Weiner,
1989
; Zamore and Green,
1991
; Potashkin et al.,
1993
), the defective
phenotypes of ts prp2 mutants are not the same as mutations in other
components of the spliceosome. Notably prp2 is identical to
mis11, which was identified as a mutation that caused high frequency
loss of mini-chromosomes (Takahashi et al.,
1994
), demonstrating that Prp2
also plays an important role in genome stability. Therefore, it appears that,
in addition to a physical interaction, these two proteins play a common role
in genome integrity.
We propose two possibilities for Wat1 function in mRNA metabolism. One is
transcription; Wat1 may be involved in mRNA synthesis per se. In this case
Prp2 would be implicated in transcription as well as splicing. We have
performed gel filtration chromatography to examine whether a Wat1-containing
complex is co-fractionated with Tbp1 or RNA polymerase II. It appears that
these two proteins do not form a tight complex with Wat1 (I. O. and T. T.,
unpublished). The Wat1-containing complex may therefore play a regulatory role
in transcription reactions. Recent analysis shows that mRNA processing such as
splicing is orchestrated with transcription elongation steps (Proudfoot,
2000). The second possibility
is the mRNA stabilisation pathway. It has been shown that mRNA stability is
regulated by a series of pathways, such as the exosome degradation (Mitchell
et al., 1997
; van Hoof and
Parker, 1999
) and 5'
capping and polyadenylation of mRNA (Jacobson and Peltz,
1996
). RNA stability and
splicing are intimately connected with each other (Serin et al.,
2001
). Furthermore,
accumulating evidence supports the interrelationships of the pathways of mRNA
decay and translation (Olivas and Parker,
2000
). The cytoplasmic
localisation of Wat1 is consistent with a role of this protein in mRNA
turnover (K. G. unpublished). In this scenario, Prp2 may play an additional
role in inhibition of mRNA decay. Systematic yeast two-hybrid analysis
indicates that components of RNA metabolism interact with each other and also
with those involved in chromatin structure (Schwikowski et al.,
2000
). Further identification
and characterisation of Wat1-interacting proteins will give clues about the
biochemical pathways, which require Wat1.
![]() |
ACKNOWLEDGMENTS |
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