From the Institute of Microbiology and Immunology,
National Yang-Ming University, Shih-Pai 112, Taiwan,
Republic of China and the § Institute of Molecular Biology,
Academia Sinica, Nankang 115, Taiwan, Republic of China
Received for publication, August 2, 2000, and in revised form, September 25, 2000
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ABSTRACT |
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The yeast Saccharomyces cerevisiae
Prp19p protein is an essential splicing factor and a spliceosomal
component. It is not tightly associated with small nuclear RNAs
(snRNAs) but is associated with a protein complex consisting of at
least eight proteins. We have identified two novel components of the
Prp19p-associated complex, Ntc30p and Ntc20p. Like other identified
components of the complex, both Ntc30p and Ntc20p are associated with
the spliceosome in the same manner as Prp19p immediately after or
concurrently with dissociation of U4, indicating that the entire
complex may bind to the spliceosome as an intact form. Neither Ntc30p
nor Ntc20p directly interacts with Prp19p, but both interact with another component of the complex, Ntc85p. Immunoprecipitation analysis
revealed an ordered interactions of these components in formation of
the Prp19p-associated complex. Although null mutants of
NTC30 or NTC20 showed no obvious growth
phenotype, deletion of both genes impaired yeast growth resulting in
accumulation of precursor mRNA. Extracts prepared from such a
strain were defective in pre-mRNA splicing in vitro,
but the splicing activity could be restored upon addition of the
purified Prp19p-associated complex. These results indicate that Ntc30p
and Ntc20p are auxiliary splicing factors the functions of which may be
modulating the function of the Prp19p-associated complex.
Splicing of pre-mRNA requires five small nuclear RNAs
(snRNAs)1 and a large number
of protein factors, which assemble into a large ribonucleoprotein
complex called the spliceosome (for reviews, see Refs. 1-6).
Spliceosome assembly is a multistep process that involves sequential
binding of snRNAs to the pre-mRNA in an order of U1, U2, then U4/U6
and U5 as a preformed tri-snRNP particle. A subsequent conformational
rearrangement results in dissociation of U1 and U4, accompanied by new
base pair formation between U2 and U6 and between U6 and the 5' splice
site, leading to the formation of the active spliceosome on
which the catalytic reactions take place.
Functional studies of snRNAs have revealed their important roles in
recognition and alignment of splice sites mediated through base pair
interactions between snRNAs and the intron sequences during spliceosome
assembly. Although numerous protein splicing factors have been
identified, their functional roles are not well understood. The
DEx(D/H) box proteins are among the best characterized protein factors
and have been shown RNA unwindase activity (6-10). It is
generally believed that these proteins play essential roles in
modulating structural change of the spliceosome during spliceosome assembly by either unwinding RNA base pairing or by hydrolyzing ATP to
provide energy required for conformational rearrangement (6). In
addition, a U5 protein with strong sequence similarity to ribosomal
translocase EF-2 was demonstrated to have GTP binding activity
and implicated in the structural rearrangement of RNA (11).
We have previously shown that the yeast Saccharomyces
cerevisiae Prp19p protein is an essential splicing factor and is
associated with a protein complex consisting of at least eight protein
components (12). Prp19p is not tightly associated with snRNAs but is
associated with the spliceosome immediately following or concurrent
with dissociation of U4 from the spliceosome, suggesting a possible role in mediating conformational rearrangement or stabilizing the
rearranged structure of the spliceosome during U4 dissociation (13).
Two components of the Prp19p-associated complex have been identified.
Ntc85p was identified by sequencing the components of the affinity
purified complex (Ntc stands for PRP nineteen complex) (14). It is essential for pre-mRNA splicing
and may play a role in promoting binding of the Prp19p-associated
complex to the spliceosome. Snt309p was identified by screening
synthetic lethal mutants to the prp19 mutation (Snt stands
for synthetic lethal to PRP
nineteen) (15). Although the SNT309
gene is not required for yeast growth, Snt309p, through interaction
with Prp19p, plays an important role in modulating interactions of
Prp19p with other associated components to stabilize the
Prp19p-associated complex (16). Both proteins are associated with the
spliceosome immediately after or simultaneously with dissociation of
U4. We report here the identification of genes complementing synthetic lethal mutants snt304 and snt384. We show that
these two genes encode two other components of the Prp19p-associated
complex, Ntc30p and Ntc20p, respectively. Both Ntc30p and Ntc20p are
also associated with the spliceosome in the same manner as Prp19p, indicating that the entire complex may bind to the spliceosome as an
intact unit.
Strains--
The strains used were as follows: YSCC1,
M at a prc1 prb1 pep4 leu2 trp1 ura3
PRP19-HA; YSCC3, Mata prc1 prb1 pep4 leu2 trp1
ura3 PRP4-HA PRP19-MYC; SEY6210.5,
Mata/Mat Oligonucleotides--
The oligonucleotides used were as
follows: 20-1, GGCCGAATTCTTAGTTGTCTTGTT; 20-2, GGCCGGATCCTAGGCGAGCTAGGT; 20-3, GGCCGGATCCCCAGTATGCCCTCTCT; 30-1, GGCCTCGCGAATGCACAGGTATCT; 30-2, GGCCTCGCGAGGCACTATTTCGA; 30-3, GGCCGGATCCTCAAAATGAGTAGAA; 30-4, GGCCGGATCCTGCATGATTCATATA.
Plasmids--
The construction of plasmids was as follows.
pCC30: the 4-kb ScaI DNA fragment was inserted into the
SmaI site of plasmid vector pRS416.
pCC31: the 1-kb DNA fragment containing the NTC30 open
reading frame (ORF) and a 300-bp
downstream sequence retrieved from the yeast genome by PCR using
primers 30-3 and 30-4 was digested with BamHI and inserted
into the BamHI site of plasmid vector pGEM-1.
pCC311: the 1-kb BamHI fragment from pCC31 was inserted into plasmid
vector pEG202.
pCC312: the 1-kb BamHI fragment from pCC31 was inserted into
plasmid vector pACT2.
pCC301: the PCR product with primers 30-1 and 30-2 using pCC30 as a
template was digested with NruI and then self-ligated.
pCC302: the 1.8-kb fragment containing the His3 gene was
end-repaired and inserted into the NruI site of pCC301.
pWT20: the 1.2-kb DNA fragment containing the NTC20 ORF and
400-bp upstream and 450-bp downstream sequences retrieved from the
yeast genome by PCR with oligonucleotides 20-1 and 20-2 was ligated
with the EcoRI- and BamHI-digested pRS416.
pWT21: the 870-bp DNA fragment containing the NTC20 ORF and
450-bp downstream sequence retrieved from the yeast genome by PCR with
oligonucleotides 20-2 and 20-3 was digested with BamHI and
inserted into the BamHI site of plasmid vector pGEM-1.
pWT201: the 170-bp SacI-XhoI fragment with the
NTC20 gene was replaced with the 2-kb LEU2 fragment.
pWT211: the 870-bp BamHI DNA fragment from pWT21 was
inserted into the BamHI site of plasmid vector pEG202.
pWT212: the 870-bp BamHI DNA fragment from pWT21 was
inserted into the BamHI site of plasmid vector pACT2.
Cloning of the SNT304 and SNT384 Genes--
The
SNT304 and SNT384 genes were isolated by
complementation of the synthetic lethal phenotype of the
snt304 and snt384 mutants with a YCp50-based
Sau3A genomic library obtained from M. Rose and P. Novick as
described (15). The ends of the isolated DNA fragments were sequenced
and compared with the yeast genome data base to identify the regions of
the fragments in the genome. DNA fragments containing speculated ORFs
were then subcloned for complementation analysis.
Construction of Gene Replacement--
The
ntc30::HIS3 allele was created by replacing the
entire ORF of NTC30 with a 1.8-kb DNA fragment of the
His3 gene. The ORF was deleted by reverse PCR with primers
30-1 and 30-2 using plasmid pCC30 as a template. The resulting plasmid
pCC301 was digested with NruI and ligated with a blunt-ended
1.8-kb HIS3 fragment. This action yielded plasmid
pCC302, which was digested with XbaI and XhoI to
isolate the ntc30::HIS3 fragment for
transformation into a diploid strain SEY6210.5. Correct integration was
confirmed by Southern blot analysis. The
ntc20::LEU2 was created by replacing the
SacI-XhoI fragment of plasmid pWT20 with a 2-kb
DNA fragment of the LEU2 gene. The resulting plasmid pWT201
was digested with EcoRI and BamHI to isolate the
ntc20::LEU2 fragment for transformation into
SEY6210.5. Haploid strains harboring disrupted NTC30 or
NTC20 genes were isolated by sporulation of the heterozygous
dipolid strains followed by dissection of tetrads.
Splicing Reactions and Immunoprecipitation--
Splicing assays
were performed according to Lin et al. (17) using uncapped
actin pre-mRNA as the substrate. Immunoprecipitation was carried
out as described by Tarn et al. (18).
Northern Blot Analysis--
Total yeast RNA was isolated as
described by Vijayraghavan et al. (19). RNA was
electrophoresed in 5% polyacrylamide-8M urea gels and then
electroblotted onto GeneScreen membranes in 25 mM
NaPO4 (pH 6.5) at 4 °C overnight. Northern
hybridization was performed according to Vijayraghavan et
al. (19) using the CRY1 gene as a probe.
Two-hybrid Assays--
The NTC30, NTC20, NTC85,
PRP19, and SNT309 genes were fused to the LexA-DNA
binding domain in plasmid pEG202 and the GAL4-activation domain in
plasmid pACT2, and each pair of plasmids was transformed into yeast
strain EGY48 together with the Identification of NTC30 and NTC20--
To identify components of
the Prp19p-associated complex, we performed a screen for mutants that
showed synergistic effects to a temperature-sensitive allele of
prp19 using the ade2/ade3 sectoring
system, isolating 15 such mutants (15). Cloning of the gene
complementing the snt309 mutant phenotype identified Snt309p
as Ntc25p of the Prp19p-associated complex (15). We further cloned
genes conferring mutations snt304 and snt384 by complementation of their nonsectoring phenotype. SNT304 and
SNT384 were found to correspond to ORFs YJR050w and YBR188c,
respectively. YJR050w was recently reported by two-hybrid
screening to be Isy1p as a protein interacting with Syf1p (20). It is a
protein of 235 amino acid residues with a calculated molecular weight
of 28,024. YBR188c is a previously unidentified gene containing 140 amino acid residues of molecular weight 15,968. Neither protein sequence contains any discernible motif.
To see whether SNT304 and SNT384 encode
components of the Prp19p-associated complex, we raised antibodies
against recombinant His-tagged Snt304p and Snt384p for immunoblot
analysis. The Prp19p-associated complex was isolated by affinity
chromatography (12), fractionated by SDS-polyacrylamide gel
electrophoresis, and then subjected to Western blot analysis using
anti-Snt304p and anti-Snt384p antibodies. Fig.
1 shows that the anti-Snt304p antibody
reacted with Ntc30p (lane 3), and the anti-Snt384p antibody
reacted with Ntc20p (lane 6) of the Prp19p-associated
complex. Preincubation of individual antibody with recombinant Snt304p
or Snt384p abolished such reactions (lanes 4 and
7). Pre-immune sera from both rabbits also gave no reaction
(lanes 2 and 5). These results indicate that
Snt304p is Ntc30p and Snt384p is Ntc20p of the Prp19p-associated
complex. In fact, Ntc20p was also identified to be ORF YBR188c by
independent sequencing of the components of the affinity-purified
Prp19p-associated complex.
Ntc30p and Ntc20p Are Spliceosomal Components and Are Associated
with the Spliceosome in the Same Manner as Prp19p--
We previously
showed that Prp19p is not tightly associated with spliceosomal snRNPs,
but is associated with the spliceosome during or after dissociation of
U4 from the spliceosome (13, 18). Two other components of the
Prp19p-associated complex, Snt309p and Ntc85p/Cef1p, also associate
with the spliceosome in the same manner, suggesting that the
Prp19p-associated complex may bind to the spliceosome as an integral
complex (14, 15). To see whether Ntc30p and Ntc20p also associate with
the spliceosome during the splicing reaction, anti-Ntc30p and
anti-Ntc20p antibodies were used for immunoprecipitation of the
spliceosome. Splicing reactions were carried out under normal
conditions, and the reaction mixtures were precipitated with the
anti-Ntc30p or anti-Ntc20p antibody conjugated to protein A-Sepharose.
As shown in Fig. 2A, precursor
RNA, splicing intermediates, and the intervening sequence, but
only a small amount of the mature message, were precipitated by the
anti-Ntc30p antibody (lane 4), indicating precipitation of
the spliceosome. In the absence of the antibody (lane 2) or with the pre-immune serum (lane 3), no RNA was precipitated.
Preincubation of the antibody with the recombinant Ntc30p protein also
resulted in no precipitation of RNA (lane 5). Fig.
2B shows the same result when immunoprecipitation was
carried out with the anti-Ntc20p antibody. These results indicate that
both Ntc30p and Ntc20p are also spliceosomal components.
To see whether Ntc30p and Ntc20p also bind to the spliceosome at the
same time as Prp19p, we performed the ATP titration experiment as
before (14, 15). Fig. 3A shows
the scheme of spliceosome assembly. We previously demonstrated that
dissociation of U4 from the spliceosome is very sensitive to ATP
concentration (13). Under normal conditions (1-2 mM ATP),
U4 is dissociated from the spliceosome rapidly after binding of the
tri-snRNPs. As a consequence, only very small amount of the U4
containing splicing complex A2-1 is detected. Dissociation of U4 is
blocked at lower ATP concentrations with increasing amounts of A2-1
accumulated on decreasing ATP concentrations. It was demonstrated
before that Prp19p is not associated with the spliceosome at low
concentrations of ATP and is not present in A2-1 (13). We took
advantage of this feature to analyze the steps of spliceosome
assembly with which Ntc30p and Ntc20p are associated. In this
experiment, we constructed a strain in which Prp19p is tagged with the
c-Myc epitope, and Prp4p is tagged with HA. Prp4p was shown to bind to
the 5' portion of U4 (21) and to be dissociated from the spliceosome
with U4. Therefore, the anti-HA antibody could be used to follow U4 and anti-Myc antibody to follow Prp19p.
Splicing reactions were carried out in extracts prepared from such a
strain at different ATP concentrations, and the reaction mixtures were
subjected to immunoprecipitation with anti-Myc, anti-HA, anti-Ntc30p,
and anti-Ntc20p antibodies. As shown in Fig. 3B, the extract
showed high splicing activity at 1 mM (lane 1)
and 0.5 mM ATP (lane 7), low activity at 0.1 mM ATP (lane 13), but gave no spliced products
or intermediates at 0.05 mM ATP (lane 19). The
anti-Myc antibody precipitated the spliceosome efficiently from the
reaction mixtures at 1 and 0.5 mM ATP (lanes 3 and 9). At 0.1 mM ATP, less splicing
intermediates and products but more pre-mRNA were precipitated
(lane 15). The pre-mRNA precipitated presumably reflects
complex A1. At 0.05 mM ATP, no significant amount of the
RNA was precipitated (lane 21). This finding is consistent
with our previous results of precipitation using the anti-Prp19p
antibody or the anti-HA antibody when Prp19p was tagged with HA (13,
14). In contrast, the anti-HA antibody, binding to Prp4p-HA,
precipitated only residual amounts of pre-mRNA at 1 mM
ATP (lane 4), more at 0.5 mM ATP (lane
10), and much larger amounts at 0.1 and 0.05 mM ATP
(lanes 16 and 22). In all cases, no splicing intermediates
or products were precipitated, reflecting the fact that U4 is
dissociated prior to catalytic steps of the splicing reaction.
Precipitation with anti-Ntc30p and anti-Ntc20p antibodies gave a
similar pattern as with the anti-Myc antibody. Spliceosome-associated
RNAs were precipitated with higher efficiency at high ATP
concentrations (lanes 5, 6, 11, 12, 17, and
18), but negligible amounts were precipitated at 0.05 mM ATP (lanes 23 and 24). This
indicates that like Prp19p, both Ntc30p and Ntc20p are associated with
the spliceosome immediately after or concurrently with dissociation of U4.
Null ntc30/ntc20 Mutants Were Defective in Growth and
Splicing--
Among the two previously identified Prp19p-associated
components, even though SNT309 was not required for
cell viability (15), NTC85 was essential for yeast growth
and for pre-mRNA splicing (14). To see whether NTC30 and
NTC20 are required for cellular growth, null alleles of
NTC30 (
Because both Ntc30p and Ntc20p are spliceosomal components, growth
defect of
As Ntc30p and Ntc20p are components of the Prp19p-associated complex, a
splicing deficiency in Sequential Interactions of Components in the Formation of the
Prp19p-associated Complex--
Two previously identified components of
the Prp19p-associated complex, Snt309p and Ntc85p, were shown to
interact directly with Prp19p (14, 15). To reveal the interactions
between Ntc30p, Ntc20p, and other components of the Prp19p-associated
complex, we performed two-hybrid assays. Prp19p, Snt309p, Ntc85p,
Ntc30p, and Ntc20p were fused to both the LexA-DNA binding domain and the GAL4 activation domain, and interactions between each pair of
proteins were analyzed by assaying the
We have previously demonstrated that Snt309p plays a role in modulating
interaction of Prp19p with other associated components (16). In the
absence of Snt309p, Prp19p becomes only loosely associated with other
components. In addition, the association of Ntc85p with Ntc30p and
Ntc20p was also weakened, but association between Ntc30p and Ntc20p was
not affected (16). This indicates that Snt309p, although not
interacting directly with Ntc85p, affects interactions between Ntc85p
and Ntc30p and between Ntc85p and Ntc20p. A possible explanation for
such a distant effect is that formation of the Prp19p-associated
complex may involve sequential interactions of the Prp19p-associated
components in an order of binding of Snt309p to Prp19p, followed by the
binding of Ntc85p, and then Ntc30p and Ntc20p. In this case, depletion
of Ntc30p or Ntc20p or both should have no effect on the association of Prp19p with Snt309p and Ntc85p.
To test this theory, we prepared splicing extracts from
By screening synthetic lethal mutants to the temperature-sensitive
alleles of PRP19, we have identified three genes,
SNT309, NTC30, and NTC20, that encode
components of the Prp19p-associated complex. None of these genes is
essential for cell viability. Yeast strains deleted of the
SNT309 gene gave a temperature-sensitive phenotype and
accumulated pre-mRNA at the nonpermissive temperature (15).
Deletion of the NTC30 or NTC20 gene alone did not
show obvious growth phenotype. It is possible that Ntc30p or Ntc20p may
play a less important role than Snt309p in the splicing reaction or in
maintaining the integrity of the Prp19p-associated complex. Alternatively, NTC30 and NTC20 might be
functionally redundant to other yeast genes. The genome of S. cerevisiae is thought to have undergone a duplication event some
100 million years ago, immediately followed by random deletion of
individually duplicated genes from one or the other chromosome
(22). Fifty-five duplicated regions were identified in the whole yeast
genome as a consequence of such duplication and deletion events (22).
Among them, the COR region of chromosome 10, on which
NTC30 resides, is ancestrally related to the ARC region of
chromosome 5 (23). NTC30, originally named UTR3
as an unidentified transcribed region, is related to UTR5 in
the ARC region (23). Although the protein sequences of these two genes
do not share high homology, the possibility that they are functionally
redundant cannot be ruled out. By tagging Utr5p with the HA epitope, we
tested the possibility of whether Utr5p is a spliceosomal component or
a component of the Prp19p-associated complex. Immunoprecipitation
analysis revealed no association of Utr5p with Prp19p or with the
spliceosome during the splicing reaction (data not shown). Thus, Utr5p
is unlikely to be a functional homolog of Ntc30p.
On the other hand, NTC30 and NTC20 could be
functionally redundant to each other based on the following
observations. First, despite the fact that deletion of either the
NTC30 or NTC20 gene showed a negligible growth
phenotype, deletion of both genes severely impaired cellular growth.
Furthermore, Ntc30p and Ntc20p showed similar patterns of interaction
with other identified components of the Prp19p-associated complex. Both
proteins interact with Ntc85p (Fig. 5) and
Ntc40p,2 but neither
interacts with Prp19p or Snt309p. Detailed biochemical analyses of
these proteins are required for a direct proof.
We showed previously that Snt309p interacts strongly with Prp19p, and
through this interaction, it modulates interactions of Prp19p with
other associated components to form a stable complex. In the absence of
Snt309p, the association of Prp19p or Ntc85p with other components is
impaired, and the complex is dissociated into at least three parts, one
containing Prp19p, one containing Ntc85p, and one containing Ntc30p and
Ntc20p (16). Neither Ntc30p nor Ntc20p interacts directly with Prp19p
or Snt309p, but both interact with Ntc85p. It is interesting that
although Snt309p does not interact with Ntc85p, Ntc30p, or Ntc20p, it
affects the interactions between these components and between Ntc85p
and Prp19p. In contrast, neither Ntc30p nor Ntc20p affects association
of Ntc85p with Prp19p and Snt309p. This finding suggests that formation of the Prp19p-associated complex may involve sequential interactions of
the components in the following order: Snt309p binds to Prp19p first,
followed by binding of Ntc85p, then Ntc30p and Ntc20p. The fact that
Ntc30p and Ntc20p do not interact with each other but remain associated
even after dissociation from Ntc85p (16) suggests that they may
interact with another factor(s) to form a subcomplex regardless of the
presence of Ntc85p. A scheme of interactions between the known
components of the Prp19p-associated complex is shown in Fig.
7.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
leu2/leu2 ura3/ura3 his3/his3
trp1/trp1 suc2/suc2 lys2/LYS2 ADE2/ade2; YD30,
Mata/Mat
leu2/leu2 ura3/ura3 his3/his3
trp1/trp1 suc2/suc2 lys2/LYS2 ADE2/ade2
NTC30/NTC30::HIS3; YD20,
Mata/Mat
leu2/leu2 ura3/ura3
his3/his3 trp1/trp1 suc2/suc2 lys2/LYS2 ADE2/ade2 NTC20/TC20::LEU2; YD3020,
Mata/Mat
leu2/leu2 ura3/ura3 his3/his3
trp1/trp1 suc2/suc2 lys2/LYS2 ADE2/ade2 NTC30/NTC30::HIS3 NTC20/NTC20::LEU2; EGY48, Mat
ura3 his3 trp1
LexAop-leu2 ade2.
-galactosidase reporter plasmid
pSH18-34. Two-hybrid assays were carried out according to procedures
described in the manual for the Matchmaker system (CLONTECH).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Western blotting of the components of the
Prp19p-associated complex identified Snt304p as Ntc30p and Snt384p as
Ntc20p. The Prp19p-associated components were fractionated by
SDS-polyacrylamide gel electrophoresis and stained with silver
(lane 1) or probed with anti-Snt304p (lanes 2-4)
or anti-Snt384p (lanes 5-7) antibodies. Pre,
pre-immune serum; Imm, immune serum.
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Fig. 2.
Immunoprecipitation of the spliceosome with
anti-Ntc30p and anti-Ntc20p antibodies. A, the splicing
reaction mixtures (20 µl) were precipitated with the anti-Ntc30p
antiserum (lane 4), or pre-immune serum (Pre,
lane 3) or without serum (lane 2). Antiserum was
also preincubated with recombinant Ntc30p prior to precipitation
(lane 5). Lane 1 is 2 µl of the reaction
mixture. B, the same as A, except the anti-Ntc20p
antiserum and Ntc20p were used. RXN, reaction;
PAS, protein A-Sepharose.
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Fig. 3.
A, a scheme of the spliceosome assembly
showing sequential binding of snRNAs and Prp19p. B,
immunoprecipitation of the spliceosome using extracts in which Prp19p
was tagged with the c-Myc epitope and Prp4p tagged with the HA epitope.
The splicing reaction was carried out at 1 mM (lanes
1-6), 0.5 mM (lanes 7-12), 0.1 mM (lanes 13-18), and 0.05 mM
(lanes 19-24) of ATP, and the reaction mixtures were
subjected to immunoprecipitation with anti-Myc (lanes 3,
9, 15, and 21), anti-HA (lanes 4,
10, 16, and 22), anti-Ntc30p (lanes 5,
11, 17, and 23), and anti-Ntc20p (lanes
6, 12, 18, and 24) antibodies.
RXN, reaction; PAS, protein A-Sepharose.
NTC30) and NTC20 (
NTC20) were constructed. In these constructs, the entire ORF of NTC30
was replaced with a DNA fragment of the HIS3 gene, and a
170-bp fragment within the ORF of NTC20 was replaced with a
DNA fragment of the LEU2 gene. Tetrad dissection of diploid
strains containing one copy of the wild type and one copy of
NTC30 or
NTC20 revealed that neither NTC30 nor
NTC20 was essential for yeast growth, although
NTC30 grew
slightly less well than NTC20 or the wild-type cells (data not shown).
However, deletion of both NTC30 and NTC20
resulted in severe growth defect as shown in Fig.
4A. Dissection of a diploid strain containing one copy of the wild-type and one copy of
NTC30 and
NTC20 yielded two types of spores. One type, accounting for approximately 70% of dissected spores, grew normally. The other type
gave slow growth phenotype and yielded minute colonies, which were all
leucine and histidine prototrophy on replica plating. This result
suggests that cells from which both NTC30 and
NTC20 genes were deleted (
NTC30/
NTC20), although still
viable, were impaired in growth.
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Fig. 4.
A, tetrad analysis revealed a growth defect in
NTC30/
NTC20 cells. B, Northern blot analysis revealed
accumulation of pre-mRNA at a low level in
NTC30 cells and at a
higher level in
NTC30/
NTC20 cells. Wild-type,
NTC30,
NTC20,
NTC30/
NTC20, and prp2 cells were grown at
25 °C until log phase, and half of the culture was shifted to
37 °C and grown for 1-2 h before harvesting. RNA was isolated from
each culture, and 10 µg of total RNA was loaded in each lane.
Lanes 1 and 2, wild type; lanes 3 and
4,
NTC30; lanes 5 and 6,
NTC20;
lanes 7 and 8,
NTC30/
NTC20; lanes
9 and 10, prp2. C, the splicing
activity of
NTC30/
NTC20 extract could be restored on addition of
the Prp19p-associated complex. Lane 1, wild-type extract
(WT); lane 2,
NTC30/
NTC20 extract;
lane 3,
NTC30/
NTC20 extract plus the Prp19p-associated
complex (PRP19 CPX).
NTC30/
NTC20 cells may be a consequence of splicing deficiency. To understand whether pre-mRNA splicing is defective in
NTC30/
NTC20 cells, RNA was isolated for Northern blot analysis using the CRY1 gene as a probe. Fig. 4B shows
that as in the prp2 mutant (lanes 9 and
10), accumulation of pre-mRNA was seen in
NTC30/
NTC20 cells (lanes 7 and 8) and, to a
much lesser extent, in
NTC30 cells at 25 and 37 °C (lanes
3 and 4). Pre-mRNA accumulation was not seen in the
wild type (lanes 1 and 2) and barely detected in
NTC20 cells (lanes 5 and 6). This indicates
that Ntc30p and Ntc20p, although not essential for pre-mRNA
splicing, may play auxiliary roles in the splicing reaction.
NTC30/
NTC20 cells may reflect a deficiency
in the function of the Prp19p-associated complex. To test that
assumption, extracts were prepared from
NTC30/
NTC20 cells
and assayed for splicing. As shown in Fig. 4C, extracts prepared from
NTC30/
NTC20 cells gave very low splicing activity (lane 2). However, the splicing activity was restored to
almost the wild-type level (lane 1) upon addition of the
purified Prp19p-associated complex (lane 3). This indicates
that the splicing deficiency of the
NTC30/
NTC20 extract was
caused by a malfunction of the Prp19p-associated complex and that
Ntc30p and Ntc20p may play roles in sustaining the full activity of the
Prp19p-associated complex.
-galactosidase activity. As
shown in Fig. 5, neither Ntc30p nor
Ntc20p interacted with Prp19p or Snt309p. Neither did they interact
with each other. However, they both interacted with Ntc85p. The
interaction of Ntc85p with Ntc20p was seen only when Ntc20p was fused
to the DNA binding domain and Ntc85p was fused to the activation domain but was not detected when fusion was in an opposite way. Ntc30p showed
very strong self-interaction similar to Ntc85p and Prp19p (12,
14). No self-interaction of Ntc20p was detected.
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Fig. 5.
Two-hybrid assays revealed
interactions of Ntc85p with Ntc30p and Ntc20p. Ntc30p,
Ntc20p, Ntc85p, Prp19p, and Snt309p were fused to both the LexA-DNA
binding domain (BD) and the GAL4 activation domain
(ACT). Interactions between Ntc30p and Ntc20p with other
proteins were revealed by the blue coloring caused by
activation of -galactosidase.
NTC30,
NTC20, and
NTC30/
NTC20 strains. These extracts were
subjected to immunoprecipitation with the anti-Ntc85p antibody followed by Western blotting to reveal components associated with Ntc85p. As
shown in Fig. 6, the anti-Ntc85p antibody
precipitated both Prp19p and Snt309p in all of these extracts
regardless of the absence of Ntc30p and/or Ntc20p, indicating that
association of Ntc85p with Prp19p and Snt309p was not affected by
Ntc30p or Ntc20p or their combination. This result is in
contrast to that of depleting Snt309p, which greatly destabilizes the
association of Prp19p with all other associated components. These
results suggest that association of Prp19p with Snt309p and Ntc85p may
be a prerequisite for the stable association of Ntc30p and Ntc20p with
the Prp19p-associated complex.
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Fig. 6.
Immunoprecipitation analysis revealed
sequential interactions of the components of the Prp19p-associated
complex in formation of the complex. The splicing extract was
precipitated with the anti-Ntc85p antibody and the precipitates
subjected to Western blot analysis using anti-Prp19p, anti-Snt309p,
anti-Ntc85p, anti-Ntc30p, and anti-Ntc20p antibodies. WT,
wild type.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
A scheme for interactions of the components
in the Prp19p-associated complex. Prp19p, Ntc85p, and Ntc30p are
shown as multisubunit homo-oligomers because of observed
self-interactions in the two-hybrid analysis. Prp19p was demonstrated
to be in a tetrameric form. No self-interaction was detected for
Snt309p or Ntc20p.
Like other components of the Prp19p-associated complex, both Ntc30p and Ntc20p are also associated with the spliceosome during the splicing reaction (14, 15). Furthermore, they also associate with the spliceosome in the same manner as Prp19p and other associated components. These results strongly suggest that the Prp19p-associated complex is added to the spliceosome as an integral complex and that the associated components may function in a coordinate fashion.
Ntc30p has recently been reported as Isy1p from the two-hybrid
screening of Syf1p-interacting proteins (20). A sequence search
identified Isy1p homologs in Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and human
(20), suggesting that the protein is conserved between the lower and higher eukaryotes. A similar search identified a protein with only a
limited homology to Ntc20p in S. pombe. Two human EST
sequences were also found to share homology with Ntc20p but only at low level. Thus, Ntc20p appears to be less conserved than Ntc30p throughout its evolution and may be functionally less important, although neither is essential for vegetative growth; this is consistent with the
observed phenotype that NTC20 cells showed no detectable growth
defect or pre-mRNA accumulation, whereas
NTC30 cells grew slightly slower and accumulated a small amount of pre-mRNA (Fig. 4B).
In the report by Dix et al. (20), Isy1p, when fused to
protein A, was demonstrated to be associated with the spliceosome by
precipitation of the splicing reaction mixture with IgG-agarose. This
finding is consistent with our result that anti-Ntc30p antibody also
precipitated the spliceosome from the reaction mixture. A similar
immunoprecipitation analysis revealed association of Isy1p-protein A
with a small fraction of U5 and U6 snRNAs in the splicing extract in
the absence of splicing (20). These authors suggest that only a small
portion of Isy1p was associated with U5 and U6, which may be the
product of disruption of U4/U6 base pairing stimulated by Brr2p (20).
By immunoprecipitation with the anti-HA antibody using Prp19p-HA
extracts, we have previously shown that Prp19p, and likely its
associated complex, is not tightly associated with spliceosomal snRNAs
(18). As a component of the Prp19p-associated complex, it seems
unlikely that Ntc30p is associated with snRNAs. Nevertheless, Ntc30p
may be associated with distinct complexes other than Prp19p. We
therefore performed an immunoprecipitation analysis with the
anti-Ntc30p and anti-HA antibodies using Ntc30p-HA extracts. However,
no precipitation of snRNAs was detected (data not shown). Similar
experiments with the anti-Ntc20p antibody also detected no
co-precipitation of snRNAs (data not shown). A possible explanation for
the discrepancy between our results and those of Dix et al.
is the use of different derivatives of Ntc30p. We used both native and
HA-tagged Ntc30p, whereas they used a fusion form with protein A. A
fusion protein may have a subtle difference from the native form and
exhibit slightly different properties, e.g. increased
affinity with certain proteins.
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ACKNOWLEDGEMENT |
---|
We thank S. Elledge and R. Brent for vectors and strains of the two-hybrid system and M. Tam for synthesis of oligonucleotides. We also thank P. Lin for reading the manuscript
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FOOTNOTES |
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* This work was supported by a grant from the Academia Sinica and by Grant NSC88-2312-B-001-014 from the National Science Council (Taiwan).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.
¶ Present address: School of Medical Technology, Chung Shan Medical and Dental College, Taichung 408, Taiwan, Republic of China.
To whom correspondence should be addressed. Tel.:
886-2-2789-9200; Fax: 886-2-2788-3296; E-mail:
mbscc@ccvax.sinica.edu.tw.
Published, JBC Papers in Press, October 3, 2000, DOI 10.1074/jbc.M006958200
2 C.-H. Chen, W.-Y. Tsai, and S.-C. Cheng, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein; ORF, open reading frame; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s).
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