Received 11 May 1999/Returned for modification 23 June
1999/Accepted 9 July 1999
SF3b is a U2 snRNP-associated protein complex essential for
spliceosome assembly. Although evidence that SF3b contains the spliceosomal proteins SAPs 49, 130, 145, and 155 has accumulated, a
protein-mediated association between all of these proteins has yet to
be directly demonstrated. Here we report the isolation of a cDNA
encoding SAP 130, which completes the cloning of the putative SF3b
complex proteins. Using antibodies to SAP 130 and other putative SF3b
components, we showed that SAPs 130, 145, and 155 are present in a
protein complex in nuclear extracts and that these proteins associate
with one another in purified U2 snRNP. Moreover, SAPs 155 and 130 interact with each other (directly or indirectly) within this complex,
and SAPs 49 and 145 are known to interact directly with each other.
Thus, together with prior work, our studies indicate that SAPs 49, 130, 145, and 155 are indeed components of SF3b. The Saccharomyces
cerevisiae homologs of SAPs 49 and 145 are encoded by essential
genes. We show here that the S. cerevisiae homologs of SAPs
130 and 155 (scSAP 130/RSE1 and scSAP 155, respectively) are also
essential. Recently, the SF3b proteins were found in purified U12
snRNP, which functionally substitutes for U2 snRNP in the minor
spliceosome. This high level of conservation, together with the prior
observation that the SF3b proteins interact with pre-mRNA very close to
the branch site, suggest that the SF3b complex plays a critical role
near or at the spliceosome catalytic core.
 |
INTRODUCTION |
Many proteins essential for
spliceosome assembly and splicing have been identified, and numerous
human homologs of essential yeast splicing factors are now known (for
reviews, see references 19, 20, 24, and
29). Among the best characterized of these are the
components of U2 snRNP (for reviews, see references 19, 20, and 29). In mammals, functional 17S U2
snRNP can be assembled from 12S U2 snRNP and two essential splicing
factors, SF3a and SF3b (5, 6). SF3a has been purified to
homogeneity and contains three proteins (SF3a60, SF3a66 and SF3a120)
(5, 6). SF3b has been purified through multiple
chromatographic steps but has not been purified to homogeneity (5,
6). The components thought to constitute SF3b were identified by
comparing purified 17S U2 snRNP and the spliceosomal complex A (for
reviews see references 14 and
19). The abundant proteins common to both of these complexes are referred to as SF3b 53, 120, 150, and 160 in 17S U2 snRNP
and SAPs 49, 130, 145 and 155, respectively, in the spliceosome (we use
the latter nomenclature here) (2, 6, 19). Further evidence
that at least two of these proteins are components of SF3b came from
the observation that SAPs 49 and 145 interact directly with each other
(7). In addition, SAPs 49, 145, and 155, as well as all
three SF3a subunits, can be UV cross-linked to the region surrounding
the branch site in the spliceosomal complex A (11, 12).
Thus, these proteins are all located next to one another in functional
spliceosomal complexes, consistent with the notion that they are
present in a complex. Despite all of the circumstantial evidence that
SAPs 49, 130, 145, and 155 correspond to SF3b, it remains to be
established whether any or all of these proteins are indeed components
of a single protein complex.
All of the mammalian SF3a components and three of the putative SF3b
components (SAPs 49, 145, and 155) have been cloned (19). In
addition, yeast counterparts of SF3a have been identified and shown to
be essential for viability (for a review, see reference 19). In contrast to SF3a, none of the putative SF3b
components were identified in the early genetic screens for yeast
splicing factors. However, the likely Saccharomyces
cerevisiae homologs of SAPs 145 and 155, scSAP 145 and scSAP 155, were identified in the GenBank database on the basis of their
similarity to the corresponding mammalian proteins (7, 12,
26). One of these proteins, scSAP 145, was subsequently found to
be the same as CUS1, a protein identified as a suppressor of a U2 snRNA
mutation (27). scSAP 145 is essential for A complex assembly
in yeast (27). scSAP 49/HSH49 was also identified in the
database and shown to be essential for viability in yeast
(15). Yeast SAPs 49 and 145, like their mammalian
counterparts, interact directly with each other via protein-protein
interactions and thus are presumed to be components of a yeast SF3b
complex (10, 15). It is not yet known whether scSAP 155 is
essential in yeast or whether a yeast counterpart of SAP 130 exists.
Here we report the isolation of a cDNA encoding SAP 130. Using
antibodies to this protein, as well as antibodies to other putative
SF3b components, we showed that SAPs 130, 145, and 155 are present in a
protein complex and that SAPs 130 and 155 interact (directly or
indirectly) with each other within this complex. Together with previous
work, our data provide strong evidence that SAPs 49, 130, 145, and 155 are components of SF3b. We have also completed the description of the
yeast SF3b counterparts by identifying the S. cerevisiae
homolog of SAP 130 and showing that it and scSAP 155 are essential in
yeast. Thus, together the data indicate that the spliceosomal proteins
SAPs 49, 130, 145, and 155 are components of a highly conserved protein
complex essential for splicing.
| |
MATERIALS AND METHODS |
Isolation of SAP 130 cDNA.
Spliceosomal complex A3' was
produced in large amounts by incubating biotinylated AD3'ENH pre-mRNA
(which lacks a 5' splice site but contains an exonic enhancer) in
large-scale splicing reactions (11 ml) (3, 9). The A3'
complex was isolated by gel filtration followed by binding to avidin
agarose. Total protein from the purified A3' complex was then
fractionated by sodium dodecyl sulfate (SDS) polyacrylamide gel
electrophoresis and the SAP 130 band was excised from the Coomassie
brilliant blue-stained gel. Two sequences (NVSEELDRTPPEVSK and
KLEDIRTRYAF) were obtained by microsequencing (W. Lane, Microchemistry
Facility, Harvard University). These sequences are encoded by a partial
cDNA in the GenBank database (accession no. T92977). On the basis of this sequence, oligonucleotide probes were designed for screening human
bacteriophage cDNA libraries. A partial cDNA encoding 718 amino
acids from the carboxyl terminus of SAP 130 was isolated from this
screen. To isolate the 5' terminus of the SAP 130 cDNA, we used rapid
amplification of cDNA ends and PCR amplification (Marathon Ready cDNA;
Clontech). The full-length SAP 130 cDNA was created by joining the 5'
and the 3' cDNA clones.
Western analysis and immunoprecipitation.
Rabbit polyclonal
antibodies were raised against the SAP 130 carboxyl-terminal peptide
sequence, VSKKLEDIRTRYAF (HRP Inc). The cap (4) and B"
(13) monoclonal antibodies and SAP 155 (26), SAP
145 (23), and hPrp17 (31) rabbit polyclonal
antibodies have been described. SF3a rabbit polyclonal antibodies will
be described elsewhere (9a). For Western blots, spliceosomal
complexes were isolated by gel filtration followed by biotin-avidin
affinity purification (2, 21). Total proteins were
fractionated on SDS-6% polyacrylamide gels and then immobilized on
polyvinylidene difluoride membranes. Polyclonal antibodies were used at
a 1/1,000 dilution, and horseradish peroxidase-conjugated goat
anti-rabbit secondary antibody was used at 1/5,000. Blots were blocked
in 5% nonfat dry milk in phosphate-buffered saline containing 0.1% Tween 20. Proteins were detected with an ECL kit (Amersham).
Immunoprecipitation of U2 snRNP with the cap or B" antibody was carried
out by coupling the antibody to protein G-Sepharose and then rotating
with nuclear extract for 4 h. After extensive washing with 250 mM
NaCl-20 mM Tris (pH 7.8), proteins were eluted with SDS, RNase A, or 1 M urea as indicated below.
Immunoprecipitations of the RNase and urea eluates were carried out by
rotating with the indicated antibodies, followed by washing with 250 mM
NaCl-20 mM Tris (pH 7.8).
Disruption of scSAPs 130 and 155 in S. cerevisiae.
Disruption of scSAP 130 was carried out by using standard homologous
recombination procedures (22). Briefly, ~0.5 kb of the 5'
flanking region and ~0.5 kb of the 3' flanking region of the SAP 130 gene were amplified from total yeast genomic DNA by PCR with
appropriate primers. These flanking regions were then cloned on either
side of the LEU gene to generate the plasmid pscSAP
130
::LEU. This plasmid was digested with
AlwNI on the 5' end and StuI on the 3' end of the
SAP 130-flanking regions, and the DNA was used to transform a wild-type
diploid yeast strain. For disruption of scSAP 155, 1.229 kb of the 5'
flanking region and 761 bp of the 3' flanking region of the SAP 155 gene were amplified from total yeast genomic DNA by using PCR and
appropriate primers. These flanking regions were then cloned on either
side of the LEU gene to generate the plasmid pscSAP
155::LEU. This plasmid was digested with
ApaI on the 5' end and XbaI on the 3' end of the
SAP 155 flanking regions, and the DNA was used to transform a wild-type
diploid yeast strain. Transformants were selected on plates lacking
leucine, and correct integration of the LEU gene into the
SAP 130 locus was verified by Southern analysis. The transformants were
sporulated and tetrads were dissected by standard procedures.
Nucleotide sequence accession number.
The accession number
for the full-length SAP 130 cDNA is AJ001443.
| |
RESULTS |
Isolation of a human SAP 130 cDNA.
To isolate the cDNA
encoding SAP 130, we purified spliceosomal complex A in large
quantities (3). The proteins in this complex were
fractionated by SDS-polyacrylamide gel electrophoresis, and peptide
sequences were obtained from tryptic digestion products of SAP 130. These sequences were used to isolate a full-length cDNA (see Materials
and Methods). The SAP 130 cDNA is predicted to encode a
1,217-amino-acid protein with a molecular mass of 130 kDa and an
isoelectric point of 5.10 (Fig. 1). The
initiator methionine is preceded by a well-conserved Kozak sequence,
and the reading frame is closed upstream of this methionine, indicating that the cDNA encodes the full-length SAP 130 protein (18)
(data not shown).
View larger version (95K):
[in this window]
[in a new window]
|
FIG. 1.
Predicted amino acid sequence of SAP 130 cDNA. The amino
acid sequence predicted from the human SAP 130 cDNA sequence is shown.
The two peptide sequences obtained from microsequencing are underlined
once and twice, and the peptide used for making antibodies is boxed.
|
|
To further characterize the SAP 130 cDNA, we raised rabbit polyclonal
antibodies to a C-terminal peptide (Fig. 1). This antibody is highly
specific for SAP 130, as it detects only a single 130-kDa band in total
HeLa cell nuclear extracts (Fig. 2A). The
protein produced by in vitro translation of the SAP 130 cDNA comigrates with the protein detected on Western blots by SAP 130 antibodies (Fig.
2A). SAP 130 antibodies also detect the SAP 130 protein in
affinity-purified spliceosomal complexes (Fig. 2B and C). This protein
associates with spliceosomes by 10 min of assembly and remains bound
throughout the splicing time course (Fig. 2B and C). The SAP 130 antibody also specifically immunoprecipitates the in vitro translation
product of the SAP 130 cDNA (data not shown). Together, these
observations indicate that we have isolated a full-length cDNA encoding
the spliceosomal protein SAP 130. The SAP 130 antibodies do not
immunoprecipitate the spliceosome or U2 snRNP from splicing extracts,
indicating that the epitope (14 residues at the C terminus [Fig. 1])
is not accessible in these complexes (data not shown).
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 2.
SAP 130 antibodies detect a single ~130-kDa band in
nuclear extract and spliceosomal complexes. (A) The in vitro
translation product of the SAP 130 cDNA comigrates with a protein in
the nuclear extract that is detected by SAP 130 antibodies. (B)
32P-labeled adenovirus major late pre-mRNA was incubated
under splicing conditions for the times indicated and then total RNA
was fractionated on a 15% denaturing gel. Splicing intermediate and
products are indicated. (C) SAP 130 antibodies were used to probe a
Western blot of purified spliceosomal complexes assembled for the
indicated times under splicing conditions. IVT, in vitro translation.
|
|
SAPs 130, 145, and 155 exist in a protein complex in nuclear
extracts and in U2 snRNP.
As mentioned in the introduction, SAPs
49, 130, 145, and 155 are components of 17S U2 snRNP and putative
components of the essential splicing factor SF3b (5, 6). To
determine whether these proteins associate with one another in splicing
extracts and thus could correspond to the SF3b complex, we carried out coimmunoprecipitation assays. The antibodies used were directed against
the snRNP-specific trimethyl cap, the U2 snRNP-specific protein B",
SAPs 130, 145, and 155, and hPrp17 (see Materials and Methods). As
shown in Fig. 3A, all of the spliceosomal
snRNPs were immunoprecipitated by anticap antibodies, and U2 snRNP
alone was immunoprecipitated by anti-B", anti-SAP 145, and anti-SAP 155. No snRNPs were immunoprecipitated by anti-SAP 130 (see above) or
the negative control, anti-hPrp17.
View larger version (49K):
[in this window]
[in a new window]
|
FIG. 3.
SAPs 130, 145, and 155 are part of a protein complex in
nuclear extracts. (A) The indicated antibodies were used for
immunoprecipitations from total nuclear extracts (NE). Total RNA was
fractionated on an 8% denaturing gel. The snRNAs and tRNA are
indicated. (B) An aliquot of each immunoprecipitate was fractionated on
an SDS-6% polyacrylamide gel, and then Western blots were probed with
the indicated antibodies ( ). (C) Same as panel B except that nuclear
extract was incubated with 2 µl of RNase A (10 mg/ml) prior to the
immunoprecipitations.
|
|
Western analysis showed that SAPs 155, 145, and 130 were present in all
of the immunoprecipitates that contained U2 snRNA (Fig. 3B). Thus, as
expected, these proteins are components of U2 snRNP. To determine
whether these proteins exist in a protein complex independently of U2
snRNA, we preincubated nuclear extracts in RNase A, which completely
digested the snRNAs (data not shown). Immunoprecipitations were then
carried out with the antibodies described above. As shown in Fig. 3C,
SAPs 155, 145, and 130 were no longer detected in the anticap or
anti-B" immunoprecipitates. In contrast, all three proteins are
detected in the anti-SAP 145 and anti-SAP 155 immunoprecipitates. These
data indicate that SAPs 130, 145, and 155 exist in a protein complex
independently of U2 snRNA.
To determine whether SAPs 130, 145, and 155 interact with each other in
a discrete protein complex within the purified U2 snRNP particle, we
immunopurified 17S U2 snRNP from nuclear extracts using immobilized
anti-B" antibody. The proteins were eluted from the antibody by
incubation with RNase A (Fig. 4A). The
major proteins in the eluate correspond to SF3a and the putative SF3b
proteins (see below; also data not shown). When the eluate was used for immunoprecipitations with anti-SAP 155 (Fig. 4B, lanes 2 and 6) or
anti-SAP 145 (data not shown) antibodies, SAPs 145, 155, and 130 were
detected on Western blots of the immunoprecipitates. These three
proteins were not detected when the eluate was immunoprecipitated with
antibodies to SF3a (Fig. 4B, lanes 3 and 7) or the negative control
antibody, anti-hPrp17 (Fig. 4B, lanes 4 and 8). Thus, SAPs 130, 145, and 155 are present in a specific protein complex which is associated
with U2 snRNP. The protein complex does not associate with SF3a in the
absence of U2 snRNA.
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 4.
SAPs 130 and 155 interact with each other in the protein
complex. (A) Antibodies to the U2 snRNP protein B" were used to
immunoprecipitate U2 snRNP from nuclear extract. Total proteins were
eluted with RNase A and fractionated on an SDS-9% polyacrylamide gel.
The abundant SAPs are indicated and were identified on two-dimensional
gels and on Western blots (panel B and data not shown). The asterisk
designates a band of unknown identity. However, it is unlikely to be a
component of SF3b since it does not coimmunoprecipitate with SF3b
antibodies (data not shown). (B) The eluate from panel A was used for
immunoprecipitations with the antibodies () indicated on the top of
each lane, and Western blots were probed with the antibodies indicated
to the left of each blot. (C) Same as panel A except that proteins were
eluted with 1 M urea. (D) The urea eluate was used for
immunoprecipitations with the SAP 155 and SAP 145 antibodies. The bound
(IP) and unbound (FT) proteins were fractionated on an SDS-6%
polyacrylamide gel, and Western blots were probed with the indicated
antibodies.
|
|
To further define interactions among the SF3b proteins within U2 snRNP,
we immunopurified U2 snRNP using the B" antibody and then eluted the
proteins with 1 M urea. This eluate contained the set of proteins
detected in the RNase eluate (compare Fig. 4A and C). When the urea
eluate was used for immunoprecipitations with anti-SAP 155, we found
that SAPs 155 and 130 coimmunoprecipitated (Fig. 4D, lane 1). In
contrast, SAPs 130 and 155 were found in the flowthrough when anti-SAP
145 was used for the immunoprecipitation (Fig. 4D, lanes 3 and 4).
Consistent with these data, SAP 145 was found in the flowthrough when
anti-SAP 155 was used for immunoprecipitation (Fig. 4D, lanes 5 and 6)
whereas SAP 145 was found in the immunoprecipitate when anti-SAP 145 antibodies were used (Fig. 4D, lanes 7 and 8). We conclude that SAPs
130 and 155 interact more tightly with each other than with SAP 145.
Together, the data presented above indicate that SAPs 130, 145, and 155 associate with each other via protein-protein interactions. These
proteins are present as a protein complex in purified U2 snRNP, and
SAPs 130 and 155 interact more tightly with each other than with SAP
145 within this complex. Further studies are needed to determine
whether the interaction between SAPs 130 and 155 is direct or indirect
(see Discussion).
scSAPs 130 and 155 are essential for viability in yeast.
Previously, the S. cerevisiae homologs of SAP 145/CUS1
(YMR240c) and SAP 49/HSH49 (YOR319w) were shown to be essential in yeast (15, 27). These and other data (see the introduction) indicate that yeast also contains the SF3b complex (15, 27). To obtain further evidence that this is the case, we asked whether yeast contains a SAP 130 homolog. The greatest similarity to the human
SAP 130 gene in the database was an S. cerevisiae open
reading frame that we designated scSAP 130. During the course of our
study, Chen and coworkers (8) independently identified scSAP
130 as a protein, RSE1 (YML049c), in a screen of temperature-sensitive mutants defective in endoplasmic reticulum-to-golgi transport. Characterization of the temperature-sensitive RSE1 gene revealed that
it is a splicing factor (8). Taken together, these data indicate that scSAP 130/RSE1 is the ortholog of SAP 130.
SAP 130 also resembles the large subunit of a human heterodimer,
designated damage-specific DNA-binding protein (DDB), that has been
implicated in DNA repair (8, 16, 17). Human SAP 130 is 27%
identical and 40% similar to scSAP 130 and 20% identical and 29%
similar to DDB127 (Fig. 5). The size of
the three proteins is conserved, and the similarity is distributed
throughout their lengths. Yeast SAP 130 is more related to human SAP
130 than to DDB (Fig. 5). Thus, it is likely that yeast SAP 130 is the
functional homolog of human SAP 130 and not of DDB. Consistent with
this conclusion, Chen and coworkers (8) showed that RSE1
mutants have normal DNA repair after treatment with UV light. Using
antibodies to DDB, we detected this protein in nuclear extracts but not
in either purified spliceosomes or the purified hnRNP complex H (data not shown). Thus, DDB does not appear to be a general spliceosomal protein. It is possible that the similarities between these proteins are due to a common function that remains to be identified.
View larger version (122K):
[in this window]
[in a new window]
|
FIG. 5.
Alignment of SAP 130 with scSAP 130 and human DDB. The
alignment was done by the Clustal method (DNASTAR Inc.). Residues that
are identical in hsSAP 130 and the other two proteins are shown in
white on black. The overall identities of scSAP 130 and hsDDB to hsSAP
130 are 27 and 20%, respectively.
|
|
Recently, we identified the likely homolog of SAP 155 (YMR288w) in
yeast (26). To determine whether scSAPs 130 and 155 are essential for viability in yeast, we constructed diploid yeast strains
in which one copy of the genes was disrupted by replacement with the
leu2 selectable marker (22). Southern analysis of
total yeast genomic DNA confirmed the replacement of one allele by the Leu2 gene (Fig. 6A and B [panels I and
II]). Tetrad analysis demonstrated a 2:2 segregation of nonviable and
viable spores (Fig. 6C [panels I and II]), and the viable spores did
not grow on plates lacking leucine (Fig. 6D [panels I and II]).
Together, these data indicate that scSAP 130 and SAP 155 genes are both
essential for viability in yeast.
View larger version (40K):
[in this window]
[in a new window]
|
FIG. 6.
The scSAP 130 and scSAP 155 genes are essential in
S. cerevisiae. (Panel I) (A) Structure of scSAP 130 and
scSAP 130::LEU alleles. The sizes of
AlwNI and StuI restriction fragments are
indicated. The probe is shown below the region with which it
hybridizes. (B) Southern analysis of total genomic DNA from a normal
diploid strain (WT) and diploid strains transformed with the scSAP
130::LEU allele (lanes 1 and 2). The sizes (in
kilobases) of markers and restriction fragments are shown. (C) Tetrad
analysis. The scSAP 130::LEU strain was
sporulated, and 13 individual tetrads were dissected. (D) Viable spores
are Leu . Fifteen viable spores were patched onto a plate
lacking leucine. The diploid strain was patched as a control. (Panel
II) (A) Structure of SAP 155 and scSAP 155::LEU
alleles. The sizes of the StuI and XhoI
restriction fragments used to identify each allele are indicated. (B)
Southern analysis of total genomic DNA from a normal diploid yeast
strain (wt) and diploid strains transformed with the scSAP
155::LEU allele (lanes 1 through 5). The sizes
(in kilobases) of the markers and restriction fragments are shown. The
differences in band intensities between lanes is most likely due to
different levels of DNA loaded on the gel. (C) Tetrad analysis. The
scSAP 155::LEU strain was sporulated, and 15 individual tetrads were dissected. (D) Viable spores are
Leu. Eighteen viable spores were patched onto a plate
lacking leucine. The diploid strain was patched as a control.
|
|
| |
DISCUSSION |
Here we report the isolation of a cDNA encoding the human
spliceosomal protein SAP 130, which completes the cloning of the major
U2 snRNP proteins in humans. Our data show that SAP 130, together with
SAPs 145 and 155, exists in a protein complex in nuclear extracts. We
do not have antibodies to the U2 snRNP protein SAP 49. However, this
protein interacts very tightly with SAP 145 (7). Thus, the
simplest interpretation of the data is that SAPs 49, 130, 145, and 155 are present in the same protein complex. Although functional studies
are required to prove that this protein complex corresponds to the
essential splicing factor SF3b, it is highly likely that this is the
case. This contention is based on several observations. Functional 17S
U2 snRNP is composed of 12S U2 snRNP together with SF3a and SF3b
(5). Purified 17S U2 snRNP contains SAPs 61, 62, and 114, which are known to be the components of SF3a (5, 6). 17S U2
snRNP also contains SAPs 49, 130, 145, and 155 in equal stoichiometries
(1). Our data show that these proteins are present in a
protein complex within purified U2 snRNP. Thus, it is highly likely
that this protein complex corresponds to the U2 snRNP-associated SF3b complex.
Previous work showed that purified 17S U2 snRNP contains two additional
proteins (35 and 92 kDa) (1), but these proteins are present
at lower levels than the other SF3a and SF3b proteins. Indeed, the 35- and 92-kDa proteins are not evident in our immunopurified U2 snRNP
(Fig. 4A and C). Thus, whether they are essential components of U2
snRNP or SF3b remains to be determined.
Previous studies showed that SAPs 145 and 49 interact with each other
through direct protein-protein interactions (7). Our data
show that SAPs 130 and 155 interact with each other (directly or
indirectly). However, it is not known how these two protein complexes
(SAP 145-SAP 49 and SAP 155-SAP 130) associate. One possibility is that
these complexes interact directly to form SF3b. However, we cannot rule
out the alternative possibility that a protein not yet detected
mediates the interaction, or that the 35- or 92-kDa protein mentioned
above is involved. We note that we were unable to detect direct
interactions between SAPs 155 and 130 or between SAP 155-SAP 130 and
any of the other U2 snRNP proteins using far-Western analysis. Thus,
further work is needed to determine how the SF3b proteins interact with
each other.
Our study also provides new evidence that the counterpart of human SF3b
is present in S. cerevisiae. Prior work identified scSAPs
145 and 49 and showed that they interact directly and that they are
essential in yeast (12, 15, 27). We have shown here that
scSAPs 130 and 155 are also essential in yeast, and recent work showed
that scSAP 130 is a splicing factor (8). Studies with yeast
showed that scSAP 145 suppresses lethal mutations in U2 snRNA near the
region that base-pairs to the branch site (30). Likewise, in
mammals, all of the SF3a and -b subunits interact with the 5' end of U2
snRNA near the region that base-pairs with the branch point sequence
(1). All of the mammalian SF3a and -b subunits, except SAP
130, also UV cross-link to pre-mRNA around that branch site prior to
both catalytic steps I and II. One function for these interactions
appears to be to anchor U2 snRNP tightly to the pre-mRNA
(12). The SF3b proteins are also part of the U12 snRNP,
which is the equivalent of U2 snRNP in the minor-abundance human
spliceosome (28). This spliceosome functions to splice a
rare class of introns (25). Thus, the high degree of
conservation of SF3b, together with its positioning near the catalytic
center of the spliceosome, indicates that this complex is likely to
play key roles in the establishment and/or functioning of the catalytic
center of the spliceosome for both steps of the splicing reaction. The
availability of recombinant SF3b subunits in both yeast and mammals
should now allow these possibilities to be tested directly.
| 1.
|
Behrens, S. E.,
K. Tyc,
B. Kastner,
J. Reichelt, and R. Luhrmann.
1993.
Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions.
Mol. Cell. Biol.
13:307-319[Abstract/Free Full Text].
|
| 2.
|
Bennett, M.,
S. Michaud,
J. Kingston, and R. Reed.
1992.
Protein components specifically associated with prespliceosome and spliceosome complexes.
Genes Dev.
6:1986-2000[Abstract/Free Full Text].
|
| 3.
|
Bennett, M., and R. Reed.
1993.
Correspondence between a mammalian spliceosome component and an essential yeast splicing factor.
Science
262:105-108[Abstract/Free Full Text].
|
| 4.
|
Bochnig, P.,
R. Reuter,
P. Bringmann, and R. Luhrmann.
1987.
A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact, class U, small nuclear ribonucleoproteins as well as with 7-methylguanosine-capped RNAs.
Eur. J. Biochem.
168:461-467[Medline].
|
| 5.
|
Brosi, R.,
K. Groning,
S. E. Behrens,
R. Luhrmann, and A. Kramer.
1993.
Interaction of mammalian splicing factor SF3a with U2 snRNP and relation of its 60-kD subunit to yeast PRP9.
Science
262:102-105[Abstract/Free Full Text].
|
| 6.
|
Brosi, R.,
H. P. Hauri, and A. Kramer.
1993.
Separation of splicing factor SF3 into two components and purification of SF3a activity.
J. Biol. Chem.
268:17640-17646[Abstract/Free Full Text].
|
| 7.
|
Champion-Arnaud, P., and R. Reed.
1994.
The prespliceosome components SAP 49 and SAP 145 interact in a complex implicated in tethering U2 snRNP to the branch site.
Genes Dev.
8:1974-1983[Abstract/Free Full Text].
|
| 8.
|
Chen, E. J.,
A. R. Frand,
E. Chitouras, and C. A. Kaiser.
1998.
A link between secretion and pre-mRNA processing defects in Saccharomyces cerevisiae and the identification of a novel splicing gene, RSE1.
Mol. Cell. Biol.
18:7139-7146[Abstract/Free Full Text].
|
| 9.
|
Chiara, M. D., and R. Reed.
1995.
A two-step mechanism for 5' and 3' splice-site pairing.
Nature
375:510-513[Medline].
|
| 9a.
| Das, R., and R. Reed. Unpublished data.
|
| 10.
|
Fromont-Racine, M.,
J. C. Rain, and P. Legrain.
1997.
Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens.
Nat. Genet.
16:277-282[Medline].
|
| 11.
|
Gozani, O.,
J. Potashkin, and R. Reed.
1998.
A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site.
Mol. Cell. Biol.
18:4752-4760[Abstract/Free Full Text].
|
| 12.
|
Gozani, O.,
R. Feld, and R. Reed.
1996.
Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A.
Genes Dev.
10:233-243[Abstract/Free Full Text].
|
| 13.
|
Habets, W. J.,
M. H. Hoet,
B. A. De Jong,
A. Van der Kemp, and W. J. Van Venrooij.
1989.
Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally-induced mouse monoclonal antibodies.
J. Immunol.
143:2560-2566[Abstract].
|
| 14.
|
Hodges, P. E., and J. D. Beggs.
1994.
RNA splicing. U2 fulfils a commitment.
Curr. Biol.
4:264-267[Medline].
|
| 15.
|
Igel, H.,
S. Wells,
R. Perriman, and M. Ares, Jr.
1998.
Conservation of structure and subunit interactions in yeast homologues of splicing factor 3b (SF3b) subunits.
RNA
4:1-10[Abstract].
|
| 16.
|
Kazantsev, A.,
D. Mu,
A. F. Nichols,
X. Zhao,
S. Linn, and A. Sancar.
1996.
Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.
Proc. Natl. Acad. Sci. USA
93:5014-5018[Abstract/Free Full Text].
|
| 17.
|
Keeney, S.,
G. J. Chang, and S. Linn.
1993.
Characterization of a human DNA damage binding protein implicated in xeroderma pigmentosum E.
J. Biol. Chem.
268:21293-21300[Abstract/Free Full Text].
|
| 18.
|
Kozak, M.
1986.
Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes.
Cell
44:283-292[Medline].
|
| 19.
|
Kramer, A.
1996.
The structure and function of proteins involved in mammalian pre-mRNA splicing.
Annu. Rev. Biochem.
65:367-409[Medline].
|
| 20.
|
Reed, R.
1996.
Initial splice-site recognition and pairing during pre-mRNA splicing.
Curr. Opin. Genet. Dev.
6:215-220[Medline].
|
| 21.
|
Reed, R.
1990.
Protein composition of mammalian spliceosomes assembled in vitro.
Proc. Natl. Acad. Sci. USA
87:8031-8035[Abstract/Free Full Text].
|
| 22.
|
Rothstein, R.
1991.
Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast.
Methods Enzymol.
194:281-230[Medline].
|
| 23.
|
Seghezzi, W.,
K. Chua,
F. Shanahan,
O. Gozani,
R. Reed, and E. Lees.
1998.
Cyclin E associates with components of the pre-mRNA splicing machinery in mammalian cells.
Mol. Cell. Biol.
18:4526-4536[Abstract/Free Full Text].
|
| 24.
|
Staley, J. P., and C. Guthrie.
1998.
Mechanical devices of the spliceosome: motors, clocks, springs, and things.
Cell
92:315-326[Medline].
|
| 25.
|
Tarn, W. Y., and J. A. Steitz.
1997.
Pre-mRNA splicing: the discovery of a new spliceosome doubles the challenge.
Trends Biochem. Sci.
22:132-137[Medline].
|
| 26.
|
Wang, C.,
K. Chua,
W. Seghezzi,
E. Lees,
O. Gozani, and R. Reed.
1998.
Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis.
Genes Dev.
12:1409-1414[Abstract/Free Full Text].
|
| 27.
|
Wells, S. E.,
M. Neville,
M. Haynes,
J. Wang,
H. Igel, and M. Ares, Jr.
1996.
CUS1, a suppressor of cold-sensitive U2 snRNA mutations, is a novel yeast splicing factor homologous to human SAP 145.
Genes Dev.
10:220-232[Abstract/Free Full Text].
|
| 28.
|
Will, C.,
C. Schneider,
R. Reed, and R. Luhrmann.
1999.
Identification of both shared and distinct proteins in the major and minor spliceosomes.
Science
284:2003-2005[Abstract/Free Full Text].
|
| 29.
|
Will, C. L., and R. Luhrmann.
1997.
Protein functions in pre-mRNA splicing.
Curr. Opin. Cell Biol.
9:320-328[Medline].
|
| 30.
|
Yan, D., and M. Ares, Jr.
1996.
Invariant U2 RNA sequences bordering the branchpoint recognition region are essential for interaction with yeast SF3a and SF3b subunits.
Mol. Cell. Biol.
16:818-828[Abstract].
|
| 31.
|
Zhou, Z., and R. Reed.
1998.
Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing.
EMBO J.
17:2095-2106[Medline].
|
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
