From the Departments of Biology and
§ Chemistry, University of Kentucky,
Lexington, Kentucky 40506-0225
Received for publication, October 22, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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Spliceosome assembly follows a well conserved
pathway of subunit addition that includes both small nuclear
ribonucleoprotein (snRNP) particles and non-snRNP splicing factors.
Clf1p is an unusual splicing factor composed almost entirely of direct
repeats of the tetratricopeptide repeat (TPR) protein-binding motif.
Here we show that the Clf1p protein resides in at least two
multisubunit protein complexes, a small nuclear RNA-free
structure similar to what was reported as the Prp19p complex (nineteen
complex; NTC) and an RNP structure that contains the U2, U5, and U6
small nuclear RNAs. Thirty Ccf (Clf1p complex
factor) proteins have been identified by mass spectroscopy
or immune detection as known or suspected components of the yeast
spliceosome. Deletion of TPR1 or TPR2 from an epitope-tagged Clf1p
protein (i.e. Clf1 The spliceosome is composed of five small nuclear RNAs
(snRNAs)1 and over 70 distinct polypeptides (reviewed in Refs. 1 and 2; see references in
Refs. 3 and 4). Numerous studies have demonstrated that in
vitro spliceosome assembly progresses through an ordered sequence
that is largely conserved from yeast to humans. By tracking assembly
through small nuclear ribonucleoprotein (snRNP) association with
pre-mRNA, it has been shown that the U1 snRNP binds independently
other snRNP particles in an ATP-independent step said to commit the
pre-mRNA to the splicing pathway. U2 snRNP is recruited next in an
ATP-dependent step that forms the prespliceosome. Finally,
the U4, U5, and U6 snRNAs are jointly recruited as the exceptionally
large U4/U6.U5 tri-snRNP particle to produce the spliceosome. Together,
the U1/U2 snRNP-dependent prespliceosome and the U4/U6.U5
tri-snRNP particle account for all of the snRNAs and the majority of
proteins known to act in splicing. A more limited number of non-snRNP
splicing factors bind directly to the pre-mRNA substrate to promote
snRNP addition or promote conformational changes within the splicing
complex following snRNP addition (1-3).
Regulated pre-mRNA splicing events are driven by proteins that act
to promote or block the recruitment of constitutive splicing factors
(4, 5). The major snRNP addition steps are clear targets of this
regulation. Whereas comparatively few pre-mRNAs appear regulated in
yeast, this system does offer a valuable means to characterize the
basic pathway for splicing factor association and hence to identify
critical steps in assembly and possible targets for regulation. The
addition of the U1 and U2 snRNP particles to the splicing complex
occurs in part through well characterized snRNA interactions at the
premRNA 5' splice site and branch point regions, respectively, and
is supported by a number of protein-based contacts, reviewed in Refs. 6
and 7. The U5 and U6 snRNAs also bind the splicing substrate, although
these interactions appear to occur subsequent to stable recruitment of
the U4/U6.U5 tri-snRNP particle to the prespliceosome. In yeast,
mutations that prevent stable U4/U6.U5 tri-snRNP assembly
(e.g. Prp6p; see Refs. 8 and 9) block spliceosome formation,
yet little is known in yeast or in mammals of the contacts that
actually promote U4/U6.U5 tri-snRNP particle/prespliceosome association.
CLF1/SYF3 (henceforth referred to by the Stanford
Genome Database standard name, CLF1) was identified as a
putative Saccharomyces cerevisiae RNA-processing factor
based on the presence of multiple crooked neck-like tetratricopeptide
repeats (TPRs) (10). This protein binding motif was first recognized as
a distinct TPR subtype in the characterization of the Drosophila
melanogaster crooked neck (crn) gene product (11) and
appears restricted to RNA-processing proteins (10, 12). Crooked neck
and its homologs, including the yeast Clf1p protein, are ~80 kDa in
mass and composed largely of direct iterations of the TPR motif. Loss
of function crooked neck mutations in Drosophila
are lethal when homozygous (11) (and references within) and influence
alternative splice site selection when heterozygous (13). In humans,
the crooked neck protein is a stable component of the spliceosome,
recruited at or near the time of U4/U6.U5 tri-snRNP addition (14). In
yeast, genetic and biochemical studies implicate Clf1p in a number of steps in spliceosome assembly and in both catalytic steps in splicing (10, 15-18). This broad spectrum of Clf1p interactions is consistent with the 15 TPR repeats of Clf1p serving as a platform for the recruitment of splicing factors during spliceosome assembly.
Clf1p is present in the nineteen complex (NTC), a structure that
contains Prp19p and at least eight other proteins but no RNA (see Ref.
18 and references within) (19). Yeast extracts metabolically depleted
of Clf1p block the prespliceosome to spliceosome transition even in the
presence of stable U4/U6.U5 tri-snRNP complexes (10), suggesting that
Clf1p acts in the recruitment or retention of the U4/U6.U5 tri-snRNP
particle. Curiously, whereas Clf1p functions prior to stable
spliceosome formation, the NTC is reported to bind the splicing complex
later, during or after the U4 snRNA release from the mature spliceosome
(20). This inconsistency and results from the Schizosaccharomyces
pombe system that cast doubt on the RNA-free status of the NTC
(21) stimulated a detailed analysis of Clf1p function in the
spliceosome assembly pathway.
The genetic and biochemical data presented here show that Prp19p and
Clf1p reside in at least two resolvable states, a complex free of snRNA
similar to the NTC and an RNP complex reminiscent of an endogenous late
stage or postcatalytic spliceosome. Removal of TPR elements from the
Clf1p amino terminus results in the conditional release of several
essential splicing factors and blocks the recruitment of Prp19p to the
splicing apparatus. These observations provide evidence that the
N-terminal TPR elements of Clf1p recruit splicing factors required for
the successful integration of the two large RNP halves of the spliceosome.
Yeast Strains and Plasmids--
Details on the construction of
the clf1::HIS3 yeast deletion mutant
and the GAL1::clf1(697),
Ycplac22-CLF1-HA, and Ycplac22-clf1
PRP46 was amplified from genomic DNA with oligonucleotides
46-1 and 46-2 and cloned into Ycplac111 (25). The TAP PCR fragment was
inserted into a SmaI site engineered in the 3'-end of the PRP46 coding sequence. The Ycplac111-PRP46-TAP
plasmid was transformed into yeast heterozygous for a PRP46
deletion obtained from the ATCC. Haploid strains that exclusively
expressed the plasmid-borne PRP46-TAP allele were isolated
from meiotic offspring.
Splicing and Spliceosome Assembly Assays--
The analysis of
cellular pre-mRNA splicing was conducted as described (26). To
deplete Clf1p from yeast, the
GAL1::clf(697) strain was grown to
early log phase in galactose-based YP medium (23), harvested by
centrifugation, washed with 1 culture volume of sterile water, and then
incubated in glucose-based YP medium for 6-8 h. Splicing extracts were
prepared by grinding yeast cell pellets in liquid nitrogen as described
(27). Pre-mRNA substrates were prepared by in vitro
transcription of pSPrp51A (RP51A) (28) or pT7 Clf1-TAP, clf1 Mass Spectrometry--
The purified Clf1-TAP complex sample was
resolved on a 5-10% gradient SDS-polyacrylamide gel in a Tris-Tricine
buffer system (34). After electrophoresis, the gel was stained with
silver (35), and the protein-containing bands were excised and digested with trypsin (36). The tryptic peptides were fractionated with a Luna
C18 (1 × 50 mm, 3-µm particle size) column (Phenomenex, Torrance, CA) using an acetonitrile/water (0.1% formic acid) gradient at a flow rate of 35 µl/min. The LC column was connected to the electrospray interface of an LCQ Classic mass spectrometer
(ThermoFinnigan, San Jose, CA). Mass spectra were acquired using
data-dependent analysis (full scan automatically followed
by tandem mass spectrometry of the most intense ion detected by the
data system). Alternatively, the Clf1-TAP complexes were assayed by
DALPC without gel fractionation using strong cation exchange and C18
(Whatman) packing material on a LCQ Deca mass spectrometer
(ThermoFinnigan, San Jose, CA). In all cases, the tandem mass spectra
were converted to mass/intensity lists and searched against the
nonredundant OWL data base using SEQUEST and the nonredundant NCBI data
base using MASCOT. The number of unique peptides identified for each
protein was as follows: Clf1p, 26; Prp8p, 21; Brr2p, 10; Rse1p, 3;
Snu114p, 5; Hsh155p, 4; Syf1p, 22; Cef1p, 26; Prp28p, 2; Prp19P, 14;
Prp46p, 6; Prp45p, 4; Cwc2p, 3; Cwc23p, 4; Ecm2p, 4; Isy1p, 5; Smb1p,
2; Syf2p, 2; Snt309p, 8; Smd1p, 3; Ntc20p, 4; Smd2p, 7; Smd3p,
3; Lsm2p, 1; Sme1p, 1; Smx3p, 1; Prp22p, 13; Cus2p, 2. Proteins without
confirmed links to splicing that co-purified with Clf1-TAP in one or
more preparations include Aad15p, Adh1p, Ade6p, Bud31p, Cin8p, Imp2p, Lem3p, Nop1p, Pdc1p, Ptc7p, Rgd2p, Rpl8ap, Rlp18bp, Rlp24p, Rpl28p, Rpl15p Rpl18bp, Rps14p, Rps19ap, Rpl24p, Rps17p, Rps13p, Rpl13p, Rps6p,
Scc2p, Smc3p, Ssa2p, Ssa4p, Sum1p, Tef2p, Tdh1p, Tdh3p, Ulp1p,
Zms1p, and the product of open reading frames YGL034C, YKR022C,
YIL080W, and YLR424W.
Clf1p Resides in at Least Two Distinct Multisubunit
Complexes--
To learn more of Clf1p function in spliceosome
assembly, proteins associating with this essential splicing factor were
purified from yeast. As a first step, Clf1p was modified to include a
TAP affinity cassette (22) at the nonessential C terminus (10). The
CLF1-TAP strain and a negative control strain that expresses a hemagglutinin (HA)-tagged protein that does not bind the TAP affinity
resin were labeled with [35S]methionine and cysteine for
4 h prior to tandem affinity purification. PAGE analysis reveals
numerous proteins present in the Clf1-TAP but absent in the Clf1-HA
control sample (Fig. 1A).
Thirty of the Ccf (Clf1p complex
factor) proteins were identified as known or suspected
splicing factors by mass spectroscopy (Table
II). In several cases, two or three Ccf
proteins co-migrate in a single band, leading to an underestimation of
the number of Ccf factors by one-dimensional electrophoresis. The
validity of the Ccf assignments was spot-checked with antibodies
against specific proteins and through the use of gene-specific deletion
strains (Table I; see "Experimental Procedures"). For the sake of
consistency, the established nomenclature will be used for all
previously identified Ccf splicing factors.
In order to investigate the subunit diversity of the Clf1p complex(es),
the 35S-labeled affinity-purified proteins were resolved on
a 15-40% glycerol gradient, and alternate fractions were compared
(Fig. 1B; note, for clarity, where multiple proteins are
present in a single band, only one is labeled). Fraction 1 contains
mostly background proteins, a pattern similar to that observed with the control extract in Fig. 1A. Superimposed on this pattern in
fraction 3 and extending through fraction 11 are Clf1p, Syf1p, Ccf8p,
Prp19p, and Isy1p (but presumably not the co-migrating Lea1p protein; see snRNA analysis, discussed below). Cef1p co-migrates with Clf1p at
~84 kDa (due to the presence of residual TAP sequence) but can be
resolved with the use of Clf1p TPR deletion mutants or when an
alternatively tagged strain is used for purification (see below). Other
than the undetectable levels of certain low molecular weight proteins
that label poorly (e.g. Syf2p and Snt309p), this banding pattern closely resembles that of the NTC and will be referred
to as the Clf1p-NTC to designate the protein used for purification. Two
unidentified proteins, Ccf8p, which may be equivalent to Ntc81p (37),
and a previously unreported protein of ~220 kDa (Ccf25p) are
also present in these fractions. A small amount of Prp8p and Brr2p are
reproducibly observed in this region of the gradient as well.
The Sm/Lsm core snRNP proteins are absent in fractions 1-11.
With the intriguing exception of Ccf8p, the Clf1p-NTC complex proteins
become markedly enriched in fractions 13-15. Here too are the
U5-snRNP-associated proteins Prp28p and Snu114p (as well as Prp8p and
Brr2p), Prp46p, the co-migrating Prp45p/Slu7p and Cwc2p/Cwc23p/Ecm2p
protein sets, the uncharacterized protein Ccf14p, the Sm/Lsm proteins,
and four protein bands not observed prior to gradient separation
(Ccf25p-Ccf28p; see Table II). Ccf26p has been
identified by Western blot as Prp22p, a protein that interacts genetically with Clf1p and other components of the Clf1p-RNP complex (16, 38). In addition to the mentioned proteins, fractions 17-23 also
contain the U2 snRNP-specific proteins Rse1p and Hsh155p, presumably
Lea1p, the Ccf29p band (Cus2p), and factors found in the lighter
snRNP-enriched gradient fractions. Of the proteins identified by mass
analysis, only Prp16p was not observed as an obvious gel band. The
Clf1p profile from whole cell extract (Fig. 1C) is similar
to that of the purified complex with discreet pools in the lower and
upper regions of the gradient, although the high concentration of total
protein in fractions 3-5 partially inhibits Clf1p transfer (see Fig.
2E for a related image). The
presence of Clf1-TAP in fractions 7-11 of the whole cell extract
argues against the Clf1p-NTC arising from dissociation of the larger complex during the lengthy affinity purification steps. The Clf1-TAP peak enriched for U2 as well as U5 and U6 snRNP proteins (Fig. 1,
B and C, roughly fractions 17-19) overlaps the
position of the U4/U5.U6 snRNP particle (Fig. 1D) and
corresponds to a complex of ~40 S.
The Clf1p-RNP Complex--
A subset of the known U2 and U5 snRNP
proteins co-purify with the Clf1p complex. Recently, Cef1p-bearing
multi-snRNP complexes were reported to be RNase-resistant (39). In
contrast, treatment of the Clf1-TAP extract with RNase A
and T1 prior to purification causes release of Lsm2p, Smb1p, Smd2p,
Sme1p, and Smx3p core snRNP proteins and a reduction in the
Smd1p/Ntc20p band intensity (Fig. 2A). Multiple other
proteins, including the U2- and U5-specific snRNP proteins, remain
Clf1-TAP-associated. The RNase treatment regime used degrades all snRNA
detectable by Northern blot (data not shown). Consequently, it appears
that snRNA contacts stabilize core snRNP protein association with the
Clf1p complex, whereas the bulk of the remaining components associate
though protein-based contacts. It is possible, however, that small RNA
fragments persist in the Clf1p complex and contribute to this stabilization.
Consistent with the snRNP protein composition, the U2, U5, and U6
snRNAs (but not U1 or U4 snRNA) are enriched in the affinity-purified Clf1-TAP complexes (Fig. 2B). Nonspecific binding to the TAP
affinity resin is very low, since no detectable snRNAs are recovered
with a control extract that lacks a TAP-tagged protein (i.e.
Clf1-HA). The bulky TAP epitope enhances snRNA recovery, since previous experiments performed with alternative CLF1 alleles failed
to recover snRNA (10) or recovered no U2 snRNA and nearly background levels of U5 and U6 snRNA (15). The amount of snRNA recovered with
Clf1-TAP is less than what is commonly observed with snRNP-specific proteins, however. Soluble Clf1-TAP protein recovery is typically 30-70% (Fig. 2C), and only 2-5% of the U2, U5, and U6
snRNAs co-purify.
As shown above, the Clf1p present in whole cell lysates distributes
broadly across a 15-40% glycerol gradient. When these gradient
fractions are used for immune precipitation, the majority of
Clf1-TAP-associated snRNA is recovered from the poly-snRNP fraction
(fractions 15-19) with no detectable free U6 snRNP (Fig. 2D, fractions 5-7; see Fig. 1D) and little if
any U4/U6 di-snRNP (fractions 9-11), or free U2 snRNP or U5 snRNP
(fractions 7-13) precipitating. This recovery pattern indicates that
Clf1p does not bind individual snRNP complexes but resides in a more
complex RNP structure. Curiously, whereas Prp19p is reported as
predominately RNA-free in extracts (see Ref. 18 and references within)
(19), both Clf1-TAP and Prp19p are recovered from the RNP fractions with antibodies against the TAP epitope (Fig. 2E). This
apparent discrepancy was resolved when representative gradient
fractions were reassayed with the anti-Prp19p antibody. In this case,
Prp19p is efficiently recovered from gradient fractions with little
snRNA (other than U6) is present (e.g. Fig. 2F,
factions 4 and 7; see Fig. 1D) but poorly from the
snRNP-containing fractions (e.g. fractions 10 and 17). As
with the earlier reports, no snRNA was recovered with the anti-Prp19p
antibody (data not shown). Thus, whereas Clf1p and Prp19p reside within
RNA-free and RNP complexes, the RNP structure inhibits Prp19p recovery
by immune precipitation.
Influence of TPR Structure on Clf1p Complex Integrity--
Yeast
with deletions of the first or second Clf1p TPR repeat have growth and
splicing defects that become more pronounced at elevated temperatures,
presumably due to changes in Clf1p stability or activity (10) (data not
shown). To investigate protein abundance, lysates from
CLF1-TAP, clf1
TAP affinity purification was repeated at different salt concentrations
to learn whether the N-terminal TPR deletions described above
destabilize the Clf1p complexes (Fig. 3B). For the wild type
Clf1-TAP complex, the recovered protein set is equivalent at 200 and
300 mM NaCl and shows reduced levels of selected proteins at 450 mM NaCl. With the Clf1
Rse1p and Hsh155p are sensitive to dissociation at 450 mM
NaCl in the wild type extracts (Fig. 3B) and missing from
gradient fractions 13-15 of the Clf1p-RNP even under low salt
conditions (see Fig. 1B). Even so, Rse1p, Hsh155p, and
Snu114p appear more sensitive to dissociation in the Clf1 Clf1 Clf1p Is Required for Prp19p Recruitment to the
Spliceosome--
The results presented above show that brief heat
treatment destabilizes the Clf1
When radiolabeled pre-mRNA is used for in vitro
splicing, lariat intermediate, excised intron, and low levels of
pre-mRNA are recovered with the anti-TAP antibody from wild type
extracts with or without heat treatment (Fig. 5B).
The presence of pre-mRNA in the complex shows that Clf1p resides in
the precatalytic spliceosome, whereas the absence of mRNA shows
that Clf1p persists at least through the Prp22p-dependent
step of mRNA release. The same RNA species are recovered with the
Clf1 Premessenger RNA splicing is an amazingly faithful process given
the limited information content of splice site consensus sequences.
Stepwise spliceosome assembly may offset the limitations of restricted
sequence conservation, since this process advances only when previously
bound factors are present in the correct position, orientation, and
spacing. Whereas the recent identification of unexpected poly-snRNP
complexes and interactions (e.g. see Refs. 41-44) suggests
that the details of spliceosome assembly may be less rigidly
constrained than formerly envisioned, the basic view that substrate
selection is context-dependent and occurs through a dynamic
process of splicing factor association is very likely correct. Here we
show that the poly-TPR protein, Clf1p, plays a critical role in the
union of the two large RNP "halves" of the spliceosome, namely the
U1/U2-dependent prespliceosome and the U4/U6.U5 tri-snRNP
particle. Whereas inactivation of the yeast U4/U6.U5 tri-snRNP protein,
Prp31p (45), or the removal of the mammalian SR-like U4/U5.U6 tri-snRNP
protein sp110/START1 or p65 (46) blocks spliceosome formation, to our
knowledge this is the first example of a non-snRNP yeast splicing
factor influencing the prespliceosome to spliceosome transition.
The Clf1p-NTC contains proteins previously reported in the Prp19p-NTC
(see Ref. 18 and references within) (19), uncharacterized proteins of
~74 kDa (Ccf8p) and 220 kDa (Ccf25p), and, surprisingly, low
levels of the normally snRNP-associated Prp8p and Brr2p. In addition to
residing in the NTC state, Clf1p and Prp19p are also present in the
Clf1-RNP. The presence of Prp19p in this RNP complex provides
explanation for earlier observations that U6 snRNA levels decrease and
"free" U4 snRNA levels increase after prp19-1p inactivation, characteristics tightly correlated with the perturbation of U6-bearing snRNP complexes (26, 47). The Clf1p-RNP is refractory to precipitation with an antibody against Prp19p, consistent with the inability of this
antibody to deplete Prp19p activity from extracts (19) and accounting
for the earlier suggestion that Prp19p exists largely in an RNA-free
state in cell extracts (19, 20). Prp19p does not appear to bind Clf1p
directly, and of the many protein-protein interactions identified among
Clf1p-NTC components only Cef1 binds both Prp19p and Clf1p (18). Since
Prp19p and Cef1p interact in two-hybrid and far Western assays (37)
and, as shown here, dissociate in parallel from Clf1 In contrast to what was recently reported for related
Cef1p/Cdc5p RNP complexes (21), we find that the Clf1-RNP is
RNase-sensitive, since all detectable core snRNP proteins are released
by this treatment. Many factors do remain bound to Clf1p after RNase
digestion, however, showing that the Sm and Lsm proteins do not provide
extensive stabilizing contacts. In addition to the majority of proteins present in Cef1p/Cdc5p RNP complexes, we observe the Prp16p and Prp28p DExD/H-box proteins, Sme1p, and the Lsm2p core snRNP protein in
the Clf1p-RNP. The relative Clf1p-NTC/Clf1p-RNP abundance is highly
reproducible in S. cerevisiae, and we have observed nothing to indicate that the Clf1p-RNP complex can be dissociated into the
RNA-free state by simple manipulation of salt or temperature. Curiously, no equivalent to the RNA-free NTC was found in fission yeast
Cdc5p complexes (39). Given the conservation of the spliceosome assembly pathway, we think it unlikely that Clf1p-NTC exists in budding
yeast but not in fission yeast. Rather, the steady state abundance of
this RNA-free NTC complex may be lower in the fission yeast, or Cdc5p
may preferentially associate with the RNP form of this complex in
S. pombe.
Pre-mRNA, splicing intermediates, and excised intron co-precipitate
with Clf1-TAP, supporting genetic and biochemical studies that suggest
Clf1p-associated proteins act from the earliest stages of spliceosome
assembly (e.g. Mud2p and Prp40p) through the final step in
splicing and product release (e.g. Prp16p, Prp17p, and Prp22p) (see Refs. 10, 15-18, and 38 and references within). Cheng and
co-workers (20) report that Prp19p binds spliceosomes with or soon
after the release of U4 snRNA. This conclusion appears inconsistent
with the observations that Prp19p requires Clf1p to bind the
spliceosome and that the removal of Clf1p activity impairs stable
association of the U4/U5.U6 tri-snRNP particle. A possible trivial
explanation for this discrepancy is that Prp19p is present in earlier
complexes but, similar to what was found with the Clf1p-RNP, is not
antibody-accessible. We do find, however, that whereas assembly is
arrested at the prespliceosome stage with RP51A
pre-mRNA, certain other pre-mRNAs, including the
rp51 Abelson and colleagues (42) have reported the isolation from yeast of a
complex containing all five snRNAs. The "penta-snRNP" protein
constitution is evocative of a splicing complex prior to substrate
association. The A (prespliceosome), B (spliceosome), and C (splicing
intermediate bearing) splicing complexes characterized in mammalian
extracts have likewise been analyzed (48-50). Whereas differences in
sample origin and data base completeness complicate the direct
comparison of these structures, the reported subunit compositions are
generally consistent with the proposed time of complex function. For
instance, early acting proteins, such as U1 snRNP factors and many
U4/U6.U5 proteins, are present in the yeast penta-snRNP and in the A/B
complexes but reduced or absent in the complex C and the Clf1p-RNP
structures. Late acting factors, such as the second step proteins
Slu7p, Prp16p, and Prp17p and the mRNA release protein, Prp22p, are
absent from the earlier complexes but found in Clf1p RNP complex C
and/or mammalian C complexes.
The protein and snRNA composition of the Clf1p-RNP is consistent with
this complex being an endogenous late stage or postcatalytic spliceosome. The inhibition of pre-mRNA splicing by temperature inactivation of several early acting splicing factors blocks the formation of the Clf1p-RNP, consistent with a
"splicing-dependent" origin2
and arguing against this structure
arising as an artifact of protein isolation. If truly a late stage
spliceosome, transition from complex C to the Clf1p-RNP would be
accompanied minimally by the release of the cap binding proteins and
the remaining U4/U6.U5-specific proteins. Whereas substrate RNAs would
probably be in low abundance or missing altogether, it might be
possible to trap such molecules through the use of mutants defective in
mRNA or intron release (51, 52). What of the Clf1p-NTC? With the
possible exception of Ccf8p, all Clf1p-NTC factors are present in the
more complex Clf1p-RNP state. Given this situation, the Clf1p-NTC
probably represents either a precursor of the Clf1p-RNP or a splicing
complex at the terminal stage of dissociation. Whereas a careful
kinetic analysis of complex assembly is needed to distinguish between these alternative origins of the Clf1p-NTC, some support for the latter
model is provided by the presence of the stable snRNP proteins Brr2p
and Prp8p in the snRNA-free Clf1p-NTC structure.
2-TAP) destabilizes Clf1p complexes
assembled in vivo, causing the release of the Cef1p and
Prp19p NTC factors and decreased association of the Rse1p, Snu114p, and
Hsh155p snRNP proteins. In vitro, temperature inactivation
of Clf1
2p impairs the prespliceosome to spliceosome transition and prevents Prp19p recruitment to the splicing complex. These and related data support the view that the poly-TPR Clf1p splicing factor promotes the functional integration of the U4/U6.U5 tri-snRNP particle into the U1-, U2-dependent prespliceosome.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 plasmid
constructs were previously published (10).
Ycplac22-clf1
1 was prepared similarly by inverse PCR with
oligonucleotides
1-1 and
1-2 (Table
I). The TAP epitope was amplified from
plasmid pBS1479 (22) with oligonucleotides TAP1 and TAP2 and blunt
end-ligated into a SnaBI site created by PCR mutagenesis
(with oligonucleotides SNA1 and SNA2) at the 3'-end of the
CLF1 coding sequence. The Ycplac22-CLF1-TAP and
deletion constructs were transformed into SY101 yeast (MATa,
ura3-53, lys2-801a, ade2-101, trp1
1, his3-
200, leu2-
,
clf1::HIS3,
pSY1[GAL1::clf1(679), URA3], selected
on galactose-based complete medium without tryptophan (23).
Subsequent assays for mutant gene activity were done on YPD
medium made with 2% glucose or on complete medium with 1 µg/ml 5-fluoroorotic acid (24). Haploid stains deleted for
ISY1, SYF2, ECM2, and NTC20
were obtained from the ATCC (Manassas, VA). CLF1-TAP was
transformed into this mutant set as a XbaI-SphI
DNA fragment cloned into the URA3-marked plasmid, Ycplac33
(25).
Oligonucleotides used in this study
2
(rp51
2) (29) with nucleotide triphosphates including [32P]UTP or biotin 16-UTP (Roche Molecular Biochemicals)
and a 5-fold molar excess of cap analog (New England Biolabs).
Streptavidin-agarose affinity purification of spliceosomes and snRNA
analyses were performed as described (30) scaled down to an 80-µl
splicing reaction. Spliceosome assembly was assayed by native gel
electrophoresis, and pre-mRNA splicing was assayed by denaturing
gel electrophoresis as described (31). Clf1
2-TAP extracts
were temperature-inactivated by incubation at 37 °C for 5 min or at
34 °C for 30 min and used without additional manipulation.
Complementation experiments were performed by incubation of 50-100,000
cpm of labeled pre-mRNA substrate with the temperature-inactivated
extract for 5 min under splicing conditions in a volume of 8.5 µl
followed by the addition of 1.5 µl of Clf1p complex (approximately 3 ng of protein) together with a 50-fold molar excess of unlabeled
pre-mRNA.
1-TAP, clf1
2-TAP, and Prp46-TAP
Purifications--
To isolate preparative quantities of the Clf1-TAP
complex, 50 liters of yeast were harvested by centrifugation at an
A600 of 2-3. The cell pellets were washed twice
with 2 liters of sterile water and resuspended in 500 ml of buffer A
(10 mM Hepes, pH 7.9, 10 mM KCl, 200 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM
benzamidine, 0.5% Nonidet P-40). The yeasts were frozen in liquid
nitrogen and then broken in a Waring blender as described (32). The
lysate was cleared by centrifugation at 20,500 rpm in a Beckman Ti45
rotor for 30 min followed by 84 min at 33,500 rpm in a Beckman Ti45
rotor. The cleared lysate was then dialyzed against buffer D (10 mM Hepes, pH 7.9, 50 mM KCl, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 20% glycerol) for 3 h. The extract
was adjusted to 200 mM NaCl, 0.1% Nonidet P-40 immediately
prior to TAP affinity purification, performed as described (22), except
that 2 ml of rabbit IgG agarose (Sigma) was used for 500 ml of lysate.
Analytical scale preparation of 35S-labeled TAP complexes
was performed by adding 1 mCi of Trans-35S (ICN) to the
equivalent of 10 ml of culture at an A600 of 1.0 for 4-5 h at 30 °C as described (33). For temperature shifts, the
clf1
1-TAP and CLF1-TAP cultures were incubated
to 37 °C for the final 1 h of labeling. The cell pellets were
collected by centrifugation and washed once with water, and proteins
were extracted by vortexing the pellet for 4 min with glass beads in
buffer A. TAP purification was then performed as described (22), scaled down for use with the reduced volumes. Where indicated, NaCl was increased to 300 or 450 mM before cell breakage.
RNase-treated samples were incubated at room temperature for 15 min
with 22 µg/ml RNase A and 444 units/ml of RNase T1 (Ambion) prior to
TAP purification. The recovered protein samples were precipitated with
6% trichloroacetic acid, resolved on a 5-10% gradient (or 8%
nongradient) SDS-polyacrylamide gel with BenchMarkTM
molecular weight markers (Invitrogen), and visualized with a Typhoon
PhosphorImager (Amersham Biosciences). Alternatively, the
samples were fractionated on a 15-40% glycerol gradient prepared in
50 mM Tris-HCl, pH 7.4, 20 (or 200) mM NaCl,
and 5 mM MgCl2 prior to protein and snRNA analyses.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Clf1p-bearing protein complexes.
A, proteins from 35S-labeled yeast cultures were
purified by TAP selection and resolved by PAGE. Clf1-HA does not bind
the TAP affinity resins; consequently, proteins found in this lane
represent nonspecific background. Proteins enriched by affinity
selection are labeled Ccf (for Clf1p
complex factors). The numbers at the
left indicate the masses in kDa of protein standards.
B, affinity-purified Clf1p complex fractionated on a
15-40% glycerol gradient. Ccf proteins not clearly resolved in the
unfractionated sample (T) are shown on the right.
Bands corresponding to Cef1p (Ccf7p) and Syf2p (Ccf17p) label
poorly and were not observed in this experiment. The fractions
containing nonspecific (NS) proteins, the Clf1p-NTC, and the
Clf1p-RNP fractions enriched in U5 and U6 proteins (Clf1p-(U2),
U5, U6) and U2, U5, and U6 proteins (Clf1p-U2, U5, U6)
are indicated below. Where two or more proteins are believed
to co-migrate only a single protein is labeled, and this is marked with
an asterisk (although it is possible that not all proteins
are present in each gradient fraction). The sedimentation coefficients
presented correspond to the U6 snRNP (7 S), thyroglobulin (19 S), the
U4/U6.U5 tri-snRNP particle (25 S), and the 40 and 80 S ribosomal
particles. The migration of proteins in the input sample (T)
is somewhat distorted by a gel artifact relative to fraction 1. C, Western blot of total cellular protein resolved on a
15-40% glycerol gradient and probed with IgG to identify Clf1-TAP.
D, Northern analysis of snRNA from whole cell lysate
resolved on a 15-40% glycerol gradient. Lane T,
total, unfractionated RNA. The peak positions of the previously
identified snRNP complexes are indicated below.
Ccf protein identities
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Fig. 2.
Spliceosomal snRNAs co-purify with
Clf1p. A, the TAP-specific Ccf bands are shown with
proteins released from the complex by RNase treatment indicated by
solid bars at the right. Where
multiple proteins co-migrate, only one is labeled and indicated by an
asterisk. B, complexes isolated by TAP
purification from Clf1-TAP yeast and a Clf1-HA negative control strain
were probed for the U1, U2, U4, U5, and U6 spliceosomal snRNAs.
Total, total unfractionated extract; Unbound, RNA
remaining in the supernatant after immune precipitation;
Bound, RNA present in the immune pellet. Approximately 2%
of the total and unbound samples were loaded relative to the immune
pellet. C, Western blot of Clf1-TAP present in cell extract
(Total), recovered by tandem affinity purification
(Bound), and remaining in the supernatant
(Unbound) resolved on an 8% polyacrylamide/SDS gel.
D, Clf1-TAP whole cell extract was fractionated on a
15-40% glycerol gradient, and alternate fractions were assayed for
snRNA content after immune precipitation with IgG. RNA from the total
unfractionated extract (T) is compared with the immune
pellets numbered from the top of the gradient tube. E, mouse
IgG was used to immune precipitate Clf1-TAP from alternative gradient
fractions. The immune pellets were then probed with the anti-Prp19p
polyclonal antibody that binds the TAP epitope of Clf1-TAP as well as
Prp19p. F, glycerol gradient fractions 1, 4, 7, 10, and 17 were immune precipitated with the -Prp19p antibody and assayed by
Western blot for Prp19p. Affinity-purified Clf1-TAP complex is
presented in the first lane (Complex) for comparison.
Ab, the
-Prp19p antibody.
1-TAP, and
clf1
2-TAP cultures were assayed by Western blot before
and after shift to the restrictive temperature. Clf1-TAP and the
deletion derivatives co-migrate on this minigel system and are found at
similar levels (Fig. 3A). In
addition to the full-length proteins, a protein ~20 kDa smaller is
present in each sample but absent in an untagged extract (Fig. 3A and data not shown). The levels of this putative decay
intermediate vary between extract preparations but are reproducibly
greater with the TPR deletion strains. Nevertheless, as the full-length Clf1p (or TPR deletion derivative) levels decrease only slightly with
temperature shift, it is unlikely that splicing inhibition observed in
the clf1
1-TAP or clf1
2-TAP cultures results
from lower Clf1p abundance. Furthermore, clf1
1-TAP and
clf1
2-TAP are recessive mutations and do not act as
dominant negative inhibitors of splicing. By this measure, it appears
that the N-terminal deletion derivatives of Clf1p are stable but less
active in splicing.
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Fig. 3.
N-terminal TPR deletions
destabilize Clf1p complexes. A, Western analysis of
yeast proteins from CLF1-TAP, clf1 1-TAP, and
clf1
2-TAP cultures grown continuously at the permissive
temperature (30 °C) and after a 2-h shift to the restrictive
temperature (37 °C). The positions of the putative N-terminal
degradation products are indicated with an asterisk.
B, 35S-labeled Clf1p complexes were isolated in
200, 300, and 450 mM NaCl and resolved on a 5-10%
polyacrylamide/SDS gel. Proteins that show enhanced salt sensitivity
due to the Clf1
2-TAP mutation are indicated by bars at
the right. The asterisks indicate where two or
more Ccf bands co-migrate. The overall darker band intensities in the
lower salt Clf1
2-TAP cultures reflect minor experimental variation
in the labeling efficiency. C, 35S-labeled
proteins from Clf1-TAP and Clf1
1-TAP complexes isolated from
cultures shifted to 37 °C for 1 h prior to harvest. The
positions of the Clf1
1p and the Clf1
1p degradation
products are indicated on the right, and proteins that show
enhanced temperature sensitivity with the Clf1
1-TAP mutation are
indicated by an asterisk. D,
35S-labeled proteins from a wild type Prp46-TAP culture
isolated at 200, 300, and 450 mM NaCl. The positions of
Prp46p, the temperature-sensitive proteins, and the stably bound but
co-migrating Clf1p/Ccf8p proteins are indicated at the
right.
2-TAP complex, the Cef1p
and Prp19p proteins are reproducibly more sensitive to dissociation at
300 and 450 mM NaCl. Since Cef1p co-migrates with Clf1p,
Cef1p release was monitored with complexes isolated using a Prp46-TAP
construct (Fig. 3D). The pattern of recovered proteins is
nearly identical with Clf1-TAP and Prp46-TAP, but when Prp46-TAP is
used Clf1p and Cef1p are well resolved. Cef1p remains stably bound at
450 mM NaCl in the wild type complex but fully dissociates
from the Clf1
2-TAP complex at 300 mM NaCl.
2-TAP
complex (Fig. 3B). Rse1p, Hsh155p, Cef1p, Prp19p, and to a
lesser degree Snu114p are also more readily dissociated from the
Clf1
1-TAP complex isolated from yeast shifted to the restrictive
temperature of 37 °C prior to harvest (Fig. 3C).
Therefore, the splicing defects resulting from the N-terminal TPR
deletions are correlated with the selective loss of both snRNP and
non-snRNP components from Clf1p complexes. Importantly, all of the
Clf1
1-TAP/Clf1
2-TAP-sensitive proteins are essential; the failure
to properly assimilate any one into the spliceosome would probably
block pre-mRNA splicing.
2-TAP Inactivation Inhibits the Productive Addition of the
U4/U6.U5 to the Prespliceosome--
To investigate the
nature of the Clf1
2-TAP splicing defect, extracts were prepared from
Clf1
2-TAP yeast and assayed for spliceosome assembly and splicing
in vitro. Extract prepared at the permissive temperature
supports spliceosome assembly on RP51A pre-mRNA through
the previously described prespliceosome (complex III), precatalytic spliceosome (complex
I), and spliceosome (complex II)
states (31) (Fig. 4A). In
contrast, when this extract is briefly preincubated at the restrictive
temperature, spliceosome assembly stops at the prespliceosome stage. As
expected, the untreated sample supports pre-mRNA splicing (Fig.
4B), whereas the temperature-inactivated sample does not
(lanes 1 and 2). Splicing is restored
by the addition of purified Clf1p complex in the presence of saturating
amounts of unlabeled pre-mRNA substrate. This amount of cold
competitor blocks de novo assembly on labeled pre-mRNA
(lane 5). Consequently, the splicing complexes
formed in the Clf1
2-TAP inactive extract, while incomplete, are
functional and can be chased through the splicing pathway. The Clf1p
complex itself does not support pre-mRNA splicing (lane
4), and the inactivation conditions do not impair splicing
or spliceosome assembly in wild type extracts (e.g. Refs. 30
and 40; data not shown). Accordingly, conditions that destabilize the
Clf1
2-TAP complex structure inhibit the productive recruitment of
the U4/U6.U5 tri-snRNP particle to the prespliceosome.
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Fig. 4.
Temperature inactivation of
Clf1 2-TAP blocks splicing and impairs
spliceosome assembly. A, time course of spliceosome
assembly on 32P-labeled RP51A pre-mRNA
incubated in Clf1
2-TAP extract maintained at 23 °C (
) or
shifted to 37 °C (+) for 5 min prior to substrate addition. The
bands corresponding to the prespliceosome (III), the snRNP
complete spliceosome (I), and the spliceosome after U4 snRNA
dissociation (II) are indicated at the left.
B, assay of RP51A pre-mRNA splicing in the
Clf1
2-TAP extract before heat inactivation (lane
1) and after heat inactivation in the presence
(lane 3) and absence (lane
2) of purified Clf1p complex. Complementation was performed
by preincubating the labeled pre-mRNA with the heat-denatured
extract for 5 min followed by the addition of cold RNA. Lane
4 contains purified complex incubated with pre-mRNA
under standard splicing conditions. In lane 5,
the labeled and unlabeled RNAs were premixed and added directly to the
Clf1
2-TAP extract. The positions of the lariat intermediate
(LI), excised intron (I), pre-mRNA
(P), mRNA (M), and released 5' exon
(5'E) are indicated. In order to resolve the very
small 5' exon RNA, the samples were run a second time on a higher
percentage polyacrylamide gel, imaged, and inserted below
the solid bar at the bottom.
2-TAP complex and impairs spliceosome
formation. It was not clear, however, whether proteins released from
the Clf1
2-TAP complex could be independently bound by the splicing complex. To address this, splicing complexes were assembled in vitro with or without active Clf1p and then assayed for the
presence of the essential Prp19p maturation factor. Biotin-substituted RP51A pre-mRNA was incubated in extract for 30 min under
splicing conditions, and then the assembled complexes were recovered by streptavidin chromatography. Bands corresponding to Clf1-TAP and Prp19p
were observed in complexes assembled with the splicing-competent Clf1-TAP extract (Fig. 5A,
lanes 1 and 2). The spliceosome
association of Prp19p requires Clf1p function, however, since metabolic
depletion of Clf1p by transcriptional repression of the
GAL1::clf1(697) fusion gene
(lane 5; see "Experimental Procedures")
blocks Prp19p recovery. Unfortunately, a nonspecific protein enriched
on the streptavidin matrix co-migrates with Clf1-TAP and is bound by the Prp19p antibody. This band is prominent in all samples including controls where the pre-mRNA lacks biotin (lane
4) and where no TAP-tagged protein is present
(lane 5). To circumvent this problem, Clf1p
association with the spliceosome was tested by probing the recovered
protein with a TAP-specific antibody (lanes
11-13) and through the use of the Clf1
2-TAP derivative,
which migrates ahead of the background band (lanes
7-10). Similar to what was observed with the Clf1p-depleted
samples, temperature inactivation of Clf1
2-TAP blocks Prp19p
association. Equivalent results were observed with an alternate
splicing-competent splicing substrate, rp51
2
(lanes 3, 6, 8, and
10; see "Discussion"). Curiously, under conditions where
Prp19p recruitment to the spliceosome is blocked, Clf1
2-TAP still
associates with the splicing complex (compare lanes
7 and 8 with lanes 9 and
10).
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Fig. 5.
Temperature inactivation of
Clf1 2-TAP activity blocks Prp19p recruitment
to the spliceosome but not its own association. A,
Western blot of immune precipitated TAP complex (lanes
1 and 11) and spliceosomes recovered with
biotin-labeled pre-mRNA (lanes 2,
3, 5-10, 12, and 13) or
with a no-biotin control substrate (lane 4).
Splicing complexes were assembled in wild type extract
(lanes 2-4 and 12), extract
metabolically depleted of Clf1p (lanes 5,
6, and 13), and Clf1
2-TAP extract before
(lanes 7 and 8) and after
(lanes 9 and 10) temperature
inactivation. NS, a nonspecific band enriched by
streptavidin chromatography and bound by the anti-Prp19p antibody.
Samples present in lanes 1, 2, and
5 were rerun in lanes 11-13 and
probed with mouse protein A followed by an alkaline
phosphatase-conjugated goat anti-mouse antibody that does not detect
the background band. B, in vitro splicing
reactions were assembled before or after temperature shift (23 and
37 °C, respectively) on radiolabeled RP51A pre-mRNA
and then immune precipitated with mouse IgG agarose. This antibody
binds the TAP epitope present in samples 1-8 but missing in the
untagged control (lanes 9 and 10).
T, total unfractionated RNA; IP, immune pellet;
LI, lariat intermediate; I, circular excised
intron; P, pre-mRNA; M, mRNA;
5'E, upstream exon. The asterisk marks
the position of the linear excised intron that can be seen in several
lanes.
2-TAP extract when maintained at 23 °C (albeit at lower
levels). Heat inactivation of Clf1
2-TAP prevents splicing but, as
noted above, does prevent this protein from associating with splicing
complexes. Consistent with this, the amount of RP51A
pre-mRNA present in the heat-treated Clf1
2-TAP complex
(lane 8) is well above the background
(lane 10) and equal to or greater than that found
in the splicing competent extracts (lanes 2,
4, and 6). Together these data show that the
Clf1p is a stable constituent of the spliceosome and demonstrate that
the N-terminal TPR domain of Clf1p is required for recruitment of the
essential Prp19p splicing factor to the yeast splicing apparatus.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-TAP or
Clf1
2-TAP inactivated complexes, Cef1p is a prime candidate for a
protein that tethers Prp19p to Clf1-NTC.
2 deletion derivative used here assemble
snRNP-complete but catalytically inactive splicing complexes in the
absence of Clf1p. This shows that the prespliceosome and U4/U5.U6
tri-snRNP can interact, albeit nonproductively, in the absence of
Clf1p. And whereas the molecular basis of pre-mRNA substrate
discrimination is unknown, the rp51
2 arrest point shows
that Clf1p is also needed after the stable addition of the U4/U6.U5
tri-snRNP addition, a time when Prp19p is proposed to function. We note
that Prp19p fails to stably bind to the spliceosome when Clf1
2-TAP
is inactivated even with substrates that permit spliceosome formation,
for example, rp51
2 (see Fig. 5A).
Consequently, the rp51
2 arrest point reflects, at least in part, a defect in Prp19p recruitment.
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ACKNOWLEDGEMENTS |
---|
We thank Soo-Chen Cheng, Beate Schwer, and Bertrand Seraphin for providing various yeast strains and antibodies used in this study. We thank Martha Peterson and William Pierce for valuable comments on this manuscript and preliminary characterization of the Clf1p complex by mass spectroscopy, respectively.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM42476 (to B. C. R.) and by proteomic support through National Science Foundation Grant EPS-0132295 (to B. L.).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.
¶ To whom correspondence should be addressed. Tel.: 859-257-5530; Fax: 859-257-1717; E-mail: rymond@uky.edu.
Published, JBC Papers in Press, December 31, 2002, DOI 10.1074/jbc.M210839200
2 Q. Wang and B. C. Rymond, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: snRNA, small nuclear RNA; RNP, ribonucleoprotein; snRNP, small nuclear RNP; TPR, tetratricopeptide repeat; NTC, nineteen complex; HA, hemagglutinin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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