MOLECULAR AND CELLULAR BIOLOGY, Feb. 2003, p. 1368–1378
0270-7306/03/$08.00?0 DOI: 10.1128/MCB.23.4.1368–1378.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 23, No. 4
Dual Roles for Spt5 in Pre-mRNA Processing and Transcription
Elongation Revealed by Identification of
D. L. Lindstrom,1S. L. Squazzo,1N. Muster,2T. A. Burckin,1K. C. Wachter,1†
C. A. Emigh,1J. A. McCleery,1J. R. Yates III,2and G. A. Hartzog1*
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064,1
and Department of Cell Biology, Scripps Research Institute, La Jolla, California 921302
Received 16 September 2002/Returned for modification 22 October 2002/Accepted 14 November 2002
During transcription elongation, eukaryotic RNA polymerase II (Pol II) must contend with the barrier
presented by nucleosomes. The conserved Spt4-Spt5 complex has been proposed to regulate elongation through
nucleosomes by Pol II. To help define the mechanism of Spt5 function, we have characterized proteins that
coimmunopurify with Spt5. Among these are the general elongation factors TFIIF and TFIIS as well as Spt6
and FACT, factors thought to regulate elongation through nucleosomes. Spt5 also coimmunopurified with the
mRNA capping enzyme and cap methyltransferase, and spt4 and spt5 mutations displayed genetic interactions
with mutations in capping enzyme genes. Additionally, we found that spt4 and spt5 mutations lead to accu-
mulation of unspliced pre-mRNA. Spt5 also copurified with several previously unstudied proteins; we dem-
onstrate that one of these is encoded by a new member of the SPT gene family. Finally, by immunoprecipitating
these factors we found evidence that Spt5 participates in at least three Pol II complexes. These observations
provide new evidence of roles for Spt4-Spt5 in pre-mRNA processing and transcription elongation.
Synthesis of mRNA is a multistep process of transcription
and pre-mRNA processing. The study of mRNA synthesis has
proceeded largely through a reductionist approach, with
mRNA production viewed as a series of reactions connected by
their substrates and products. It is becoming clear that many
RNA-processing reactions occur during transcription elonga-
tion (reviewed in references 34 and 45).
However, our understanding of the elongation phase of tran-
scription is incomplete. In vitro, two general transcription elon-
gation factors, TFIIF and TFIIS, are sufficient to stimulate in
vivo rates of elongation on naked DNA templates (25). In
contrast, elongation on nucleosome-bound templates is ineffi-
cient, even in the presence of TFIIF and TFIIS, suggesting a
requirement for other factors (9, 25, 26). Several factors have
been implicated in the regulation of transcription elongation
through chromatin. Among these is the yeast Spt4-Spt5 com-
plex, known as DSIF in human cells (21, 57). DSIF/Spt4-Spt5
can inhibit and promote elongation of RNA polymerase II (Pol
II) on cellular genes and is required for the stimulation of
transcription elongation by human immunodeficiency virus
type 1 Tat in vitro (24, 28, 57, 64). A second elongation factor,
Spt6, interacts genetically with SPT4, SPT5, and TFIIS and
also promotes Tat function in vitro (21, 51, 64). Consistent with
their playing a role in elongation, chromatin immunoprecipi-
tation experiments show that the Spt5 and Spt6 proteins asso-
ciate with transcribed genes in yeast and Drosophila (2, 27, 44).
Finally, genetic and biochemical studies of Spt4, Spt5, and Spt6
in yeast have led to the proposal that they function by affecting
chromatin structure (6, 51). A third protein complex, FACT,
composed of the human Spt16 and SSRP1 proteins, promotes
elongation by Pol II through nucleosomes in vitro (40, 41). Its
yeast homolog, the CP (or SPN) complex, is composed of two
tightly associated subunits, Pob3 and Cdc68/Spt16 (the name
Cdc68 will be used here to avoid confusion of Spt6 with Spt16),
as well as a weakly associated HMG box protein Nhp6 (7, 8,
18). Mutations in SPT4, SPT5, SPT6, CDC68, POB3, and
NHP6 lead to similar mutant phenotypes, and these genes also
display numerous genetic interactions with each other (re-
viewed in references 22 and 60). Thus, although direct evi-
dence is lacking, the overlapping genetic and biochemical be-
haviors of these Spt proteins suggest that they may collaborate
to carry out a common or overlapping set of functions in vivo.
Recent observations suggest a functional interplay between
Spt4-Spt5 and the C-terminal heptapeptide repeats (CTD) of
Pol II. The CTD serves as a scaffold for factors involved in
transcription and processing. For example, the mRNA capping
enzyme, polyadenylation factors, and certain splicing proteins
all associate with the CTD of transcribing RNA Pol II. Fur-
thermore, perturbation of the CTD or addition of CTD pep-
tides affects splicing in vitro and in vivo, suggesting that the
CTD may affect the efficiency of processing reactions (re-
viewed in references 34 and 45). Biochemical studies show that
P-TEFb, a CTD kinase that regulates elongation, works in
conjunction with DSIF and possibly FACT (56, 58). In addi-
tion, we have recently shown that SPT4 and SPT5 display an
extensive set of genetic interactions with the CTD and enzymes
that modify the CTD’s phosphorylation status, including pro-
tein kinases similar to P-TEFb (31, 39). Finally, the human and
Schizosaccharomyces pombe Spt5 proteins interact with the
capping enzyme (43, 59). These studies show that Spt4-Spt5 is
* Corresponding author. Mailing address: Department of Molecu-
lar, Cell, and Developmental Biology, University of California, Santa
Cruz, CA 95064. Phone: (831) 459-5826. Fax: (831) 459-3139. E-mail:
† Present address: Molecular and Cellular Biology Program, Uni-
versity of Washington, Seattle, WA 98195.
a candidate for an elongation regulator that mediates interac-
tions between the elongating polymerase and processing events
linked to the CTD.
A mechanistic understanding of Spt4-Spt5 function requires
a knowledge of the proteins that associate with this complex.
Here we describe affinity purification of Spt5 from yeast ex-
tracts. Using mass spectrometry, we identified a large number
of proteins that copurified with Spt5. Many of these interac-
tions were subsequently verified by coimmunoprecipitation
and genetic analysis. We show that Spt5 associates with Pol II
and the general elongation factors TFIIF and TFIIS, as well as
with Spt6, Cdc68, and Pob3. Furthermore, we demonstrate
that Spt5 coimmunopurifies with the yeast capping enzyme and
cap methyltransferase and that spt4 and spt5 mutations cause
splicing defects in yeast. In addition, we show that Spt5 copu-
rifies and genetically interacts with a recently identified Spt6-
interacting protein, Iws1. Through extensive coimmunopre-
cipitation analyses we provide evidence that Spt5 participates
in at least three different protein complexes with Pol II. These
observations provide new evidence of close connections be-
tween pre-mRNA processing and transcription elongation and
suggest important roles for Spt4-Spt5 in both processes.
MATERIALS AND METHODS
Media and genetic methods. Strain construction and other genetic manipula-
tions were carried out by standard methods (46). Yeast media were made as
described previously (46). All GHY and FY Saccharomyces cerevisiae strains
used in this study (Table 1) are isogenic to S288C (61). C-terminal tagging of
Tfg1, Fcp1, and Iws1 with 13 copies of the Myc epitope was performed by a
PCR-based method (33). The dilution spotting growth assay (Fig. 4) was carried
out as described previously (31), and the results presented are representative of
those obtained for multiple transformants in at least two separate transforma-
tions. IWS1 was mutagenized by PCR amplifying the IWS1-MYC::TRP1 allele
from genomic DNA by using primers OGH268 (5?-CAGCGGCCGCCCAAAT
GCCAGATCATTG) and OGH269 (5?-GTGCGGCCGCTGGGTATCGAATC
CAAGC). This PCR product, composed of the Iws1 open reading frame fused to
the Myc tag, followed by TRP1 and then by sequences 3? to IWS1, was trans-
formed into strain FY119, and Trp?transformants were screened for mutant
phenotypes. Integration of the PCR product at the IWS1 locus was confirmed by
genetic linkage analysis and plasmid complementation. Strains carrying Spt4
tagged at its C terminus with three copies of the Flag epitope were derived from
a strain provided by Steve Hahn.
Plasmids. The CET1 plasmids pRS316-CET1 and pRS313-CET1 and deriva-
tives of pRS313-CET1 carrying cet1 mutations (52) and the pRS315-based plas-
mids pSB995 (HA-Abd1) and pSB996 (HA-Ceg1) (29) have been described
previously. Plasmid pYST138 was used to create the TUB2 probe for Northern
blot analysis (50). Plasmid pGH193, used to create the HIS4 probe for Northern
blot analysis, is a Bluescript derivative containing the HIS4 open reading frame.
TABLE 1. Strains
MATa his4-912? lys2-128? leu2?1 ura3-52 trp1?63 spt5-194
MAT? his4-912? lys2-128? leu2?1 ura3-52 spt5-242
MAT? his3?200 lys2-128? leu2?1 ura3-52 trp1?63 spt4-3
MAT? his4-912? lys2-128? leu2?1 spt5-194
MATa his4-912? lys2-128? leu2?1 spt4?2::HIS3
MAT ? his3?200 lys2-128? ura3-52 spt5-194
MATa his4-912? lys2-128? leu2?1 HA-SPT6
MAT? his4-912? lys2-128? leu2?1 trp1?63 SPT5-MYC
MAT? his4-912? lys2-128? leu2?1 SPT5-FLAG
MAT? his4-912? lys2-128? trp1?63 spt5-4
MAT? his4-912? lys2-128? leu2?1 ura3-52 trp1?63 iws1-7-MYC::TRP1
MAT? his4-912? lys2-128? leu2?1 ura3-52 trp1?63 iws1-13-MYC::TRP1
MAT? his4-912? lys2-128? leu2?1 SPT5-FLAG trp1?63 iws1-13-MYC::TRP1
MAT? his4-912? lys2-128? leu2?1 ura3-52 trp1?63 SPT5-FLAG FCP1-MYC::TRP1
MAT? his4-912? lys2-128? leu2?1 ura3-52 trp1?63 SPT5-FLAG TFG1-MYC::TRP1
MAT? his4-912? lys2-128? leu2?1 SPT5-FLAG HA-SPT6
MAT? his4-912? lys2-128? leu2?1 trp1?63 SPT5-FLAG HA-SPT6 IWS1-MYC::TRP1
MATa his4-912? lys2-128? leu2?1 ura3-52 trp1?63 HA-SPT6 IWS1-MYC::TRP1
MATa lys2(-128? or ?0) leu2(?0 or ?1) ura3(?0 or -52) trp1?63 HA-SPT6
MAT? his4-912? lys2(-128? or ?0) leu2(?0 or ?1) ura3(?0 or -52) trp1?63
MATa his3-11 can1-100 trp1-1 leu2-3,112 ura3-52 ade2-1 bar1 Gal?
MAT? his4-912? lys2-128? leu2?1 ura3-52 trp1?63
MATa his4-912? lys2-128? leu2?1 ura3-52
MATa his3?200 lys2-128? leu2?1 ura3-52 trp1?63
MATa ura3-52 ade2-101 his3 his7 prp4-1
MATa his4-912? lys2-128? spt5-4
MATa his3 lys2-128? ura3 ceg1-250
MATa his3 lys2-128? ura3 ceg1-250 spt5-194
MATa his3 lys2-128? ura3 trp1?63 ceg1-250 spt4-3
MAT? ura3-52 leu2?1 trp1?63 abd1?::TRP1 [pRS316-ABD1]
MATa his3?200 leu2-3,112 ura3-52 ceg1?1::HIS3 [pRS316-CEG1]
MATa his3?200 lys2-128? leu2?1 ura3-52 trp1?63 spt4-3 cet1?1::TRP1 [pRS316-CET1]
MATa his3?200 lys2-128? leu2?1 ura3-52 trp1?63 spt5-242 cet1?1::TRP1 [pRS316-CET1]
MATa his3?200 lys2-128? leu2?1 ura3-52 trp1?63 spt5-194 cet1?1::TRP1 [pRS316-CET1]
MATa his3?200 lys2-128? leu2?1 ura3-52 trp1?63 cet1?1::TRP1 [pRS316-CET1]
MAT? his4-912? his3?200 leu2 ura3 trp1 can1 HA-SPT6 CDC68-MYC::KanMX
MAT? ade2 can1 his3 leu2 trp1 ura3 POB3-MYC::KanMX NHP6A-HA::URA3
MAT? ade2 can1 his3 leu2 trp1 ura3 CDC68-MYC::KanMX NHP6A-HA::URA3
Hartzog lab collection
Hartzog lab collection
Hartzog lab collection
Hartzog lab collection
Hartzog lab collection
Hartzog lab collection
Hartzog lab collection
Hartzog lab collection
Hartzog lab collection
Hartzog lab collection
VOL. 23, 2003ROLES FOR Spt5 IN ELONGATION AND PRE-mRNA PROCESSING1369
Immunoprecipitation and immunoblotting. Cells were grown to mid-log phase
in yeast extract-peptone-dextrose (YPD) unless otherwise noted, harvested, and
frozen in liquid nitrogen. Frozen cell pellets were ground into a fine powder
under liquid nitrogen with a mortar and pestle. Protein lysates were prepared as
previously described in lysis buffer (30 mM HEPES [pH 7.4], 200 mM potassium
acetate, 1 mM magnesium acetate, 1 mM EGTA, 0.05% Tween 20, 10% glyc-
erol) containing protease inhibitors (31). For immunoprecipitations with the
antihemagglutinin (anti-HA) polyclonal or anti-Myc 9E10 antibodies, 6 ?g of
purified immunoglobulin G was prebound to 12 ?l of protein A-agarose beads
(Bio-Rad) overnight at 4°C. The beads were washed three times with 0.5 ml of
lysis buffer, and immunoprecipitations were performed as previously described
(31). Proteins were eluted from the beads with lysis buffer containing 1.0 M
potassium acetate. Immunoblotting was performed as previously described (31).
Approximately 1% of the total crude extracts was immunoblotted for comparison
to eluates. The anti-Spt4 and anti-Spt5 polyclonal antibodies, the anti-Myc an-
tibody (9E10; Santa Cruz Biotechnology), and the anti-Pol II antibodies B3 and
8WG16 have been previously described (15, 21, 53). The affinity-purified anti-
HA rabbit polyclonal antibody was produced as previously described (37). The
anti-Spt6 antibody was a gift from Clyde Denis (14). The anti-TFIIS antibody was
a gift from Caroline Kane. The anti-Abd1 antibody was a gift from Steve Bura-
towski (52). The anti-Cdc68 and anti-Pob3 antibodies were gifts from Tim For-
mosa (63). The anti-Nhp6 antibody was a gift from David Stillman.
In contrast to the data presented here, we and others were previously unable
to detect robust Spt5-Spt6 interactions in yeast by coimmunoprecipitation (21,
51). We have found that Spt5 and Spt6 only weakly coimmunoprecipitate from
extracts of yeast cells prepared by bead beating, as was reported previously (21,
51). In contrast, Spt5 and Spt6 show a robust interaction when coimmunopre-
cipitated from extracts prepared by grinding in a mortar and pestle under liquid
nitrogen (see Fig. 3 and 4) (D. L. Lindstrom and G. A. Hartzog, unpublished
Gel filtration chromatography. Gel filtration chromatography was performed
on a Superose 6 column (Pharmacia) equilibrated in lysis buffer (described
above). For fractionation of Spt5-Flag complexes, Spt5-Flag immunoprecipitates
derived from ?10 mg of cleared lysate were injected onto the column, and 0.5-ml
fractions were collected as previously reported (49). Samples (250 ?l) of each
fraction were trichloroacetic acid precipitated, separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to ni-
trocellulose for immunoblotting.
Affinity purification of Spt5-Flag. Spt5-Flag was affinity purified by a modifi-
cation of a published method (54). Cleared whole-cell lysates (?350 mg) were
prepared from GHY617 (Spt5-Flag) or GHY611 (mock) as previously described
(31) and batch bound to 600 ?l of anti-Flag M2 agarose beads for 2 h at 4°C. The
supernatant was collected, and the beads were washed four times with 25 vol-
umes of lysis buffer. Bound proteins were eluted twice by addition of 1.5 ml of
lysis buffer supplemented with 500 ?g of Flag peptide (Research Genetics) per
ml. The beads were washed three times with 10 ml of lysis buffer, and the original
supernatant was reapplied to the beads for a second round of immunoprecipi-
tation. Eluates from both rounds of immunoprecipitation were pooled and batch
bound to 200 ?l of Bio-Rex 70 cation exchange resin (Bio-Rad) for 10 min at 4°C.
The resin was packed into a 1-ml syringe barrel and washed with 10 volumes of
lysis buffer. The column was eluted stepwise with 1.2 ml each of lysis buffer
containing 0.4 and 1.0 M potassium acetate.
Mass spectrometry. To prepare samples for mass spectrometry, Bio-Rex 70
eluates were mixed with 4 volumes of methanol, 1 volume of chloroform, and 3
volumes of water. The phases were separated by centrifugation, and the upper
phase was transferred and precipitated with 3 volumes of methanol. The precip-
itates were pelleted and dried under vacuum. Protein samples were subjected to
tryptic digestion and mass spectrometry as previously described (32).
Analysis of RNA. RNA isolation and primer extension were performed as
previously described (4), using 10 ?g of total RNA/sample. The sequence of the
primer used to detect U3A and U3B was 5?-CCAAGTTGGATTCAGTGGCTC.
Reverse transcription-PCR (RT-PCR) was performed as previously described
(13). One microgram of total RNA per 10-?l sample was used for reverse
transcription (see primer sequences below). The cDNA product was precipitated
and suspended in 10 ?l of H2O, and 1 ?l was used to seed a 20-?l PCR mixture
spiked with ?100 fmol32P end-labeled reverse primer. PCR conditions were as
follows: 94°C for 5 min followed by 20 cycles of 94°C for 1 min, annealing
temperature (see below) for 15 s, and 72°C for 1 min; the last cycle was followed
by a final incubation at 72°C for 10 min. RT-PCR primers and annealing tem-
peratures were as follows: for RPL26A, forward primer 5?-GGTAAGATTTGT
TGAAACTCG, reverse primer 5?-GCTTTTCTGTCCTTGTCCAAA, and a
54°C annealing temperature; for RPS27B, forward primer 5?-TGAAACGACTT
TCGTTTTCG, reverse primer 5?-CCTTACCACCGGTTGGAGTA, and a 50°C
annealing temperature; for RPS25A, forward primer 5?-CCCAAATTCTACTA
GAGTTCGG, reverse primer 5?-TAGCTTGCTTGGAGTGCTTG, and a 52°C
annealing temperature; and for SED1, forward primer 5?-AGAGGCTCCAAC
CACTGCTA, reverse primer 5?-ATAGCAACACCAGCCAAACC, and a 58°C
annealing temperature. PCR products were fractionated on 6% native polyacryl-
amide gels and visualized by autoradiography. Northern blot analysis was per-
formed as described previously (50). The amount of RNA in each lane was
normalized to TUB2.
Identification of proteins that copurify with Spt5. Previous
studies have shown that Spt4-Spt5 coimmunopurifies and ge-
netically interacts with Pol II and the Paf1 complex (38, 49). To
further characterize the interaction of Spt5 with Pol II, we
separated Spt5-Flag immunoprecipitates by gel filtration and
analyzed the eluates by immunoblotting (Fig. 1A). Consistent
with the idea that Spt5 assembles into high-molecular-weight
complexes with Pol IIAand Pol IIO, we observed a broad
distribution of high-molecular-weight forms of these proteins.
Similarly, Mueller and Jaehning have previously observed Pol
II and Spt5 cofractionating in high-molecular-weight com-
plexes with affinity-purified Paf1 (38).
To determine if proteins other than Pol II and the Paf1
complex associate with Spt5, we immunoprecipitated Spt5-
Flag from yeast extracts, competitively eluted it from anti-Flag
beads with the Flag peptide, and further fractionated it on a
Bio-Rex 70 column. Immunoblotting demonstrated that Spt5
bound to Bio-Rex 70 in buffer containing 0.2 M potassium
acetate, that a small amount of Spt5 eluted in 0.4 M potassium
acetate, and that the bulk of Spt5 was eluted by 1.0 M potas-
sium acetate (Fig. 1B, upper panel, and data not shown). Silver
staining showed that many proteins copurified with Spt5-Flag
(Fig. 1B, lower panel). To control for nonspecific binding to
the anti-Flag beads, this experiment was repeated with an
equal amount of an extract of a strain that lacked the Flag
epitope. We did not observe Spt5 by immunoblotting or sig-
nificant silver staining of other protein bands in these mock
purified samples (Fig. 1B).
To identify proteins that copurified with Spt5, the 1.0 M
eluates of the mock- and affinity-purified samples from the
Bio-Rex 70 column were subjected to direct analysis of large
protein complexes (DALPC) by mass spectrometry (32). In
DALPC, a purified sample of a protein complex is proteolysed
without prior separation of the constituent proteins, and the
resulting peptides are fractionated by high-pressure liquid
chromatography and analyzed on an electrospray-equipped tri-
ple-quadrupole mass spectrometer. By using the SEQUEST
program, acquired masses were correlated with peptide se-
quences predicted from the genomic sequence. This approach
has previously been used to identify subunits of the ribosome,
proteosome, and other protein complexes (32, 55).
Peptides from many proteins were found in the Spt5-Flag
fractions. To focus on those most likely to associate with Spt5
in vivo, we excluded known cytoplasmic proteins from our
analysis, since Spt5 is nuclear (50), and we also discarded heat
shock proteins, ribosomal proteins, and translation factors, as
these are often found as contaminants in proteomic studies
(23). Of the proteins that met these criteria, nine were also
found in the mock-purified fraction and were also excluded
from further analysis. The 92 proteins in 1.0 M Spt5-Flag
1370LINDSTROM ET AL.MOL. CELL. BIOL.
fraction that pass all of these criteria are presented in Table 2.
Recently, Ho et al. described the purification of a large set of
protein complexes by using Flag affinity chromatography (23).
In addition to ribosomal proteins, eight proteins that were
frequent contaminants in their protein complexes appeared in
our data set and are indicated in Table 2. Although some of the
proteins in Table 2 have not been studied and lack strong
homologs, we were able to group many by previously reported
functions or homology to proteins of known function, and
many of these are implicated in transcription.
Four potential protein complexes containing Spt4-Spt5 were
described in two recent large-scale proteomic studies of yeast
proteins (20, 23). Only one of these complexes is composed
predominantly of proteins implicated in transcription (complex
145 in reference 20). The composition of this complex was
derived from the combined results of tandem affinity purifica-
tions of Abd1 and several Pol II subunits, and 15 of the 19
proteins in the complex are represented in our data (Table 2).
No independent methods were used to confirm the composi-
tion of this complex. Spurious copurification of proteins is a
common concern in proteomic studies (23), and it is likely that
some of the proteins reported as Spt5 associated in Table 2 or
by Gavin et al. (20) do not specifically associate with Spt5-Flag.
The remainder of this report is devoted to the analysis of the
association of Spt5 with proteins reported in Table 2 that are
implicated in Pol II transcription and pre-mRNA processing.
Pol II and general transcription factors. The general tran-
scription elongation factor TFIIS and at least 10 of the 12 Pol
II subunits copurified with Spt5 (Table 2). This is consistent
with previous observations of Spt5-Pol II coimmunoprecipi-
tation and of genetic interactions of SPT4 and SPT5 with
DST1, RPO21, and RPB2, which encode TFIIS and the two
largest subunits of Pol II (21, 31, 58) (Fig. 1A and C). Because
DALPC is not quantitative, protein stoichiometry cannot be
determined (32). To examine the relationship between Spt5
and TFIIS, we probed blots of Spt5-Flag immunoprecipitates
and found that TFIIS had coprecipitated (Fig. 1C). However,
in contrast to Pol II, TFIIS did not cofractionate with Spt5 on
a gel filtration column (data not shown). Thus, TFIIS likely
associates with Spt5 in a transient or indirect manner.
All three subunits of transcription initiation and elongation
factor TFIIF copurified with Spt5 (Table 2). To confirm these
data, we immunoprecipitated a Myc epitope-tagged allele of
Tfg1 and observed that Spt5 specifically coimmunoprecipitated
with it (Fig. 1D). TFIIF interacts biochemically with the CTD
phosphatase Fcp1, and an fcp1 mutation interacts genetically
with spt4 and spt5 mutations (3, 31). Furthermore, human Fcp1
associates weakly with a complex that includes Spt5 (42).
Therefore, even though it was not found in our mass spectrom-
etry data set, we immunoprecipitated a Myc-tagged form of
Fcp1 to determine whether it coimmunopurifies with Spt5 (Fig.
1D). We failed to detect Spt5-Fcp1 coimmunoprecipitation
and conclude that Fcp1 does not associate with Spt5 under
these conditions, whereas the largest subunit of TFIIF specif-
ically coimmunopurifies with Spt5.
Spt proteins. The identification of Spt6, Cdc68, and Nhp6A/B
in the Flag-Spt5 purification was of particular interest, as these
proteins, like Spt4-Spt5, have been proposed to facilitate elon-
gation through nucleosomes (7, 18, 21, 41). Immunoblot anal-
ysis of Spt5-Flag immunoprecipitates confirmed the coimmu-
nopurification of Spt6 and Cdc68 but not Nhp6, which only
weakly associates with Cdc68 under the moderate salt condi-
tions used here (7, 18) (Fig. 1C). Cdc68 also interacts strongly
with Pob3 (8, 63). Consistent with this, we found that Pob3 also
coimmunoprecipitated with Spt5-Flag (Fig. 1C). In addition,
like Spt5, Pob3 eluted across a broad size range when Spt5-
Flag immunoprecipitates were separated by gel filtration (data
We have previously shown that Spt5 binds to Spt4 (21, 57).
Although Spt4 was not found by mass spectrometry, it clearly
coprecipitated with Spt5-Flag (Fig. 1C). Spt4’s absence prob-
ably reflects its small size and highlights the qualitative nature
of DALPC. When we immunoprecipitated a Flag-tagged de-
rivative of Spt4 from yeast extracts, we found that it coprecipi-
FIG. 1. Identification of Spt5-associated proteins. (A) Anti-Flag
immunoprecipitates from extracts of an Spt5-Flag strain (GHY617)
were size fractionated by gel filtration, and fractions were analyzed by
blotting with antibodies specific for Spt5, the hypophosphorylated
form of Rpb1 (Pol IIA), and the hyperphosphorylated form of Rpb1
(Pol IIO). Control experiments demonstrated that coprecipitation of
both Pol II isoforms with Spt5-Flag was specific (reference 31 and data
not shown). (B) Spt5-Flag purification. Extracts of an Spt5-Flag strain
and an untagged strain (mock) were incubated with anti-Flag M2
agarose beads. Spt5-Flag complexes were competitively eluted from
the beads with Flag peptide and further fractionated on a Bio-Rex 70
column. Fractions were analyzed by silver staining and blotting for
Spt5. Upper panel, anti-Spt5 Western blot. Lower panel, silver-stained
gel. (C) Anti-Flag immunoprecipitations from extracts of Spt5-Flag
and untagged (mock) strains. Proteins were eluted with Flag peptide,
separated by SDS-PAGE, and blotted with the indicated antibodies.
WCE, 30 ?g of whole-cell extract of Spt5-Flag strain. Note that in this
panel the relative proportion of whole-cell extract to eluate is five
times higher than in other immunoprecipitations. (D) Anti-Myc immu-
noprecipitations from extracts of strains carrying Fcp1-Myc or Tfg1-
Myc and a strain lacking the Myc tag (mock). Proteins were eluted in
1.0 M potassium acetate, fractionated by SDS-PAGE, and blotted for
Spt5. Strains: Spt5-Flag, GHY617; Fcp1-Myc, GHY1207; Tfg1-Myc,
GHY1208; mock in panels A to C, GHY611; mock in panel D, GHY617.
VOL. 23, 2003 ROLES FOR Spt5 IN ELONGATION AND PRE-mRNA PROCESSING 1371
tated Spt5, Pob3-Cdc68, Spt6, Pol IIO, and Pol IIAbut not
Nhp6 (Fig. 2A and data not shown). We also size fractionated
these Spt4-Flag immunoprecipitates by gel filtration and found
that Spt4 is associated with high-molecular-weight forms of
Spt5, Spt6, and Pob3 (data not shown).
Consistent with the data above, when we immunoprecipi-
tated an HA-tagged derivative of Spt6 from yeast extracts, we
found Spt4, Spt5, Cdc68, Pob3, Pol IIA, and Pol IIOin the
immunoprecipitates (Fig. 2B and C). The difference in the
strength of the Spt5-Spt6 coprecipitation reported here and
TABLE 2. Proteins that copurified with Spt5-Flag
B, shared subunit with Pol I
and Pol III
B, RPB6, shared subunit with
Pol I and Pol III
A, B, shared subunit with Pol I
and Pol III
Shared subunit with Pol I and
B, TFIIF, Rap74 homolog
ELP1, elongator subunit
B, cap methyltransferase
A, poly(A)-binding protein
3? end processing
3? end processing
aGene descriptions are from the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/) and Yeast Proteome Database (http://www
.proteome.com/databases/index.html). A, identified as a common false positive in reference 23. B, subunit of transcription complex 145 in reference 20.
Alpha DNA polymerase
Epsilon DNA polymerase
RNA polymerase I
RNA polymerase I
RNA polymerase I
Shared subunit of RNA Pol I
and Pol III
Casein kinase II
Binds cruciform DNA
A, IMP dehydrogenase
Similarity to IMP dehydrog-
YPR133C, this work
A, methionyl aminopeptidase
PUF family member
RNase L inhibitor
RNA binding protein
A, chromosome condensation
A, nucleic acid-binding protein
RING finger domain
20% identity to Prt1
NuA3 HAT complex
1372LINDSTROM ET AL.MOL. CELL. BIOL.
that observed previously (21, 51) can be explained by differ-
ences in methods of extract preparation (see Materials and
Methods). Consistent with these observations, when we immu-
noprecipitated a Myc-tagged derivative of Cdc68 from yeast
extracts, we found both Spt5 and Spt6 in the precipitates (Fig.
2D and E). Interestingly, and in contrast to Spt4, Spt5, and
Spt6, both Pob3 and Cdc68 specifically coimmunoprecipitated
with Pol IIObut not with Pol IIA(Fig. 2E). Thus, we conclude
that Spt4, Spt5, Spt6, and Cdc68-Pob3 coimmunopurify with
each other and Pol II. We further conclude that while Cdc68-
Pob3 appears to associate only with the hyperphosphorylated
form of Pol II, Spt4-Spt5 and Spt6 associate with both Pol IIA
and Pol IIO. Finally, in the immunoprecipitations of Spt4, Spt5,
and Spt6, we observed some variability in the fraction of Pob3
that coprecipitated compared to the fraction of Cdc68 that
coprecipitated (Fig. 1C and 2A and B and data not shown).
These differences likely represent experiment-to-experiment
variability, as they were not consistently observed.
Spt4-Spt5 and RNA processing. Both components of the
yeast mRNA capping enzyme, Ceg1 and Cet1 (48), copurified
with Spt5 (Table 2). We also identified Abd1, the cap methyl-
transferase, which binds Pol II independently of the capping
enzyme (35). To confirm these interactions, we performed
anti-HA immunoprecipitations of strains carrying epitope-
tagged alleles of Ceg1 or Abd1 (Fig. 3). Spt5 specifically co-
immunoprecipitated with both HA-Ceg1 and HA-Abd1. In
contrast, Pob3 did not coprecipitate with either HA-Ceg1 or
HA-Abd1 (Fig. 3). In reciprocal experiments, we observed that
Abd1 coimmunoprecipitated with Spt4-Flag, Spt5-Flag, and
HA-Spt6 (Fig. 1C and 2A and C). We also probed Cdc68-Myc
immunoprecipitates for Abd1 and did not observe significant
coimmunoprecipitation (Fig. 2D [compare to Fig. 1C and 2A
and C; note that in comparison to the other immunoprecipi-
tations, fivefold more extract relative to the eluate was loaded
on the gel in Fig. 1C]). However, because we observe a weak
cross-reactivity to immunoglobulin G migrating near the ex-
pected position of Abd1 in the gel, we cannot rule out a very
weak interaction between Abd1 and Cdc68. We conclude that
Spt4, Spt5, and Spt6 associate with the yeast capping enzyme
and cap methyltransferase, whereas Cdc68-Pob3 does not ap-
preciably associate with these proteins.
These observations are consistent with the finding of Spt5-
capping enzyme interactions in humans and S. pombe (43, 59).
Although Spt5 has mild effects on the in vitro activity of the cap
guanyltransferase in humans, it does not affect the in vitro
FIG. 2. Immunoprecipitation of Spt4, Spt6, Cdc68, and Pob3. (A)
Anti-Flag immunoprecipitation from lysates of an Spt4-Flag strain and
an untagged strain (mock). Bound proteins were eluted from the beads
in 1.0 M potassium acetate, fractionated by SDS-PAGE, and Western
blotted for the indicated proteins. Strains: mock, GHY1320; Spt4-Flag,
GHY1324. (B) Anti-HA immunoprecipitations from lysates of an HA-
Spt6 strain and an untagged strain were performed as for panel A.
Strains: mock, GHY611; HA-Spt6, GHY605. WCE, 30 ?g of whole-
cell extract. (C) Anti-HA immunoprecipitations from lysates of a HA-
Spt6 and an untagged strain were performed as for panel A. Strains:
mock, GHY1325; HA-Spt6, GHY1324. WCE, 60 ?g of whole-cell
extract. (D) Anti-Myc immunoprecipitations from extracts of Cdc68-
Myc and untagged (mock) strains were performed as for panel A. Top
panel, 60 ?g of whole-cell extract and 10% of the anti-Myc beads from
the immunoprecipitations were separated by SDS-PAGE and blotted
for the presence of Cdc68-Myc and Pob3. In contrast to the other
proteins analyzed in panels D and E, Cdc68-Pob3 complexes are stable
in 1.0 M potassium acetate and therefore remain bound to the beads
(data not shown). Bottom panel, 60 ?g of whole-cell extract and 1 M
potassium acetate eluates from the anti-Myc beads were separated by
SDS-PAGE and blotted for the presence of HA-Spt6 and Abd1. The
asterisk indicates a weak cross-reactivity to immunoglobulin G migrat-
ing near the expected position of Abd1 in the gel. Strains: Cdc68-Myc,
OY240; mock, GHY605. (E) Anti-Myc immunoprecipitations from
extracts of Pob3-Myc, Cdc68-Myc, and untagged (mock) strains were
performed as for panel A. Top panel, 30 ?g of whole-cell lysates and
1 M potassium acetate eluates were separated by SDS-PAGE and
probed for the indicated proteins. Bottom panel, 30 ?g of whole-cell
extract and 10% of the anti-Myc beads from the immunoprecipitations
were separated by SDS-PAGE and blotted with an anti-Myc antibody
for the presence of Cdc68-Myc and Pob3-Myc. Strains used: Pob3-
Myc, DY6460; Cdc68-Myc, DY6529; untagged, DK186.
FIG. 3. Spt5 but not Pob3 associates with capping enzyme and cap
methyltransferase. Anti-HA immunoprecipitations from extracts of
HA-Ceg1, HA-Abd1, and untagged (mock) strains are shown. Bound
proteins were eluted with 1.0 M potassium acetate, separated by SDS-
PAGE, and blotted for Spt5 and Pob3. WCE, 30 ?g of whole-cell ex-
tract. Eluate, 1.0 M potassium acetate elutions. Strains: HA-Ceg1,
YSB244/pSB996; HA-Abd1, YSB427/pSB995; untagged, YSB244/
VOL. 23, 2003 ROLES FOR Spt5 IN ELONGATION AND PRE-mRNA PROCESSING1373
activity of the S. pombe enzyme (43, 59). Neither set of obser-
vations indicates whether Spt5 and the capping enzyme func-
tionally interact in vivo. We used genetic analysis to begin to
address this issue. spt5-194 and spt4-3 strains were crossed to a
strain carrying the Ts?ceg1-250 allele (10). spt5-194 ceg1-250
and spt4-3 ceg1-250 double mutants both showed a decrease in
their restrictive temperature, indicating an interaction between
these genes (Fig. 4A). We used a plasmid shuffle assay to test
interactions between SPT4, SPT5, and CET1 and found that spt
cet1 double mutants displayed allele-specific synthetic lethality
and poor growth phenotypes (Fig. 4B). Thus, Spt5 interacts
genetically and coimmunopurifies with the capping enzyme in
yeast, suggesting a functional interaction between these pro-
teins in vivo.
Given the interactions between Spt5 and the capping en-
zyme in yeast and humans and previous studies showing that
the 5? cap influences the efficiency of subsequent steps of
pre-mRNA processing (16, 19, 47), we asked whether spt mu-
tations affect pre-mRNA splicing. RNA was extracted from a
series of spt4 and spt5 mutants and from a strain carrying a
mutation in the essential splicing factor PRP4 (5). Because the
spt4?, spt5-4, and spt5-194 mutants grow poorly or are inviable
at elevated temperatures, RNA was prepared from cells that
were grown at 30°C and then shifted to 39°C for 45 min prior
to harvest. Although the cold-sensitive spt5-242 mutant does
not have an obvious growth defect on rich media at elevated
temperatures, it is Spt?at both 30 and 37°C (G. A. Hartzog,
unpublished data). Thus, the spt5-242 mutation causes mutant
phenotypes at all temperatures tested. We first performed
primer extension analysis of the closely related U3A and U3B
snRNAs and observed unspliced U3 RNA in the spt5-242 and
prp4 strains (Fig. 5A). Next, we used an RT-PCR assay to
monitor levels of unspliced RPS25A, RPL26A, and RPS27B
pre-mRNAs in the spt4 and spt5 mutants (Fig. 5B). We ob-
served strong accumulation of unspliced RPL26A and moder-
ate accumulation of unspliced RPS25A for all three spt5 mu-
FIG. 4. SPT4 and SPT5 display genetic interactions with capping
enzyme. (A) Strains with the indicated genotypes were assayed by
serial dilution onto YPD plates, incubated for 2 days at the indicated
temperatures, and photographed. Note that the restrictive tempera-
ture for the ceg1-250 single mutant is 37°C (19). Strains: wild type,
FY120; spt5-194, GHY594; spt4-3, GHY96; ceg1-250, OY163; spt5-194
ceg1-250, OY215; spt4-3 ceg1-250, OY167. (B) spt cet1? mutants car-
rying a URA3 CET1 plasmid were transformed with a series of HIS3
cet1 plasmids as indicated. Transformants were grown to saturation in
SC-histidine medium and assayed by serial dilution on 5-fluoroorotic
acid plates, which allows growth only of cells that have lost the URA3
CET1 plasmid. Plates were incubated for 2 days at 30°C and photo-
graphed. Strains: cet1?1::TRP, OY227; spt4? cet1?1::TRP1, OY204;
spt5-194 cet1?1::TRP1, OY207; spt5-242 cet1?1::TRP1, OY205.
FIG. 5. Splicing defects in spt4 and spt5 mutants. RNA was pre-
pared from strains with the indicated genotypes either grown at 30°C
or grown at 30°C and then shifted to 39°C for 45 min prior to harvest.
The prp4 strain was grown at 30°C and then shifted to 37°C for 1 h
prior to harvest. (A) Primer extension analysis of the U3A and U3B
snRNAs. Note that mature U3A and U3B snRNAs are indistinguish-
able in this assay. In contrast, the unspliced U3A and U3B pre-
snRNAs give products that differ by 27 nucleotides (4). (B) RT-PCR
analysis for unspliced forms of the RPS25A, RPL26A, and RPS27B
genes. Unspliced pre-mRNAs were specifically amplified by using one
primer chosen from intron sequences and another chosen from the
second exon. As a loading control, SED1, which does not contain an
intron, was also analyzed by RT-PCR. Control reactions confirmed
that the PCRs were performed in the linear range and that the PCR
products were dependent upon reverse transcriptase (data not shown).
Strains: wild type (WT), FY120; spt5-4, OY43; spt5-194, GHY379;
spt5-242, GHY92; spt4?, GHY524; prp4, SRY4-1a.
1374LINDSTROM ET AL.MOL. CELL. BIOL.
tants, as well as moderate accumulation of unspliced RPS27B
in the spt5-194 and spt5-242 mutants (Fig. 5B). We also ob-
served small but reproducible accumulation of unspliced
RPS25A and RPS27B RNAs in the spt4? mutant (Fig. 5B).
Thus, spt4 and spt5 mutations lead to accumulation of un-
spliced forms of several yeast genes.
Characterization of Iws1. We initially chose proteins from
the mass spectrometry data for further analysis based on their
connection to Pol II transcription and processing. To examine
whether previously uncharacterized proteins in this data set
might also be involved in transcription or processing, we chose
to study YPR133C, an essential gene of unknown function
(62). While this work was in progress, YPR133C was identified
as an Spt6-interacting protein and named Iws1 (30). BLAST
searches revealed metazoan homologs of Iws1, none of which
has previously been characterized (Fig. 6A). These proteins all
have acidic N termini and a conserved C-terminal domain. The
predicted Drosophila Iws1 protein has a region enriched in
serine-arginine dipeptides. This motif is found in a number of
proteins that associate with Pol II and that have roles in pre-
mRNA splicing (34). We tagged Iws1 with the Myc epitope,
performed anti-Myc immunoprecipitations, and observed that
Spt5, Spt6, Abd1, Pol IIO, and Pol IIAspecifically coimmuno-
precipitated with Iws1-Myc (Fig. 6B). We also found Iws1 in
Spt4 and Spt6 immunoprecipitates (Fig. 2A and C). In con-
trast, neither Cdc68 nor Pob3 coprecipitated with Iws1-Myc
To genetically characterize IWS1, we isolated temperature-
sensitive alleles of Myc-tagged IWS1. Interestingly, we found
that these mutants displayed a Spt?phenotype (Fig. 6C). spt
mutations were originally identified by virtue of their ability to
suppress transcription defects caused by insertion of the long
terminal repeat of the Ty retrotransposon into certain promot-
ers (60). For example, in Spt?cells carrying the his4-912?
mutation, the HIS4 transcript is longer than normal and trans-
lationally nonfunctional, rendering the cells His?(60). In a spt
mutant, this defect is suppressed and the strain reverts to a
His?phenotype (60) (Fig. 6C). Northern blot analysis showed
that the Spt?phenotype of the iws1 mutants was due to altered
transcription of the his4-912? gene (Fig. 6D). Finally, when we
crossed an iws1-13 strain with spt4? or spt5-194 mutants, we
found that neither the iws1-13 spt4? nor the iws1-13 spt5-194
double mutant was viable (data not shown). Thus, Iws1 is a
conserved protein that coimmunopurifies with Spt5 and Pol II,
displays an Spt?phenotype when altered by mutation, and
causes synthetic lethality when combined with spt4 or spt5
mutations, results indicative of roles in Spt4-Spt5 function and
Pol II transcription.
To determine the roles that the Spt4-Spt5 complex plays in
gene expression, we sought to identify proteins that associate,
directly or indirectly, as members of a protein complex with
Spt5. Combining affinity purification and mass spectrometry,
we identified a large number of proteins that specifically co-
immunopurified with Spt5-Flag. We further confirmed a num-
ber of these observations by coimmunoprecipitation and ge-
netic analysis. Consistent with Spt5’s role in transcription
elongation, we found evidence of copurification of Pol II and
many transcription elongation factors. In contrast, we recov-
ered only two peptides for initiation factors that function ex-
clusively at the promoter, one for TFIIB and one for Taf60
(Table 2). No peptides for SRB/mediator subunits or other
general initiation factors were recovered. Thus, these data
strongly support the model that Spt5 functions as an elonga-
tion factor in vivo.
The Spt4-Spt5 complex influences RNA processing in vivo.
The copurification of capping factors with Spt5 is consistent
with previous observations with human and S. pombe cells.
However, the effect of Spt5 on capping enzyme activity in vitro
is weak (43, 59), and the relevance of the Spt5-capping enzyme
interaction is therefore uncertain. We confirmed the associa-
tion of Spt5 with the capping enzyme and also found that Spt5
coimmunopurifies with Abd1, the cap methyltransferase, which
does not directly associate with the capping enzyme (35) (Fig.
3). We also found that SPT4 and SPT5 interact genetically with
both CET1 and CEG1 (Fig. 5). These observations strongly
suggest an in vivo role for Spt4-Spt5 in pre-mRNA capping.
We also observed accumulation of several intron-containing
RNAs in spt mutants (Fig. 4). In preliminary studies using
FIG. 6. IWS1/YPR133C encodes a conserved Spt5-associated pro-
tein required for normal transcription. (A) Alignment of Iws1 and
homologs. The regions of highest identity between the proteins are
marked by shading, and the percent amino acid identity is noted. The
N-terminal SR repeats in the Drosophila homolog are also noted by
shading. The accession numbers for the human and Drosophila ho-
mologs are BAA91402 and AAF48587, respectively. Alignments and
percent amino acid identities were determined by BLAST searches (1).
(B) Anti-Myc immunoprecipitations were performed from extracts of
an Iws1-Myc strain and an untagged (mock) strain. Bound proteins
were eluted with 1.0 M potassium acetate, separated by SDS-PAGE,
and blotted for Spt5. Strains: Iws1-Myc, GHY1300; mock, GHY1280.
WCE, 30 ?g of whole-cell extract of GHY1300. (C) iws1 mutants
display an Spt?phenotype. Cells were replica plated to the indicated
media and grown for 2 days at 30°C. Strains: Spt?, GHY611; iws1-13,
GHY1202; spt5-4, GHY1073. (D) The Spt?phenotypes of iws1-7 and
iws1-13 cells are due to altered transcription. Top panel, Northern blot
analysis of HIS4 and his4-912? RNA derived from strains with the
indicated genotypes. RNA from a spt5-194 his4-912? strain was in-
cluded for comparison to the iws1 his4-912? strains. Bottom panel, as
a loading control, the blot was stripped and then rehybridized with a
TUB2 probe. Strains: lane 1, FY602; lane 2, FY119; lane 3, GHY13;
lane 4, GHY1199; lane 5, GHY1200.
VOL. 23, 2003ROLES FOR Spt5 IN ELONGATION AND PRE-mRNA PROCESSING 1375
splicing-sensitive DNA microarrays (12), we have observed
similar effects of spt mutations for at least half of all intron-
containing genes in yeast (T. A. Burckin and G. A. Hartzog,
unpublished results). This may indicate a role for Spt4-Spt5 in
splicing or possibly in the nuclear degradation of unspliced
pre-mRNAs. We did not detect any splicing factors in our mass
spectrometry data, and thus we have no evidence for a direct
interaction of Spt4-Spt5 with the splicing machinery. Even if
Spt4-Spt5 does not play a direct role in splicing, it is possible
that spt4 and spt5 mutations indirectly lead to splicing defects
as a consequence of defects in elongation or pre-mRNA cap-
ping (17, 19, 47). Regardless of the mechanism, our observa-
tions suggest an important role for Spt4-Spt5 in pre-mRNA
Is Spt5 a component of more than one complex? When
Spt5-Flag immunoprecipitates were subjected to gel filtration
chromatography, Spt5 was broadly distributed across the elu-
ates, suggesting that it assembles into several large complexes
(Fig. 1). Preliminary results indicate that treatment of Spt5-
Flag complexes with RNase does not affect association of Pol
II, Spt6, Cdc68-Pob3, or Iws1, indicating that their interactions
with Spt5, although not necessarily direct, are likely mediated
by protein interactions (D. L. Lindstrom and G. A. Hartzog,
unpublished results). An interesting question is whether these
Spt proteins form a complex before association with Pol II or
are recruited individually to elongating polymerase. For exam-
ple, the association of Spt4, Spt5, Spt6, and Iws1 with both Pol
IIAand Pol IIOsuggests that they may associate with Pol II
prior to and during processive elongation (31) (Fig. 1A and
2B). This model is consistent with chromatin immunoprecipi-
tation studies with yeast and Drosophila (2, 27, 44). In contrast,
the capping enzyme and cap methyltransferase are recruited
specifically to the phosphorylated CTD of Pol IIO(11, 36, 68),
suggesting that they may be recruited to Pol II separately from
Spt4-Spt5, Spt6, and Iws1. Similarly, we have found that Cdc68
and Pob3 associate with Pol IIObut not Pol IIA(Fig. 2E).
Furthermore, the failure of Cdc68 or Pob3 to coimmunopre-
cipitate with HA-Ceg1 or HA-Abd1 (Fig. 3) and the at best
weak coprecipitation of Abd1 with Cdc68-Myc (Fig. 2D and
data not shown) suggest that recruitment of the capping ma-
chinery and Cdc68-Pob3 are also distinct events.
In summary, we have provided evidence that Spt4-Spt5 par-
ticipates in three or more Pol II complexes. The first is a Pol
IIAcomplex with Spt4, Spt5, Spt6, and Iws1 (Fig. 7A). The
second is a Pol IIOcomplex with Spt4, Spt5, Spt6, Iws1, and
Abd1 (Fig. 7B). This complex likely includes the yeast capping
enzyme (i.e., Ceg1-Cet1), as we have found that Spt5 copre-
cipitates with HA-Ceg1 (Fig. 3). However, the potential asso-
ciation of the capping enzyme with Spt4, Spt6, and Iws1 re-
mains to be tested. The third complex (Fig. 7C) includes Pol
IIO, Spt4, Spt5, Spt6, and Cdc68-Pob3 but lacks Iws1 and the
capping apparatus. We cannot rule out the possibility of other,
lower-abundance or unstable complexes. Although it is intrigu-
ing to speculate that these complexes may share temporal
relationships during elongation, this idea remains to be tested.
None of our data indicate whether these Spt proteins di-
rectly interact or are indirectly associated with each other,
perhaps with Pol II as an intermediate. However, based upon
their shared genetic behaviors, Spt4, Spt5, Spt6, Cdc68, Pob3,
and Nhp6 have been proposed to perform similar or overlap-
ping functions in transcription elongation in the context of
chromatin (reviewed in references 22, 60, and 66). Combina-
tions of spt4, spt5, and spt6 mutations display unlinked non-
complementation and synthetic lethality, behaviors often ob-
served for genes encoding interacting proteins (51). CDC68,
POB3, and NHP6 also display genetic interactions with SPT4
and SPT5, and recent biochemical studies suggest that these
proteins may have overlapping functions (7, 18, 41, 56). To
date, evidence for Spt5-Spt6 binding has been weak and there
has been no evidence that Spt5 physically interacts, either
directly or indirectly, with Cdc68-Pob3. Our observations that
Spt5, Spt6, Cdc68, and Pob3 coimmunopurify with each other
and with Pol IIOare consistent with their genetic interactions
and suggest that these proteins cooperate with each other to
regulate transcription elongation in the context of chromatin.
Other proteins that associate with Spt5. In this work we
aimed to comprehensively identify proteins that copurify with
Spt5. In a recent large-scale proteomic study, a complex con-
taining Spt4, Spt5, TFIIF, Abd1, and Pol II was identified,
although it was not independently verified by other methods
(complex 145 in reference 20). Many of the proteins that we
have shown to associate with Spt5 here, including Spt6, Cdc68,
Pob3, Iws1, and the capping enzyme, were not found in that
study. Conversely, our mass spectrometry data did not include
three proteins, Aos1, YDL115C, and YHL021C, that were
identified in complex 145. Similarly, Krogan et al. (30) used a
TAP-Tag approach to identify a network of protein interac-
tions that overlap with those we have reported here and else-
where (49). While Krogan et al. did not identify the capping
apparatus in their work, they did note interactions between
Cdc68-Pob3 and several proteins not observed here, using low-
er-salt conditions than we have used. Previous purifications of
FIG. 7. Model of Spt5 complexes. This model is based on coimmunoprecipitation and purification data presented in Fig. 1, 2, 3, and 6 and on
previous studies demonstrating that Abd1 and the capping enzyme interact specifically with Pol IIO(11, 36, 68). The presence of Spt4, Spt6, and
Iws1 in the same complex as the capping enzyme remains to be tested directly. For this reason, the capping enzyme complex in panel B is indicated
with a dashed line.
1376LINDSTROM ET AL.MOL. CELL. BIOL.
Spt4-Spt5 from human cells have been based upon functional
assays of Pol II transcription. DSIF, the human Spt4-Spt5
complex, was purified based on its ability to inhibit transcrip-
tion in the presence of the protein kinase inhibitor DRB (57).
Subsequent work identified another multiprotein complex,
NELF, which is required for DSIF’s repressive activity. The
identities of two NELF subunits, RD and WHSC2, have been
reported, but neither has an obvious yeast homolog (65, 67).
Protein complexes that stimulate Tat activity in vitro have been
partially purified from HeLa cells and reported to contain
Spt4-Spt5, P-TEFb, Tat-SF1, nucleolin, XP-E, Pol II, the small
subunit of TFIIF (equivalent to Tfg2) and other novel polypep-
tides (28, 42, 64). Other than Pol II and TFIIF, we have not
identified homologs of these proteins here. A number of pro-
teins known to associate with Spt5 are not included in our data,
reflecting technical pitfalls of complex purifications. For exam-
ple, we recently demonstrated genetic and biochemical inter-
actions between Spt4-Spt5 and the Paf1 complex (49). While
we identified Paf1 complex members in the Spt5-Flag frac-
tions, each was also present in the mock purification, possibly
due to a Flag-mimetic epitope in Rtf1 (D. L. Lindstrom and
G. A. Hartzog, unpublished results).
Many of the Spt5-associated proteins that we identified have
not been previously characterized or are known to function in
processes other than Pol II transcription and pre-mRNA pro-
cessing. Although these proteins may have nonspecifically co-
purified with Spt5-Flag, our characterization of Iws1 (Fig. 6)
suggests that some of these factors are involved in Pol II
transcription and/or are likely to associate and functionally
interact with Spt5. Thus, analysis of these other putative Spt5-
associated proteins may reveal further clues to Spt4-Spt5 func-
This work was supported by a grant from the NIH to G.A.H.
(GM60479) and by NIH Yeast Resource Center grant RR11823-05 to
We thank Steve Buratowski, Tim Formosa, David Stillman, Caroline
Kane, Steve Hahn, Clyde Denis, Fred Winston, and Doug Kellogg for
gifts of strains, plasmids, and antibodies. We thank Manny Ares and
Caroline Kane for sharing information prior to publication. We thank
Manny Ares, John Tamkun, Fred Winston, Craig Kaplan, and mem-
bers of the Hartzog lab for helpful discussions and their comments on
1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.
Basic local alignment search tool. J. Mol. Biol. 215:403–410.
2. Andrulis, E. D., E. Guzman, P. Doring, J. Werner, and J. T. Lis. 2000.
High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes
in vivo: roles in promoter proximal pausing and transcription elongation.
Genes Dev. 14:2635–2649.
3. Archambault, J., R. S. Chambers, M. S. Kobor, Y. Ho, M. Cartier, D.
Bolotin, B. Andrews, C. M. Kane, and J. Greenblatt. 1997. An essential
component of a C-terminal domain phosphatase that interacts with tran-
scription factor IIF in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA
4. Ares, M., Jr., and A. H. Igel. 1990. Lethal and temperature-sensitive muta-
tions and their suppressors identify an essential structural element in U2
small nuclear RNA. Genes Dev. 4:2132–2145.
5. Banroques, J., and J. N. Abelson. 1989. PRP4: a protein of the yeast U4/U6
small nuclear ribonucleoprotein particle. Mol. Cell. Biol. 9:3710–3719.
6. Bortvin, A. L., and F. Winston. 1996. Evidence that Spt6p controls chromatin
structure by a direct interaction with histones. Science 272:1473–1476.
7. Brewster, N. K., G. C. Johnston, and R. A. Singer. 2001. A bipartite yeast
SSRP1 analog comprised of Pob3 and Nhp6 proteins modulates transcrip-
tion. Mol. Cell. Biol. 21:3491–3502.
8. Brewster, N. K., G. C. Johnston, and R. A. Singer. 1998. Characterization of
the CP complex, an abundant dimer of Cdc68 and Pob3 proteins that reg-
ulates yeast transcriptional activation and chromatin repression. J. Biol.
9. Chang, C. H., and D. S. Luse. 1997. The H3/H4 tetramer blocks transcript
elongation by RNA polymerase II in vitro. J. Biol. Chem. 272:23427–23434.
10. Cho, E. J., C. R. Rodriguez, T. Takagi, and S. Buratowski. 1998. Allosteric
interactions between capping enzyme subunits and the RNA polymerase II
carboxy-terminal domain. Genes Dev. 12:3482–3487.
11. Cho, E. J., T. Takagi, C. R. Moore, and S. Buratowski. 1997. mRNA capping
enzyme is recruited to the transcription complex by phosphorylation of the
RNA polymerase II carboxy-terminal domain. Genes Dev. 11:3319–3326.
12. Clark, T. A., C. W. Sugnet, and M. Ares, Jr. 2002. Genomewide analysis of
mRNA processing in yeast using splicing-specific microarrays. Science 296:
13. Davis, C. A., L. Grate, M. Spingola, and M. Ares, Jr. 2000. Test of intron
predictions reveals novel splice sites, alternatively spliced mRNAs and new
introns in meiotically regulated genes of yeast. Nucleic Acids Res. 28:1700–
14. Denis, C. L., M. P. Draper, H. Y. Liu, T. Malvar, R. C. Vallari, and W. J.
Cook. 1994. The yeast CCR4 protein is neither regulated by nor associated
with the SPT6 and SPT10 proteins and forms a functionally distinct complex
from that of the SNF/SWI transcription factors. Genetics 138:1005–1013.
15. Evan, G. I., G. K. Lewis, G. Ramsay, and J. M. Bishop. 1985. Isolation of
monoclonal antibodies specific for human c-myc proto-oncogene product.
Mol. Cell. Biol. 5:3610–3616.
16. Flaherty, S. M., P. Fortes, E. Izaurralde, I. W. Mattaj, and G. M. Gilmartin.
1997. Participation of the nuclear cap binding complex in pre-mRNA 3?
processing. Proc. Natl. Acad. Sci. USA 94:11893–11898.
17. Fong, Y. W., and Q. Zhou. 2001. Stimulatory effect of splicing factors on
transcriptional elongation. Nature 414:929–933.
18. Formosa, T., P. Eriksson, J. Wittmeyer, J. Ginn, Y. Yu, and D. J. Stillman.
2001. Spt16-Pob3 and the HMG protein Nhp6 combine to form the nucleo-
some-binding factor SPN. EMBO J. 20:3506–3517.
19. Fresco, L. D., and S. Buratowski. 1996. Conditional mutants of the yeast
mRNA capping enzyme show that the cap enhances, but is not required for,
mRNA splicing. RNA 2:584–596.
20. Gavin, A. C., M. Bosche, R. Krause, P. Grandi, M. Marzioch, A. Bauer, J.
Schultz, J. M. Rick, A. M. Michon, C. M. Cruciat, M. Remor, C. Hofert, M.
Schelder, M. Brajenovic, H. Ruffner, A. Merino, K. Klein, M. Hudak, D.
Dickson, T. Rudi, V. Gnau, A. Bauch, S. Bastuck, B. Huhse, C. Leutwein,
M. A. Heurtier, R. R. Copley, A. Edelmann, E. Querfurth, V. Rybin, G.
Drewes, M. Raida, T. Bouwmeester, P. Bork, B. Seraphin, B. Kuster, G.
Neubauer, and G. Superti-Furga. 2002. Functional organization of the yeast
proteome by systematic analysis of protein complexes. Nature 415:141–147.
21. Hartzog, G. A., T. Wada, H. Handa, and F. Winston. 1998. Evidence that
Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II
in Saccharomyces cerevisiae. Genes Dev. 12:357–369.
22. Hartzog, G. H., J. L. Speer, and D. L. Lindstrom. 2002. Transcript elongation
on a nucleoprotein template. Biochim. Biophys. Acta 1577:276–286.
23. Ho, Y., A. Gruhler, A. Heilbut, G. D. Bader, L. Moore, S. L. Adams, A.
Millar, P. Taylor, K. Bennett, K. Boutilier, L. Yang, C. Wolting, I. Donald-
son, S. Schandorff, J. Shewnarane, M. Vo, J. Taggart, M. Goudreault, B.
Muskat, C. Alfarano, D. Dewar, Z. Lin, K. Michalickova, A. R. Willems, H.
Sassi, P. A. Nielsen, K. J. Rasmussen, J. R. Andersen, L. E. Johansen, L. H.
Hansen, H. Jespersen, A. Podtelejnikov, E. Nielsen, J. Crawford, V. Poulsen,
B. D. Sorensen, J. Matthiesen, R. C. Hendrickson, F. Gleeson, T. Pawson,
M. F. Moran, D. Durocher, M. Mann, C. W. Hogue, D. Figeys, and M. Tyers.
2002. Systematic identification of protein complexes in Saccharomyces cer-
evisiae by mass spectrometry. Nature 415:180–183.
24. Ivanov, D., Y. T. Kwak, J. Guo, and R. B. Gaynor. 2000. Domains in the SPT5
protein that modulate its transcriptional regulatory properties. Mol. Cell.
25. Izban, M. G., and D. S. Luse. 1992. Factor-stimulated RNA polymerase II
transcribes at physiological elongation rates on naked DNA but very poorly
on chromatin templates. J. Biol. Chem. 267:13647–13655.
26. Izban, M. G., and D. S. Luse. 1991. Transcription on nucleosomal templates
by RNA polymerase II in vitro: inhibition of elongation with enhancement of
sequence-specific pausing. Genes Dev. 5:683–696.
27. Kaplan, C. D., J. R. Morris, C. Wu, and F. Winston. 2000. Spt5 and Spt6 are
associated with active transcription and have characteristics of general elon-
gation factors in D. melanogaster. Genes Dev. 14:2623–2634.
28. Kim, J. B., Y. Yamaguchi, T. Wada, H. Handa, and P. A. Sharp. 1999.
Tat-SF1 protein associates with RAP30 and human SPT5 proteins. Mol.
Cell. Biol. 19:5960–5968.
29. Komarnitsky, P., E. J. Cho, and S. Buratowski. 2000. Different phosphory-
lated forms of RNA polymerase II and associated mRNA processing factors
during transcription. Genes Dev. 14:2452–2460.
30. Krogan, N. J., M. Kim, S. H. Ahn, G. Zhong, M. S. Kobor, G. Cagney, A.
Emili, A. Shilatifard, S. Buratowski, and J. F. Greenblatt. 2002. RNA poly-
merase II elongation factors of Saccharomyces cerevisiae: a targeted pro-
teomics approach. Mol. Cell. Biol. 22:6979–6992.
VOL. 23, 2003ROLES FOR Spt5 IN ELONGATION AND PRE-mRNA PROCESSING1377
31. Lindstrom, D. L., and G. A. Hartzog. 2001. Genetic interactions of Spt4-Spt5 Download full-text
and TFIIS with the RNA polymerase II CTD and CTD modifying enzymes
in Saccharomyces cerevisiae. Genetics 159:487–497.
32. Link, A. J., J. Eng, D. M. Schieltz, E. Carmack, G. J. Mize, D. R. Morris,
B. M. Garvik, and J. R. Yates III. 1999. Direct analysis of protein complexes
using mass spectrometry. Nat. Biotechnol. 17:676–682.
33. Longtine, M. S., A. McKenzie III, D. J. Demarini, N. G. Shah, A. Wach, A.
Brachat, P. Philippsen, and J. R. Pringle. 1998. Additional modules for
versatile and economical PCR-based gene deletion and modification in Sac-
charomyces cerevisiae. Yeast 14:953–961.
34. Maniatis, T., and R. Reed. 2002. An extensive network of coupling among
gene expression machines. Nature 416:499–506.
35. Mao, X., B. Schwer, and S. Shuman. 1995. Yeast mRNA cap methyltrans-
ferase is a 50-kilodalton protein encoded by an essential gene. Mol. Cell.
36. McCracken, S., N. Fong, E. Rosonina, K. Yankulov, G. Brothers, D. Sid-
erovski, A. Hessel, S. Foster, S. Shuman, and D. L. Bentley. 1997. 5?-capping
enzymes are targeted to pre-mRNA by binding to the phosphorylated car-
boxy-terminal domain of RNA polymerase II. Genes Dev. 11:3306–3318.
37. Mortensen, E. M., H. McDonald, J. Yates III, and D. R. Kellogg. 2002. Cell
cycle-dependent assembly of a Gin4-septin complex. Mol. Biol. Cell 13:2091–
38. Mueller, C. L., and J. A. Jaehning. 2002. Ctr9, Rtf1, and Leo1 are compo-
nents of the Paf1/RNA polymerase II complex. Mol. Cell. Biol. 22:1971–
39. Murray, S., R. Udupa, S. Yao, G. Hartzog, and G. Prelich. 2001. Phosphor-
ylation of the RNA polymerase II carboxy-terminal domain by the Bur1
cyclin-dependent kinase. Mol. Cell. Biol. 21:4089–4096.
40. Orphanides, G., G. LeRoy, C. H. Chang, D. S. Luse, and D. Reinberg. 1998.
FACT, a factor that facilitates transcript elongation through nucleosomes.
41. Orphanides, G., W. H. Wu, W. S. Lane, M. Hampsey, and D. Reinberg. 1999.
The chromatin-specific transcription elongation factor FACT comprises hu-
man SPT16 and SSRP1 proteins. Nature 400:284–288.
42. Parada, C. A., and R. G. Roeder. 1999. A novel RNA polymerase II-con-
taining complex potentiates Tat-enhanced HIV-1 transcription. EMBO J.
43. Pei, Y., and S. Shuman. 2002. Interactions between fission yeast mRNA
capping enzymes and elongation factor Spt5. J. Biol. Chem. 277:19639–
44. Pokholok, D. K., N. M. Hannett, and R. A. Young. 2002. Exchange of RNA
polymerase II initiation and elongation factors during gene expression in
vivo. Mol. Cell 9:799–809.
45. Proudfoot, N. J., A. Furger, and M. J. Dye. 2002. Integrating mRNA pro-
cessing with transcription. Cell 108:501–512.
46. Rose, M. D., F. Winston, and P. Hieter. 1990. Methods in yeast genetics: a
laboratory course manual. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.
47. Schwer, B., and S. Shuman. 1996. Conditional inactivation of mRNA cap-
ping enzyme affects yeast pre-mRNA splicing in vivo. RNA 2:574–583.
48. Shuman, S. 2001. Structure, mechanism, and evolution of the mRNA cap-
ping apparatus. Prog. Nucleic Acid Res. Mol. Biol. 66:1–40.
49. Squazzo, S. L., P. J. Costa, D. L. Lindstrom, K. E. Kumer, R. Simic, J. L.
Jennings, A. J. Link, K. M. Arndt, and G. A. Hartzog. 2002. The Paf1
complex physically and functionally associates with transcription elongation
factors in vivo. EMBO J. 21:1764–1774.
50. Swanson, M. S., E. A. Malone, and F. Winston. 1991. SPT5, an essential gene
important for normal transcription in Saccharomyces cerevisiae, encodes an
acidic nuclear protein with a carboxy-terminal repeat. Mol. Cell. Biol. 11:
51. Swanson, M. S., and F. Winston. 1992. SPT4, SPT5 and SPT6 interactions:
effects on transcription and viability in Saccharomyces cerevisiae. Genetics
52. Takase, Y., T. Takagi, P. B. Komarnitsky, and S. Buratowski. 2000. The
essential interaction between yeast mRNA capping enzyme subunits is not
required for triphosphatase function in vivo. Mol. Cell. Biol. 20:9307–9316.
53. Thompson, N., T. Steinberg, D. Aronson, and R. Burgess. 1989. Inhibition of
in vivo and in vitro transcription by monoclonal antibodies prepared against
wheat germ RNA polymerase II that react with the heptapeptide repeat of
eukaryotic RNA polymerase II. J. Biol. Chem. 264:11511–11520.
54. Tsukiyama, T., J. Palmer, C. C. Landel, J. Shiloach, and C. Wu. 1999.
Characterization of the ISWI subfamily of ATP-dependent chromatin re-
modeling factors in S. cerevisiae. Genes Dev. 13:686–697.
55. Verma, R., S. Chen, R. Feldman, D. Schieltz, J. Yates, J. Dohmen, and R. J.
Deshaies. 2000. Proteasomal proteomics: identification of nucleotide-sensi-
tive proteasome-interacting proteins by mass spectrometric analysis of affin-
ity-purified proteasomes. Mol. Biol. Cell 11:3425–3439.
56. Wada, T., G. Orphanides, J. Hasegawa, D. K. Kim, D. Shima, Y. Yamaguchi,
A. Fukuda, K. Hisatake, S. Oh, D. Reinberg, and H. Handa. 2000. FACT
relieves DSIF/NELF-mediated inhibition of transcriptional elongation and
reveals functional differences between P-TEFb and TFIIH. Mol. Cell
57. Wada, T., T. Takagi, Y. Yamaguchi, A. Ferdous, T. Imai, S. Hirose, S. Sugi-
moto, K. Yano, G. A. Hartzog, F. Winston, S. Buratowski, and H. Handa.
1998. DSIF, a novel transcription elongation factor that regulates RNA
polymerase II processivity, is composed of human Spt4 and Spt5 homologs.
Genes Dev. 12:343–356.
58. Wada, T., T. Takagi, Y. Yamaguchi, D. Watanabe, and H. Handa. 1998.
Evidence that P-TEFb alleviates the negative effect of DSIF on RNA poly-
merase II-dependent transcription in vitro. EMBO J. 17:7395–7403.
59. Wen, Y., and A. J. Shatkin. 1999. Transcription elongation factor hSPT5
stimulates mRNA capping. Genes Dev. 13:1774–1779.
60. Winston, F. 1992. Analysis of SPT genes: a genetic approach towards analysis
of TFIID, histones and other transcription factors of yeast, p. 1271–1293. In
S. L. McKnight and K. R. Yamamoto (ed.), Transcriptional regulation. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
61. Winston, F., C. Dollard, and S. L. Ricupero-Hovasse. 1995. Construction of
a set of convenient S. cerevisiae strains that are isogenic to S288C. Yeast
62. Winzeler, E. A., D. D. Shoemaker, A. Astromoff, H. Liang, K. Anderson, B.
Andre, R. Bangham, R. Benito, J. D. Boeke, H. Bussey, A. M. Chu, C.
Connelly, K. Davis, F. Dietrich, S. W. Dow, M. El Bakkoury, F. Foury, S. H.
Friend, E. Gentalen, G. Giaever, J. H. Hegemann, T. Jones, M. Laub, H.
Liao, R. W. Davis, et al. 1999. Functional characterization of the S. cerevisiae
genome by gene deletion and parallel analysis. Science 285:901–906.
63. Wittmeyer, J., L. Joss, and T. Formosa. 1999. Spt16 and Pob3 of Saccharo-
myces cerevisiae form an essential, abundant heterodimer that is nuclear,
chromatin-associated, and copurifies with DNA polymerase alpha. Biochem-
64. Wu-Baer, F., W. S. Lane, and R. B. Gaynor. 1998. Role of the human
homolog of the yeast transcription factor SPT5 in HIV-1 Tat-activation.
J. Mol. Biol. 277:179–197.
65. Yamaguchi, Y., J. Filipovska, K. Yano, A. Furuya, N. Inukai, T. Narita, T.
Wada, S. Sugimoto, M. M. Konarska, and H. Handa. 2001. Stimulation of
RNA polymerase II elongation by hepatitis delta antigen. Science 293:124–
66. Yamaguchi, Y., T. Narita, N. Inukai, T. Wada, and H. Handa. 2001. SPT
genes: key players in the regulation of transcription, chromatin structure and
other cellular processes. J. Biochem. (Tokyo) 129:185–191.
67. Yamaguchi, Y., T. Takagi, T. Wada, K. Yano, A. Furuya, S. Sugimoto, J.
Hasegawa, and H. Handa. 1999. NELF, a multisubunit complex containing
RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell
68. Yue, Z., E. Maldonado, R. Pillutla, H. Cho, D. Reinberg, and A. J. Shatkin.
1997. Mammalian capping enzyme complements mutant Saccharomyces cer-
evisiae lacking mRNA guanylyltransferase and selectively binds the elongat-
ing form of RNA polymerase II. Proc. Natl. Acad. Sci. USA 94:12898–12903.
1378LINDSTROM ET AL.MOL. CELL. BIOL.