Molecular Biology of the Cell
Vol. 19, 4310–4318, October 2008
The THP1-SAC3-SUS1-CDC31 Complex Works in
Transcription Elongation-mRNA Export Preventing
RNA-mediated Genome Instability
Cristina Gonza ´lez-Aguilera, Cristina Tous, Bele ´n Go ´mez-Gonza ´lez,
Pablo Huertas,* Rosa Luna, and Andre ´s Aguilera
Centro Andaluz de Biologia Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC,
41092 Sevilla, Spain
Submitted April 7, 2008; Revised July 18, 2008; Accepted July 22, 2008
Monitoring Editor: Marvin P. Wickens
The eukaryotic THO/TREX complex, involved in mRNP biogenesis, plays a key role in the maintenance of genome
integrity in yeast. mRNA export factors such as Thp1-Sac3 also affect genome integrity, but their mutations have other
phenotypes different from those of THO/TREX. Sus1 is a novel component of SAGA transcription factor that also
associates with Thp1-Sac3, but little is known about its effect on genome instability and transcription. Here we show that
Thp1, Sac3, and Sus1 form a functional unit with a role in mRNP biogenesis and maintenance of genome integrity that
is independent of SAGA. Importantly, the effects of ribozyme-containing transcription units, RNase H, and the action of
human activation-induced cytidine deaminase on transcription and genome instability are consistent with the possibility
that R-loops are formed in Thp1-Sac3-Sus1-Cdc31 as in THO mutants. Our data reveal that Thp1-Sac3-Sus1-Cdc31,
together with THO/TREX, define a specific pathway connecting transcription elongation with export via an RNA-
dependent dynamic process that provides a feedback mechanism for the control of transcription and the preservation of
genetic integrity of transcribed DNA regions.
The nascent mRNA is cotranscriptionally coated with pro-
teins that assure RNA integrity, nuclear export, and down-
stream cytoplasmic steps. Formation of a mature ribonucle-
oprotein particle (mRNP) competent for export requires the
correct coupling of transcription with mRNA processing
steps such as 5?-end capping, splicing, 3?-end cleavage and
polyadenylation (Aguilera, 2005; Buratowski, 2005; Cole and
Scarcelli, 2006). Incorrectly spliced or 3?-end processed tran-
scripts are retained within the nucleus, providing evidence
that mRNA maturation and export are linked to each other
(Lei and Silver, 2002). Any failure in the formation of an
export-proficient mRNP would cause nuclear RNA reten-
tion and could trigger nuclear mRNA decay in association
with the nuclear pore complex (NPC; Galy et al., 2004) and
with transcription (Andrulis et al., 2002). Importantly, recent
reports in yeast, Drosophila and human lymphocytes have
revealed that dynamically regulated genes are recruited to
the nuclear periphery when transcription is activated (Ca-
solari et al., 2004; Faria et al., 2006; Mendjan et al., 2006;
Ragoczy et al., 2006; Taddei et al., 2006). It has been sug-
gested that the activation of genes in the nucleus is linked
to specialized NPCs that in turn facilitate efficient mRNA
export (Blobel, 1985). Supporting this hypothesis, gene
positioning at the proximity of the NPC has been shown
to be dependent on factors involved in transcription ini-
tiation and mRNA export (Cabal et al., 2006; Dieppois et
al., 2006; Kurshakova et al., 2007; Chekanova et al., 2008;
Ko ¨hler et al., 2008).
mRNP formation seems to play a key role not only in gene
expression but also in other cellular processes such as the
maintenance of genome integrity. An example of this con-
nection between mRNP formation and genetic integrity is
provided by the THO complex of Saccharomyces cerevisiae, a
conserved four-protein complex composed of stoichiometric
amounts of Tho2, Hpr1, Mft1, and Thp2 (Chavez et al., 2000),
which is recruited to active chromatin in vivo (Strasser et al.,
2002; Zenklusen et al., 2002). Null mutations of any compo-
nent of THO lead to similar phenotypes of transcription
impairment and RNA export defects (Chavez et al., 2000;
Strasser et al., 2002), the most intriguing phenotype being
their transcription-associated hyper-recombination. Analy-
sis of yeast THO mutants has led to the idea that transcrip-
tion-associated recombination (TAR) may be a consequence
of transcriptional-elongation impairment (Aguilera and Go-
mez-Gonzalez, 2008). One major cause of this phenomenon
is the cotranscriptional formation of R-loops (DNA-RNA
hybrids) formed behind the elongating RNAPII (Huertas
and Aguilera, 2003). In the current view, the THO complex
would participate in cotranscriptional formation of export-
competent mRNPs during transcription elongation prevent-
ing R-loop formation. The observation that depletion of the
ASF/SF2 splicing factor in chicken DT40 cells and human
HeLa cells also lead to genomic instability linked to R-loop
formation indicates that a number of mRNA-processing en-
zymes may contribute to prevent the formation RNA-depen-
This article was published online ahead of print in MBC in Press
on July 30, 2008.
* Present address: The Wellcome Trust/Cancer Research UK Gurdon
Institute, Tennis Court Road, Cambridge CB2 1QN, United Kingdom.
Address correspondence to: Andre ´s Aguilera (firstname.lastname@example.org).
4310© 2008 by The American Society for Cell Biology
dent structures that may trigger genome instability (Li and
THO forms, together with the RNA export proteins Sub2/
UAP56 and Yra1/Aly, a larger and conserved complex
termed TREX (Strasser et al., 2002; Rehwinkel et al., 2004).
Interestingly, yeast mutants of SUB2 and YRA1 are synthet-
ic-lethal with THO mutations and also lead to hyper-recom-
bination and gene expression defects (Fan et al., 2001; Jimeno
et al., 2002; Strasser et al., 2002). Furthermore, mutations in
the genes of the Mex67-Mtr2 export factor, the Nab2 hnRNP
or the NPC-associated Thp1 and Sac3 proteins also confer
hyper-recombination and gene expression defects (Gallardo
and Aguilera, 2001; Jimeno et al., 2002; Gallardo et al., 2003),
even though this is not a general feature of mRNA-process-
ing mutations (Luna et al., 2005). Despite some similarities
there are important differences between THO and Thp1 and
Sac3. Thus, Sub2 overexpression suppresses THO mutants,
but it inhibits growth of the Thp1 mutant. Also, Nab2 over-
expression suppresses the Thp1 mutant but has no effect on
THO mutants (Jimeno et al., 2002; Gallardo et al., 2003).
Notably, in contrast to THO, Thp1 and Sac3 associate with
nucleoporins at the nuclear basket and mediate export of
mRNPs (Fischer et al., 2002; Lei et al., 2003). In addition, Thp1
and Sac3 are found in association with Cdc31 centrin (Gal-
lardo et al., 2003; Fischer et al., 2004), which functions in the
duplication of microtubule-organizing centers, and with
Sus1, a small protein conserved from yeast to humans re-
cently identified as a novel component of SAGA histone-
modification complex involved in transcription initiation
(Rodriguez-Navarro et al., 2004; Zhao et al., 2008). The ob-
servation that Sus1 is involved in the SAGA-dependent
histone H2B deubiquitylation and maintenance of normal
H3 methylation levels (Ko ¨hler et al., 2006) and that Thp1,
Sac3, Sus1, and Ada2, a bona fide component of SAGA, act
in the repositioning and dynamic motility of SAGA-depen-
dent loci, to the nuclear periphery upon transcriptional ac-
tivation (Cabal et al., 2006; Kurshakova et al., 2007; Chek-
anova et al., 2008) suggests the possibility that Sus1 could be
a bridge protein between transcription, via SAGA, and
Given all these observations, the question emerging is
how RNA export factors, such as Thp1 and Sac3 control
genome integrity and whether they are functionally related
to THO/TREX, which is physically bound to active chroma-
tin and does not seem to be located at the nuclear periphery.
Another emerging question is whether or not the main func-
tion of Sus1 is linked to transcription initiation as part of the
SAGA complex. Here we show that Thp1, Sac3, and Sus1
form a functional unit with a role in transcription elongation
that is independent of SAGA and is linked to RNA export.
Our data reveal that the Thp1-Sac3-Sus1-Cdc31 (THSC)
complex, together with THO/TREX, define a specific path-
way connecting transcription elongation with nuclear ex-
port by an RNA-mediated dynamic process. This provides
a feedback mechanism for the control of transcription that
guarantees genetic stability of highly transcribed DNA
MATERIALS AND METHODS
Strains and Plasmids
Yeast strains used are listed in Supplemental Information Table S1 . Plasmids
pRS316L, pRS316LY?NS (Prado et al., 1997), pRS314L-lacZ, pRS314GL-lacZ
(Piruat and Aguilera, 1998), pCM184-LAUR (Jimeno et al., 2002), pCM189-
LEU2 (Gonzalez-Barrera et al., 2002), pGCYC1-402 (Rondon et al., 2003b),
pGL-ribm, pGL-Rib?, pPHO5-ribm-lacZ, pPHO5-Rib?-lacZ, pGAL:RNH1
(Huertas and Aguilera, 2003) p416-GAL1 (Mumberg et al., 1994), and
p413GALAID (Gomez-Gonzalez and Aguilera, 2007) were described previ-
ously. Plasmid Ptet-SUB2 containing the complete SUB2 coding sequence
under control of the tetO promoter was constructed by inserting the 1.3-kb
BamHI SUB2 fragment obtained by PCR using the primers 5?-ATC GCG GAT
CCA TGT CAC ACG AAG GTG AA-3? and 5?-CGC GCG GAT CCT TAA
TTA TTC AAA TAA GT-3? into pCM189 (Gari et al., 1997).
For chromatin immunoprecipitation (ChIP) experiments, strains were grown
in synthetic complete medium (SC) 2% glycerol-2% lactate to an OD660of 0.5.
The culture was split in two, and one-half was supplemented with 2% glucose
(repressed transcription) and the other with 2% galactose (activated transcrip-
tion). Samples were taken after 4 h, and ChIP assays were performed as
described (Hecht and Grunstein, 1999). Monoclonal anti-Rpb1-CTD antibody
8WG16 (Berkeley Antibody Company, Richmond, CA) and protein A-Sepha-
rose were used for RNAPII immunoprecipitation. The GFX purification sys-
tem (Amersham, Indianapolis, IN) was used for the last DNA purification
step. We used the PCR of the intergenic region at positions 9716–9863 of
chromosome V as a negative control. Real-time quantitative PCR and calcu-
lations of the relative abundance of each DNA fragment were performed as
described (Huertas et al., 2006).
In Vitro Transcription Elongation Assays
Transcription elongation was assayed in yeast whole cell extracts (WCEs) in
vitro. WCEs were prepared from yeast cells grown in rich medium YEPD at
30°C to an OD600of 1, and the reactions were carried as described previously
(Rondon et al., 2003b).
Recombination and Mutation Analysis
Recombination and mutation frequencies of the monocopy centromeric plas-
mids pRS316L, pRS316LY?NS, pRS314GL-lacZ, pRS314L-lacZ, pGL-ribm, and
pGL-Rib?described earlier were obtained as the average of three to four
median frequencies from two different transformants each and for each
genotype tested. Median frequencies were obtained as previously described
(Santos-Rosa and Aguilera, 1994) from six independent colonies per transfor-
Northern analyses were performed according to standard procedures with
32P-radiolabeled probes. Probes used were described previously (Chavez et
al., 2000). RNA analyses for the Rib?and ribmconstructs were performed as
described in Huertas and Aguilera (2003).
Overexpression of SUB2 Inhibits Growth of Mutants of
Thp1, Sac3, and Sus1, and Other mRNP Factors But Not
of SAGA Mutants
We have previously reported that overexpression of Sub2, a
component of TREX, strongly inhibited growth of thp1? cells
(Gallardo et al., 2003). Here we used this feature to obtain
new insights into the functional relationship of Thp1 and
Sac3 and other proteins involved in mRNP biogenesis. We
placed the SUB2 gene in a centromeric plasmid under the
control of the Tet promoter, which allowed the expression of
the gene in the absence of doxycycline (Ptet-SUB2), and
transformed different mutants of mRNP biogenesis factors
(Figure 1A). Figure 1B shows that growth of mutants of the
THSC complex as sac3? and sus1? were affected when SUB2
was overexpressed. The results suggest that Thp1, Sac3, and
Sus1 act as a unit that functionally interacts with Sub2. We
also analyzed two cdc31 mutants, (cdc31-1 and cdc31-115);
cdc31-1 is defective in spindle pole body (SPB) duplication,
cell integrity and morphogenesis (Sullivan et al., 1998),
whereas cdc31-115 is not affected in SPB duplication
(Ivanovska and Rose 2001) but has an mRNA export defect
(Fischer et al., 2004). Overexpression of Sub2 did not have
any effect on neither of these mutants.
Next, we analyzed the effect of SUB2 overexpression on 22
mutants in genes encoding proteins with a function in nu-
clear 5?-end cap binding, transcription termination, 3?-end
cleavage and polyA? tail addition, mRNA degradation,
mRNA export, and genes encoding mRNA-associated pro-
teins involved in other nuclear processes. As can be seen in
RNA Export and Genome Instability
Vol. 19, October 20084311
Figure 1C overexpression of SUB2 inhibited growth not only
of Thp1, Sac3, and Sus1 mutants, but also of mutants of
genes involved in other steps of mRNP biogenesis: polyade-
nylation (pap1-1), mRNA stability (rrp6?, rat1-1, and mtr4-1),
and mRNA export (mex67-5, yra1-1, nab2-1, gle1-4, and rat8-
2). Nevertheless, it had no effect in mutants of other nuclear
processes, such as protein transport (crm1-1) and the pro-
cessing of other RNA species (dbp7?) and in mutants in
genes encoding mRNA binding proteins (npl3?). Sub2 over-
expression inhibited growth of a number of mutants includ-
ing those in nucleoporins that interact with Thp1 and Sac3
(nup60?) and in nucleoporins of the Nup84 complex
(nup84?, nup133?, and nup120?), but not of other nucleo-
porins (nup2?, nup100?, nup188?, and nup170?; Figure 1C).
In addition, Sub2 overexpression inhibits growth of a mutant
of Nab2, an hnRNP (heterogeneous nuclear ribonucleoprotein)
1C). Therefore, our results suggest that the growth-defect
phenotype caused by overexpression of SUB2 is specific to a
subset of mRNP biogenesis and export factors that could
define a particular pathway.
Because Sus1 was identified as part of the SAGA histone
acetylase complex (Rodriguez-Navarro et al., 2004) and Sub2
overexpression inhibited growth of sus1? mutants, we asked
whether Sub2 overexpression also affected SAGA mutants.
Notably, we did not observe growth inhibition in mutants of
different representative genes of the functional and struc-
tural modules of SAGA (Figure 1D). This suggests that
despite the association of Sus1 with SAGA, a function of
Sus1 is directly related to Thp1 and Sac3 in mRNP biogen-
esis and export rather than SAGA.
sus1? But Not SAGA Mutants Confers
As SUS1 and CDC31 encode proteins that have been shown
to copurify with Thp1 and Sac3 (Fischer et al., 2002; Gallardo
et al., 2003; Rodriguez-Navarro et al., 2004), we wondered
whether their mutations also lead to increased TAR. Repre-
sentative mutants of the different modules of SAGA were
included in the study. For the analysis of transcription-
dependent recombination, we used the plasmid-based sys-
tem LY?NS based on 0.6-kb leu2 repeats in which transcrip-
tion has to proceed through a long and GC-rich intervening
sequence. In this system thp1? and sac3? lead to an increase
in recombination of two- to three orders of magnitude above
wild-type levels (Gallardo and Aguilera, 2001; Gallardo et
al., 2003). As control, we used the L system, identical to
LY?NS but without intervening sequences between the leu2
repeats and which is not significantly affected by thp1?.
Here we show that whereas sus1? mutant showed a clear
increase in recombination in LY?NS (5.5-fold) but no effect
in the L system, ubp8?, sgf11?, spt7?, spt8?, gcn5?, and
spt20? mutants showed low recombination levels in both the
L and LY?NS systems (Figure 2A). Besides, none of the
Cdc31 centrin mutants analyzed (cdc31-1 and cdc31-115)
showed hyper-recombination, neither at 30°C (Figure 2A)
nor at restrictive temperature 34°C (data not shown). Con-
sistent with Sub2 overexpression data (Figure 1), these re-
sults suggest that Sus1 share functions with Thp1 and Sac3
in the maintenance of genetic integrity. Although we found
no hyper-recombination in the cdc31 mutants tested,
whether or not Cdc31 has a related or more distant role to
the other subunits of the THSC complex would need to be
addressed with specifically selected cdc31 alleles, given that
Cdc31 is the only essential subunit of this complex.
Altogether, the data suggest that the hyper-recombination
phenotype of sus1? is transcription-dependent. To demon-
strate this, we determined the effect of sus1? on recombina-
tion in the L-lacZ and GL-lacZ systems carrying 0.6-kb leu2
direct repeats flanking the lacZ open reading frame under
conditions of low (GAL1 promoter in 2% glucose), medium
(LEU2 promoter), and high levels of transcription (GAL1
on mutants affected in different steps of mRNP
biogenesis and export. (A) Scheme of the plas-
mid Ptet-SUB2, containing the SUB2 gene under
the Tet promoter, which is expressed in the ab-
sence of doxycycline. (B) Growth of wild-type
W303–1A (WT), WFBE046 (thp1?), Y03517 (sac3?),
Y17455 (sus1?), and cdc31-115 and cdc31-1 strains
transformed with the plasmid Ptet-SUB2. Trans-
formants were spotted as 10-fold serial dilutions
on selective medium with and without doxycy-
cline (5 ?g/ml). (C) Effect of the overexpression
of SUB2 in mutants affected in RNA metabolism.
I, nuclear cap binding complex; II, 3?-end pro-
cessing and termination; III, nuclear mRNA deg-
radation; IV, mRNA export and RNA processing
steps; V, nucleoporins; and VI, protein nuclear
export and others. (D) Effect of the overexpres-
sion of SUB2 in SAGA mutants. Photographs
were taken after 3 d at 30°C, except for the nab2–1
mutant, which grows slowly and needed 5 d to
form colonies without doxycycline.
Effect of the overexpression of SUB2
C. Gonza ´lez-Aguilera et al.
Molecular Biology of the Cell4312
promoter in 2% galactose). As can be seen in Figure 2B and
Supplemental Figure S1, the higher the levels of transcrip-
tion the stronger the increase in recombination. The results
demonstrate that hyper-recombination in sus1? is mainly
transcription dependent as has been described for thp1 and
Transcription Elongation Is Impaired in THSC Mutants In
Vivo, But Only Slightly In Vitro
We have previously reported that thp1? and sac3? mutants
are defective in transcription through high G?C content
genes like lacZ (Gallardo et al., 2003). To test whether this is
also the case of sus1?, we analyzed gene expression in the
LAUR expression system (Jimeno et al., 2002) containing a
4.15-kb lacZ-URA3 translational fusion under the control of
the Tet promoter. As can be seen in Figure 3A, sus1? and
sac3? cells, carrying the LAUR system, were unable to form
colonies on synthetic complete medium lacking uracil
(SC-Ura-Trp), indicating that they did not express the
lacZ-URA3 fusion. Consistently, they did not produce ?-ga-
lactosidase activity (data not shown). Northern analysis
shows that whereas wild-type cells could express this con-
struct properly, sus1? and sac3? mutants showed a reduc-
tion in mRNA accumulation (Figure 3B), similar to that
observed for thp1? (Gallardo et al., 2003). Such a reduction
was not caused by an impairment of transcription initiation
at the Tet promoter because mRNA accumulation of the
LEU2 gene under the Tet promoter (pCM189-LEU2 expres-
sion system) was the same in sus1? and sac3? as in the wild
type (Figure 3B). We conclude, therefore, that sus1? lead to
similar gene-expression defects as those of thp1? and sac3?
The impairment of lacZ expression and the transcription-
associated recombination phenotypes of sus1?, thp1?, and
sac3? suggest that transcription elongation may be im-
paired, as was previously shown for THO mutants (Rondon
et al., 2003b). To determine whether THSC mutants were
impaired in transcription elongation we used our previously
reported in vitro system containing two G-less cassettes
(Rondon et al., 2003a), in which transcription-elongation ef-
ficiency is determined in whole cell extracts (WCEs) by the
values of the ratio of accumulation of the downstream
(376-nt long) versus the upstream (84-nt long) G-less RNA
fragments (Figure 4; see Materials and Methods). We assayed
in vitro transcription elongation in thp1?, sac3?, and sus1?.
In addition, we included in our analysis two mutants of
SAGA: ada2?, impaired in histone acetylation, and ubp8?,
mutated in the Ubp8-Sgf11 deubiquitinylating enzyme,
shown to control binding of Sus1 to SAGA (Ko ¨hler et al.,
2006), because recently both have been reported to be linked
to transcription elongation (Govind et al., 2007; Wyce et al.,
2007). As can be seen in Figure 4, the transcription-elonga-
tion efficiencies of thp1?, sac3?, sus1?, ada2?, and ubp8?
were above 80% of the wild-type values, in some cases close
to wild-type levels, whereas in the THO mutants used as
controls (tho2? and hpr1?) efficiencies were 60% or lower. As
transcription is coupled with mRNA export and THSC is
Recombination was analyzed in the plasmidic recombination sys-
tems L and LY?NS. A small diagram of each system used (not
drawn to scale) is shown. Repeats are shown as gray boxes, and
gray arrows indicate relevant transcripts produced by the con-
structs. Recombinants were selected as Leu?. All mutants were
analyzed together with their isogenic WT strains. Median and SD
are shown. (B) Recombination analysis of sus1? and thp1? mutants.
The frequency of Leu?recombination was determined in the plas-
mid-borne systems L-lacZ and GL-lacZ. Recombination frequencies
are plotted as a function of the transcription levels. Low transcrip-
tion refers to the GL-lacZ systems in strains cultured in 2% glucose;
medium refers to L-lacZ in 2% glucose, and high to GL-lacZ in 2%
galactose (for more details, see Supplemental Figure S1).
Recombination analysis of sus1 and SAGA mutants. (A)
capacity of BYSU-3B (sus1?) and TY13517–1D (sac3?) strains carry-
ing the Ptet::lacZ-URA3 fusion construct (plasmid pCM184–LAUR)
to form colonies on SC-Trp-Ura after 3 d at 30°C. (B) Northern
analysis of the expression of the Ptet::lacZ-URA3 and Ptet-LEU2
fusion constructs (plasmid pCM189-LEU2). RNA was isolated from
midlog phase cultures grown in SC-Trp. As a32P-labeled DNA
probe, we used the 3-kb BamHI lacZ fragment, the ClaI-EcoRV
LEU2 internal fragment, and an internal 589-base pair 25S rDNA
fragment obtained by PCR.
Transcription analysis of sus1? strain. (A) Analysis of the
RNA Export and Genome Instability
Vol. 19, October 20084313
located at the nuclear periphery in association with the NPC,
it is possible that the effect of the THSC complex on tran-
scription is relevant when coupled to the NPC, and not in
cell extracts in which coupling is disrupted.
Next, we analyzed RNAPII elongation in vivo. RNAPII
recruitment was assayed by ChIP at the 8-kb long YLR454w
gene fused to the GAL1 promoter (Mason and Struhl, 2005).
RNAPII occupancy was determined at a 5? and a 3? region of
YLR454w in thp1? and sac3? mutants, using hpr1? as a
control. Figure 5 shows that the presence of RNAPII at the
3?-end of the gene was reduced with respect to the 5?-end to
51 and 73% in thp1? and sac3? mutants, respectively. These
values were similar to those of hpr1?, indicating that the
RNAPII elongation is decreased in THSC mutants in vivo.
The Transcription and Hyper-Recombination Phenotypes
of THO/TREX and THSC Mutants Are Mediated by the
The transcription impairment and hyper-recombination
phenotypes of THO mutants have been shown to be depen-
dent on the nascent mRNA (Huertas and Aguilera, 2003). To
determine whether this was also the case for THSC and also
Sub2 mutants, this last component of the TREX complex, we
analyzed transcription in the previously described Rib?and
ribmconstructs (Figure 6A, Supplemental Figure S2), in
which a PHO5-Rib-lacZ transcriptional fusion containing ei-
ther a wild-type (Rib?) or a mutated (ribm) hammerhead
ribozyme sequence was placed under the control of the
GAL1 promoter (Huertas and Aguilera, 2003). In both con-
structs, a 2.2-kb-long mRNA is synthesized, but in the Rib?
construct the active hammerhead ribozyme cleaves the tran-
script, leading only to a short 0.6-kb-long mRNA fragment
downstream of the ribozyme. We analyzed transcription in
thp1?, used as representative mutant of the THSC complex,
and in sub2?, hpr1?, and tho2? as representative mutants of
THO/TREX. Northern analyses revealed that the thp1? mu-
tant was suppressed in the Rib?construct, as it was the case
for hpr1?, tho2?, and sub2? mutants. In such mutants only
30–40% of the transcription efficiency of the wild type can
be observed because this is the maximum level of tran-
scription reached with these types of constructs (Garcia-
Rubio et al., 2008).
Next, we asked whether ribozyme cleavage, together with
RNase H overexpression, was also capable of suppressing
hyper-recombination of THSC and other mRNP biogenesis
mutants. Hyper-recombination was assayed with the GL-
Rib?and GL-ribmrepeats systems containing PHO5, fol-
lowed by active ribozyme Rib?or inactive ribozyme ribm
sequences between 0.6-kb-long leu2 direct repeats, respec-
tively (Figure 6B). We have previously reported that the
major suppression of hyper-recombination of the hpr1?
strains was obtained when the ribozyme was active (GL-
Rib?) in the presence of highly expressed RNAse H that
would remove the RNA chain of a putatively formed R-loop
(Huertas and Aguilera, 2003). Here we show that hpr1?,
tho2?, sub2?, and thp1? strains carrying the GL-Rib?con-
THSC and SAGA mutants. (A) Scheme of the
two G-less cassette system of plasmid pGCYC1-
402 used for the analysis of in vitro transcription
elongation. RNase T1 treatment of the mRNA
driven from the GAL4-CYC1 promoter, which is
activated by purified Gal4-VP16, renders two
fragments corresponding to the G-less cassettes.
(B) In vitro transcription assays of WCEs from
BY4741 (WT), Y02937 (tho2?), SChY58a (hpr1?K),
Y01764 (thp1?), Y03517 (sac3?), Y17455 (sus1?),
Y04282 (ada2?), and Y00809 (ubp8?) strains. M is
the marker. Each reaction was stopped after 30
min, treated with RNaseT1, and run in a 6%
PAGE. Transcription reactions were made at
23°C with WCEs obtained from cells grown at
30°C. Two bands from each G-less cassette were
obtained, probably due to incomplete action of
RNaseT1. Efficiency of transcription elongation
was determined as the percentage of total tran-
scripts that reached the 376-nt G-less cassette
with respect to the transcripts that covered the
84-nt cassette. Radioactivity incorporated into
the G-less cassettes was quantified in a Fuji
FLA3000 (Tokyo, Japan) and normalized wi‘th
In vitro transcription elongation of
respect to the C content of each G-less cassette. The mean and SD of three independent experiments are shown.
mutants. ChIP analyses in wild-type, hpr1?, thp1?, and sac3? strains
carrying the GAL1p::YLR454w fusion construct located at the en-
dogenous YLR454w chromosomal locus. Strains used were WT-
YLR454 (WT), WHYL.2A (hpr1?), WThpYL-1D (thp1?), and
BSAC-4A (sac3?). The scheme of the gene and the PCR-amplified
fragments are shown. The DNA ratios in regions 5? and 3? were
calculated from the amounts of regions 5? and 3? relative to the
amounts of the intergenic region. The recruitment data shown are
referred to the value of the 5? region taken as 100%. ChIPs were
performed from three independent cultures, and quantitative PCRs
were repeated three times for each culture. Error bars, SDs.
RNAPII occupancy at the GAL1-YLR454w gene in THSC
C. Gonza ´lez-Aguilera et al.
Molecular Biology of the Cell 4314
struct and overexpressing RNaseH1 had significantly re-
duced recombination frequencies compared with those of
GL-ribm, as was previously shown for hpr1? mutants (Huer-
tas and Aguilera, 2003). Altogether the results suggest
that not only in THO complex, as shown with hpr1 and
tho2 mutants, but also in TREX, as shown with sub2, and
THSC, as shown with thp1, both the transcription impair-
ment and hyper-recombination are dependent on the nas-
THSC Inactivation Strongly Enhances the Mutator Ability
of Human Activation-induced Cytidine Deaminase Protein
Activation-induced cytidine deaminase (AID) is a specific
B-cell enzyme essential for immunoglobulin (Ig) somatic
hypermutation and class switching that acts in vitro on
single-stranded DNA, one of its in vivo targets being the S
regions of Ig genes, in which R-loops are formed (Mura-
matsu et al., 2000; Revy et al., 2000; Okazaki et al., 2002). We
have recently reported that the heterologous overexpression
of human AID is able to strongly induce both mutation and
recombination in yeast THO mutants (Gomez-Gonzalez and
Aguilera, 2007). This is explained by the fact that R loops
formed in THO mutants leave the nontranscribed chain
(NTS) as single-strand DNA (ssDNA), thereby increasing
accessibility to AID. Consistently, in mft1? cells expressing
AID, mutations were 10-fold higher in the NTS, whereas
such a strand bias was not observed in the wild type
(Gomez-Gonzalez and Aguilera, 2007).
We wondered, therefore, whether THSC inactivation by
thp1?, stimulated the action of AID, as an indirect manner to
assess whether R-loops also formed in THSC mutants. We
used the LAUR system. In this assay Ura?colonies are
selected in SC-FOA. As can be seen in Figure 7, AID expres-
sion increases the frequency of Ura?colonies fivefold in
wild-type cells and 19-fold in mft1 cells, consistent with
previously reported data (Gomez-Gonzalez and Aguilera,
2007). Noteworthy, the effect of AID was not specific to THO
mutants but was also observed in thp1? mutant, in which
AID increased mutations 58-fold. Such an increase was not
seen in spt4? strains and others mutants in factors involved
in transcription such as Spt6 and Rpb2 (data not shown).
and hyper-recombination of mRNP biogenesis and export mutants.
(A) Steady-state analyses of transcription of the ribmand Rib?fu-
sions in W303–1A (WT), SChY58a (hpr1?K), RK2–6C (tho2?), DLY23
(sub2?), and WFBE046 (thp1?) strains. The PHO5-ribm-lacZ (ribm)
and PHO5-Rib?-lacZ (Rib?) transcriptional fusions were under the
GAL1 promoter. They contain an inactive or active (respectively)
synthetically made 52-base pair ribozyme (Rib), followed by a 266-
base pair fragment of the U3 gene to prevent the cleaved RNA from
degradation, and the 369-base pairs PvuII 3?-end lacZ fragment at
the UTR of PHO5 (position ?1405). Samples where taken after
galactose addition at 0 or 120 min. One representative experiment is
shown in Supplemental Figure S2. Results from 120-min samples
were quantified and normalized with the endogenous U3 signal and
are represented as the percentage value with respect to wild type
taken as 100%. The average of three different experiments is plotted.
(B) Recombination frequencies in W303–1A (WT), SChY58a
(hpr1?K), RK2–6C (tho2?), DLY23 (sub2?), and WFBE046 (thp1?) cells
shown. All experiments were performed in 2% galactose to allow
expression of the direct repeats. Lack (?RNH1) or overexpression
(?RNH1) of RNase H1 was achieved with either p416-GAL1 or the
multicopy plasmid pGAL-RNH1 carrying RNH1 under the GAL1
promoter, respectively. Recombination frequencies are the median
value of six independent cultures. The average median value of 2–4
experiments and SD are shown.
Nascent mRNA dependency of the transcription defect
wild-type, mft1?, and thp1? mutants. Analyses of the genetic insta-
bility (mutation and recombination) phenotype in W303–1A (WT),
WHMK-1A (mft1?), and WFBE046 (thp1?) strains, using the
Ptet::lacZ-URA3 (pCM184-LAUR) fusion construct. Ura?mutants
are selected in synthetic medium (SC) with FOA. The human AID
gene, present in p413GALAID, was overexpressed in 2% galactose
medium. Median value of genetic instability frequency and SD of
3–4 different fluctuation tests are shown.
Spontaneous and AID-induced mutation frequencies in
RNA Export and Genome Instability
Vol. 19, October 20084315
Therefore, we can conclude that in THSC mutants, there is a
transcription-dependent transient accumulation of ssDNA
that facilitates AID action. This is consistent with the pres-
ence of R-loops that would leave the nontranscribed chain as
single-stranded, as in THO mutants (Go ´mez-Gonza ´lez and
We show here that Thp1, Sac3, and Sus1 form a functional
unit with a role in mRNP biogenesis and maintenance of
genomic integrity in the cell that is independent of the main
function of SAGA, but it is dependent on the nascent mRNA
molecule. The THSC complex acts in the mRNP biogenesis
pathway together with THO, Sub2, and the Mex67-Mtr2
export factor. We propose that eukaryotic transcription elon-
gation is controlled by an RNA-export–associated feedback
mechanism that prevents RNA-mediated genome instabil-
THO contributes to the formation of an optimal mRNP,
presumably facilitating the assembly of RNA-binding pro-
teins onto the nascent mRNA such Sub2 or Yra1. A defective
THO complex would lead to failure in this process that in
turn would create suboptimal mRNP that would not be
competent for export and would contribute to inhibit tran-
scription elongation and to trigger recombination, with the
concomitant formation of an R loop (Huertas and Aguilera,
2003). Here we show that THO is not the only complex in the
absence of which, failures of transcription-RNA export cou-
pling causes genome instability. Hyper-recombination and
mRNA accumulation defects were previously observed in
mutants of Sub2, Mex67, Thp1, and Sac3 (Jimeno et al., 2002;
Gallardo et al., 2003), but whether mutations in these genes
led to transcription-elongation impairment in an RNA-de-
pendent manner was not known.
It is worth noticing that Sus1 is part of two different
protein complexes, THSC and SAGA, and has been pro-
posed to act as a bridge between mRNA export and tran-
scription (Rodriguez-Navarro et al., 2004). Recent data sug-
gest that Sus1 could function in histone acetylation and
transcription in a SAGA-dependent manner and is necessary
for RNA export (Ko ¨hler et al., 2006; Zhao et al., 2008). Nev-
ertheless, our results suggest that Sus1 plays an important
role as part of the THSC complex in RNA biogenesis/export.
This is deduced from the observations that overexpression
of Sub2 inhibited the growth of sus1? cells, because thp1?
and sac3? and other RNA export factor mutants, such as
pap1, mex67, yra1, nab2, gle1,and dbp5, and nucleoporin mu-
tants such as nup60, nup84, nup133, and nup120, but not any
of the SAGA mutants tested, including ubp8 and sgf11, en-
coding the closest partners of Sus1 in the SAGA complex
(Ko ¨hler et al., 2006) and sgf73, mutated in the Sgf73 subunit
that mediate recruitment of Thp1-Sac3 to SAGA (Ko ¨hler et
al., 2008). It is likely that overexpression of Sub2 leads to an
aberrant mRNP structure causing an irreversible block of
mRNP biogenesis and export and hence growth inhibition in
THSC mutants deficient in RNA export. Besides, sus1? con-
fers a reduction in mRNA accumulation of lacZ similar to
thp1? and sac3?, whereas this transcription defect is not
observed in ubp8? and sgf11? mutants, the two SAGA sub-
units functionally linked to Sus1 (data not shown). Further-
more, sus1? mutants share the in vivo transcription impair-
recombination of thp1? and sac3? mutants; whereas SAGA
mutants show wild-type recombination phenotypes (Figure
2A and Supplemental Figure S3). Altogether, these data
suggest that Sus1 forms a functional unit with Thp1 and
Sac3 (THSC) with a role in mRNP biogenesis independent of
SAGA. Nevertheless, and as it happens with some subunits
of other protein complexes, such as Tex1 of TREX or Mft1 of
THO (Luna et al., 2005), the relevance of Sus1 in THSC seems
to be lower than that of Thp1 and Sac3, according to the
milder phenotypes of sus1 mutants. This would be in agree-
ment with the recent work on Sgf73 and the THSC complex
that indicates that this factor mediates the recruitment of
Thp1 and Sac3 to SAGA and their stable interaction with
Sus1-Cdc31 (Ko ¨hler et al., 2008).
The similarity of transcription and recombination pheno-
types of THSC mutants with those of THO and the obser-
vation that THSC plays a role in maintaining the nuclear
pore localization of genes (Cabal et al., 2006, 2008; Kursha-
kova et al., 2007) opens up the possibility that THSC could
also bind to active chromatin in a transcription-dependent
manner. Nevertheless, so far we have been unable to show
that Thp1-Sac3 is recruited to active chromatin (data not
shown). The observation that THSC mutants have a weak
effect on transcription elongation in vitro compared with
THO mutants is consistent with a role of THSC in mRNP
biogenesis that would be coupled to its function at the
nuclear pore. Our in vitro assays have been performed with
WCEs in which nuclear envelops are disrupted and the
DNA substrate is added independently and apart of NPCs.
Concordantly, the effect of a complex that interacts with the
nuclear pore as THSC (Fischer et al., 2002) may not be
properly detected with this in vitro assay, but it can be
observed in vivo assays performed with intact cells. So far
binding of Sus1 to GAL1 promoter has been reported, sug-
gesting that tethering of the DNA to the nuclear pore via
THSC could be via promoters, regardless of its transcrip-
tional state (Rodriguez-Navarro et al., 2004; Cabal et al., 2006;
Kurshakova et al., 2007), but this binding does not explain its
in vivo transcription-elongation impairment.
A key result to understand the specific transcription phe-
notypes of the mutants of this process is provided by the
analysis of the effect in different mutations of THO subunits,
Sub2 and Thp1, on ribozyme-containing transcription and
recombination assays and on hyper-mutation caused by hu-
man AID. Our study reveals that mutants in these proteins
lead to a DNA structure susceptible to the action of human
AID, as was recently shown in THO mutants (Gomez-
Gonzalez and Aguilera, 2007). This, together with the obser-
vation that AID acts preferentially on ssDNA (Chaudhuri et
al., 2003) is consistent with formation of R-loops in these
mutants, as has been shown for hpr1? (Huertas and Agu-
ilera, 2003) and the S regions of Ig genes where AID acts (Yu
et al., 2003). Therefore, in contrast to the idea that THO could
be a unique factor acting at transcription sites with a role
preventing the interaction of nascent RNA with the DNA,
other factors acting downstream on mRNP biogenesis and
export has similar effect. This implies a feedback mechanism
by which improperly formed mRNPs, presumably stacked
at the nuclear pore, have a backward effect promoting tran-
scription impairment and genetic instability. It is possible
that THSC-malfunction disrupts both RNA export and
mRNP assembly, causing transcription elongation impair-
ment via a mechanism similar to that occurring in THO
mutants and has yet to be deciphered.
Alternative mechanisms can explain the peripheral loca-
tion of activated genes. These may involve either promoter-
interacting proteins such as SAGA components or mRNP
biogenesis and processing factors such as the RNA export
factor Mex67 or the Mlp1 factor involved in mRNA surveil-
lance (Dieppois et al., 2006; Chekanova et al., 2008). Our data
suggests a model in which the biogenesis of mRNPs ends in
C. Gonza ´lez-Aguilera et al.
Molecular Biology of the Cell4316
the localization of the transcribed DNA at the proximity of
the NPC could be via the subsequent action of THO, Sub2-
Yra1, Mex67-Mtr2, and THSC in a transcription- and RNA-
mediated manner. This process would be independent of
SAGA and would prevent the generation of suboptimal
mRNPs that could react with DNA, compromising genome
integrity. The recent observation that the Mex67 export fac-
tor is recruited to chromatin in a transcription and THO-
dependent manner (Gwizdek et al., 2006) provides a new
scenario in which Mex67 may also be loaded onto the mRNP
during transcription to allow its subsequent export through
the NPC. This direct connection may explain the transcrip-
tion defects and hyper-recombination phenotype of mex67-5
mutants previously reported (Jimeno et al., 2002). Our re-
sults, therefore, define a specific pathway that controls the
fate of the mRNA from the site of transcription to the nuclear
pore as a key process in the maintenance of genome integ-
rity. Presumably, these protein factors from THO to THSC
may function on the nascent RNA in a dynamic process
starting on the DNA and finishing up in close proximity to
the NPC. To unravel why THSC has a feedback effect in
transcription elongation and genome integrity will help un-
derstand how different nuclear processes are interconnected
and the key function of these mRNP biogenesis and export
factors in maintaining genome integrity.
We thank E. Hurt (Universit¨ at Heidelberg, Heidelberg, Germany) and M.
Rose (Princeton University, Princeton, NJ) for yeast cdc31 mutants, S. Jimeno
for helpful comments on the manuscript, and D. Haun for style supervision.
This work was supported by grants from the Spanish Ministry of Science and
Education (SAF2003-00204 and BFU2006-05260) and Junta de Andalucı ´a
(CVI102 and CVI624). C.G.-A. and B.G.-G. were the recipients of (Formación
Profesorado Universitario) Ph.D. training grants from the Spanish Ministry of
Science and Education.
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