R-Loop Mediated Transcription-Associated Recombination in trf4Δ Mutants Reveals New Links between RNA Surveillance and Genome Integrity
Aguilera A (2013) R-Loop Mediated Transcription-Associated Recombination in trf4D Mutants Reveals New Links between
RNA Surveillance and Genome Integrity. PLoS ONE 8(6): e65541. doi:10.1371/journal.pone.0065541
R-Loop Mediated Transcription-Associated Recombination in trf4 D Mutants Reveals New Links between RNA Surveillance and Genome Integrity
Sandra Gavalda 0
Mercedes Gallardo 0
Rosa Luna 0
Andre s Aguilera 0
Sebastian D. Fugmann, Chang Gung University, Taiwan
0 1 Departamento de Biolog a Molecular, Centro Andaluz de Biolog a Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla , Seville , Spain , 2 Departamento de Gen e tica, Universidad de Sevilla , Seville , Spain
To get further insight into the factors involved in the maintenance of genome integrity we performed a screening of Saccharomyces cerevisiae deletion strains inducing hyperrecombination. We have identified trf4, a gene encoding a noncanonical polyA-polymerase involved in RNA surveillance, as a factor that prevents recombination between DNA repeats. We show that trf4D confers a transcription-associated recombination phenotype that is mediated by the nascent mRNA. In addition, trf4D also leads to an increase in the mutation frequency. Both genetic instability phenotypes can be suppressed by overexpression of RNase H and are exacerbated by overexpression of the human cytidine deaminase AID. These results suggest that in the absence of Trf4 R-loops accumulate co-transcriptionally increasing the recombination and mutation frequencies. Altogether our data indicate that Trf4 is necessary for both mRNA surveillance and maintenance of genome integrity, serving as a link between RNA and DNA metabolism in S. cerevisiae.
Funding: This work was supported by grants from the Spanish Ministry of Science and Research (BFU2010-16372 and Consolider Ingenio 2010 CSD2007-00015),
Junta de Andaluca (CVI-4567) and European Union (FEDER). SG was the recipient of a predoctoral training grant from the Spanish Ministry of Health. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Maintenance of genome integrity is critical for cell homeostasis.
Cells posses multiple mechanisms such as specific DNA repair
pathways or cell cycle checkpoints to deal with DNA damage and
the resulting genetic instability commonly associated with cancer
and several genetic disorders . Genomes are exposed to the
action of physical and chemical agents, and metabolic processes
that can cause lesions in the DNA. One such process is
transcription, which has been established as an inducer of genome
instability. Recombination and mutation frequencies are enhanced
by transcription, leading to transcription-associated recombination
(TAR) and transcription-associated mutation (TAM) [2,3]. Key to
understanding how transcription increases genomic instability is
the fact that single-stranded DNA (ssDNA) is chemically more
unstable than double-stranded DNA (dsDNA). Transcription itself
and changes in topology and chromatin conformation associated
with it may increase the probability of the occurrence of ssDNA.
Consistently, DNA-damaging agents show a synergistic effect with
transcription in the induction of recombination in yeast , and
mutation rates correlate with the strength of transcription and
superhelical stress . In addition to a major ssDNA accessibility,
transcription associated genomic instability could also be the result
of the collision between the transcription and replication
machineries [6,7]. A possible intermediate of
transcriptionassociated genomic instability is an R-loop structure consisting of
a RNA:DNA hybrid that displaces the non-template ssDNA
strand. R-loops are transcription by-products rarely formed in the
cell but they accumulate in a number of transcription and mRNP
mutants with a genetic instability phenotype .
During transcription, the nascent pre-mRNA associates with
mRNA-binding proteins and undergoes a series of processing steps
resulting in an export competent mRNA ribonucleoprotein
complexes (mRNP) [9,10]. Emerging evidence suggest that when
mRNP biogenesis does not occur properly the RNA can hybridize
with the DNA template, forming R-loops that would hinder
transcription elongation and block replication. One of the best
studied examples is the THO complex, which functions at the
interface transcription-mRNA export. Mutations in THO lead to
a transcription-associated hyperrecombination phenotype partially
suppressed by overexpression of RNase H, an enzyme that
degrades the RNA strand of DNA:RNA hybrids . Moreover,
in these mutants genome instability is exacerbated by the action of
the human cytidine deaminase AID that acts on the displaced
ssDNA of R-loops [12,13]. Similar R-loop-dependent
co-transcriptional genome instability is observed in mammalian and
chicken DT40 cells depleted of the ASF/SF2 splicing factor .
More recently, mutations in topoisomerase I, SenI/SENATAXIN
and Sin3 have also been reported to cause genome instability via a
common mechanism [15,16,17,18]. In addition, a number of
RNA processing factors have been shown to be relevant for the
maintenance of genome integrity by preventing R-loop
accumulation by different genetic and cellular approaches in yeast and
human cells [17,19,20].
In Saccharomyces cerevisiae, screenings based on marker stability
provide a powerful approach for studying genes that preserve
genome structure [21,22]. These screenings exploit the use of
artificial chromosome (YAC) and endogenous loci to measure
genome instability events such as gross chromosomal
rearrangements (GCR) and chromosome loss. Artificially constructed
DNA repeats have also been validated as models to study
genomic instability involving homologous recombination
[23,24]. To get further insight into the factors implicated in
the maintenance of genome integrity we performed a screening
of S. cerevisiae deletion strains for hyperrecombinant mutations,
using different systems based on differentially transcribed
DNArepeats. We identified mutations that increase recombination in
seven genes, four related with RNA metabolism, ranging from
transcription to translation. Notably, among these mutations we
found that deletion of TRF4, a polyA-polymerase of the
TRAMP complex (Trf4/5-Air1/2-Mtr4 polyadenylation) that
plays a role in RNA surveillance [25,26,27], confers a
transcription-associated hyperrecombination phenotype that is
mediated by the nascent mRNA. We provide genetic evidence
that R-loops are formed in trf4D cells, such structures being
responsible of the increase in recombination and mutation
frequencies. Our data indicate that Trf4 is necessary for the
maintenance of genome integrity, providing a link between
mRNA surveillance and DNA metabolism in S. cerevisiae.
Materials and Methods
Strains and Plasmids
Yeast strains used are listed in Table 1. Plasmids pRS314L,
pRS316L, pRS314LY, pRS316LY, pRS314SU, pRS316SU and
pRS316-LYDNS , pRS314L-lacZ, pRS314GL-lacZ ,
pGL-ribm, pGL-Rib+, pGAL:RNH1  p413GAL1,
p416GAL1  and p413GALAID  were used to determine
recombination frequencies. Plasmid pCM184-LAUR was used for
the analysis of mRNA expression levels as previously described
. Plasmids pNOPPATA1L, pNOPPATA1L-TRF4-WT and
pNOPPATA1L-TRF4-DADA kindly provided by W. Keller, have
been previously described .
Recombination and Mutation Analysis
Recombination frequencies were determined as described .
For each strain, the recombination frequencies are given as the
average and standard deviation of the median recombination
value obtained from fluctuation tests performed in 34 different
transformants using 6 independent colonies per transformant.
Recombinants were selected as Leu+ colonies for the plasmid
containing LEU2 truncated repeat systems. Recombination
analyses for the chromosomal leu2-k::ADE2-URA3::leu2-k system
(Lk-AU) were performed in wild-type and congenic mutants using
6 to 12 independent colonies grown in synthetic complete medium
SC, and recombinants were selected in SC+FOA.
Mutation frequencies were determined in wild-type and mutant
strains using the Ptet::lacZ-URA3 (pCM184-LAUR) fusion
construct. Ura_ mutants were selected in SC+FOA. The human AID
gene, present in p413GALAID, was used for overexpression in 2%
galactose medium. Median mutation frequencies were obtained by
fluctuation tests performed in 34 different transformants using 6
independent colonies per transformant.
b-galactosidase assays and Northern analyses were performed
according to previously published procedures .
New proteins involved in genome instability
To identify novel genes with a role in genome stability, we
performed a screening of S. cerevisiae deletion strains for
hyperrecombinant mutants. We analyzed a total of 610 viable
deletion strains constructed by the EUROFAN consortium. All
strains were transformed with pRS314 and pRS216 centromeric
plasmids carrying three different recombination systems, L, LY
and SU, as described previously . These systems are based on
direct (L and LY) or inverted (SU) repeats of a 0.6 kb internal
fragment of the LEU2 ORF generated with two truncated copies
of the LEU2 gene (leu2D39 and leu2D59) spaced by different DNA
sequences. Deletions (L and LY systems) and inversions (SU) were
scored as Leu+ events and quantified by fluctuation tests. Among
the strains analyzed, we found seven deletion mutants that
conferred a hyperrecombinant phenotype (Figure 1). Four out of
these mutants correspond to genes involved in RNA related
processes: MED2, a subunit of the RNA polymerase II mediator
complex ; RPL13A, a component of the large (60S) ribosomal
subunit ; LSG1, a GTPase involved in 60S ribosomal subunit
biogenesis , and TRF4, a component of the TRAMP complex
involved in RNA surveillance [25,26,27]. The other three mutants
were in TOS3, a redundant kinase that activates the Snf1/AMPK
pathway that controls nutrient and environmental stress response
; ART1, involved in regulating the endocytosis of plasma
membrane proteins , and APC9, involved in the regulation of
protein stability . Next, we measured the frequency of
directrepeat recombination in the chromosomal
leu2-k::ADE2-URA3::leu2-k system. We constructed the different mutant strains carrying
this chromosomal system and recombination leading to
uradeletions was scored. As shown in Figure 1, all mutants showed
similar recombination frequencies to those of the wild-type strain,
except trf4D. Thus we decided to focus our work on trf4D because
it showed a hyperrecombination phenotype in all direct-repeat
systems assayed, regardless of whether they were in plasmids or
trf4D mutants confer transcription-dependent
We observed that the hyperrecombination phenotype of trf4D
for the direct-repeat systems analyzed seems to be
transcriptiondependent (Figure 1). Recombination frequencies in trf4D strains
were 2.6 and 8.7 times the WT levels for the L and LY
systems, respectively. Both systems are based on the same direct
repeats (an internal fragment of the LEU2 gene) and differ in
the length of the intervening sequence (31bp for L, and 5.57kb
for LY) . As in trf4D cells the recombination frequency is
higher when there is a long DNA fragment transcribed between
the two direct repeats, we wondered if deletion of TRF4 indeed
conferred a transcription-dependent genetic instability
phenotype. To test this, we determined the effect of trf4D on
recombination in the L-lacZ and GL-lacZ systems carrying
0.6kb leu2 direct repeats flanking the lacZ ORF under conditions of
low (GAL1 promoter in 2% glucose), medium (LEU2 promoter)
and high levels of transcription (GAL1 promoter in 2%
galactose). As can be seen in Figure 2, the higher the strength
of transcription the stronger the increase in recombination.
Altogether, the data indicate a statistically significant increase in
recombination levels in trf4D cells respect to the wild-type that is
MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1-1 ura3-1
MATa ura3-52 his3D200 leu2D1 trp1D63
MATa ura3-52 his3D200 leu2D1 LYS2 trp1D63 lsg1D::KAN
MATa ura3-52 his3D200 leu2D1 LYS2 TRP1 rpl13A:: KAN
MATa ura3-52 his3D200; leu2D1 LYS2 TRP1 tos3D:: KAN
MATa ura3-52 his3D200 leu2D1 LYS2 trp1D63 apc9D:: KAN
MATa ura3-52 his3D200 leu2D1 LYS2 TRP1 art1D:: KAN
MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1-1 ura3-1 trf4D:: KAN
MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1-1 ura3-1 med2D:: KAN
MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1-1 ura3-1 trf4D:: KAN
MATa ade2 his3 trp1 ura3 leu2-k::ADE2-URA3::leu2-k
MATa ade2 his3 trp1 ura3 leu2-k::ADE2-URA3::leu2-k lsg1D:: KAN
MATa ade2 his3 trp1 ura3 leu2-k::ADE2-URA3::leu2-k rpl13AD:: KAN
MATa ade2 his3 trp1 ura3 leu2-k::ADE2-URA3::leu2-k tos3D:: KAN
MATa ade2 his3 trp1 ura3 leu2-k::ADE2-URA3::leu2-k apc9D:: KAN
MATa ade2 his3 trp1 ura3 leu2-k::ADE2-URA3::leu2-k art1D:: KAN
MATa ade2 his3 trp1 ura3 leu2-k::ADE2-URA3::leu2-k trf4D:: KAN
MATa ade2 his3 trp1 ura3 leu2-k::ADE2-URA3::leu2-k med2D:: KAN
The hyperrecombination phenotype of trf4D mutant is
mediated by the nascent mRNA
The length and high GC content of lacZ gene makes
transcription through this sequence poorly efficient in mutants
impaired in transcription elongation [40,41]. As lacZ
transcription impairment was linked in many cases to
hyperrecombination phenotype in mutants of THO and other mRNP factors
[42,43], we explored whether lacZ transcription was also
affected in trf4D mutants. For this purpose, we analyzed gene
expression in the LAUR expression system  that contains a
4.15-kb lacZ-URA3 translational fusion under the control of the
Tet promoter. Defects in lacZ expression were determined as
poor growth in the absence of uracil and as lack of
galactosidase activity. As shown in Figure 3A trf4D cells behave
as wild-type cells, suggesting that this mutant does not have a
negative effect on transcription of the lacZ-URA3 fusion.
Figure 1. Recombination analyses of med2, lsg1, trf4, rpl13A, tos3, art1 and apc9 mutants. A diagram of each recombination system (not
drawn to scale) is shown at the top. Repeats are shown as gray boxes. Arrows indicate relevant transcripts produced by the constructs. For the L, LY
and SU systems, recombination frequencies were determined in wild-type (FY1679) and mutant strains transformed with plasmids pRS314-L and
pRS314-LY carrying the leu2 direct-repeat systems, and pRS314-SU carrying an inverted repeats system. Recombinants were selected as Leu+. The
average median value and SD of 34 fluctuation tests are shown. Recombination frequencies of med2D (MGY1-2D), lsg1D (AFGL-7D), trf4D (AWT4-1C),
rpl13AD (WFDL-1D), tos3D (AFGL-2D), art1D (AFOR-1A), apc9D (WFLR-2B) and wild-type (MGY6-1A) congenic strains carrying the chromosomal
leu2k::ADE2-URA3::leu2-k system are shown. For recombination analyses, independent colonies were obtained from SC and recombinants were selected in
Moreover, northern analyses show that lacZ mRNA levels are
much higher in trf4D mutants than in wild-type cells (Figure 3B),
consistent with the previously described role of this protein in
mRNA degradation .
Our data indicate that although trf4 mutants show a
transcription-dependent hyperrecombination phenotype
(Figure 2), transcription seems not to be affected. Instead,
higher amounts of mRNAs are accumulated, probably as a
result of the defect in mRNA decay mediated by TRAMP. As
the hyperrecombination phenotypes of several mRNP mutants
depend on the nascent mRNA [11,44], we analyzed whether
this is the case for trf4 mutants. We measured recombination in
the GL-Rib+ and GL-ribm repeats systems , which contain
the sequence of the PHO5 gene followed by either an active
(Rib+) or inactive (ribm) hammerhead ribozyme respectively,
located between two 0.6-kb-long leu2 direct repeats (Figure 4A)
under the control of the inducible GAL1 promoter. Both the
Rib+ and ribm constructs synthesize a long mRNA, but upon
transcription the active hammerhead ribozyme cleaves the
transcript shortening the mRNA fragment still attached to the
polymerase . Figure 4B shows that in trf4D strains
recombination levels in the GL-Rib+ construct was lower than
in GL-ribm, close to those of the wild type. The suppression of
the hyperecombination phenotype by the ribozyme suggests that
long nascent mRNAs contribute to the genetic instability in the
absence of Trf4, therefore implicating that hyperrecombination
was mediated by the RNA molecule.
Hyperrecombination in trf4D cells does not depend on
the catalytic polyadenylation domain of Trf4
As Trf4 is a cofactor of the TRAMP complex involved in
RNA surveillance, we determined whether hyperrecombination
was dependent on its polyadenylation activity. We measured the
recombination frequency in trf4D cells carrying the
chromosomal leu2-k::ADE2-URA3::leu2-k system transformed with a
plasmid expressing either the wild-type TRF4 allele or the
polyadenylation-defective allele TRF4-DADA under the control
of the NOP1 promoter . Trf4-DADA contains two aspartate
to alanine mutations in the catalytic site of the
polyApolymerase that render the enzyme inactive .
Recombination levels were significantly reduced in trf4D cells by the
overexpression of the TRF4-DADA allele, reaching values close
to those of cells complemented with the wild-type TRF4 allele
(Figure 5). This suggests that hyperrecombination in trf4D cells
takes place through a mechanism that is independent of its
Genetic instability in trf4D is mediated by R-loops
Next we determined whether the mRNA dependency of the
hyperrecombination phenotype of trf4D cells was linked to the
cotranscriptional formation of R-loops. To address this possibility we
assayed the effect of RNase H overexpression in the trf4D mutant
carrying the direct-repeat recombination system LYDNS . As
can be seen in Figure 6 the hyperrecombination phenotype of
trf4D was suppressed by the overexpression of RNase H.
As we have previously shown that as a consequence of R-loop
formation expression of AID is able to strongly induce both
mutation and recombination in yeast THO mutants , next we
analyzed whether this was also the case for trf4D cells. As can be
seen in Figure 6 AID expression increased recombination in trf4D
mutants 8.2 times above the WT levels, which was suppressed by
RNase H overexpression, consistent with the conclusion that
Rloops are formed in trf4D mutants. To confirm this, we assayed the
effect of AID expression on the mutation frequency in trf4D. We
analyzed the frequency of Ura- mutations in the LAUR expression
system. We observed that AID expression increased the frequency
of Ura- colonies 3-fold in wild-type cells, consistent with previously
reported data . However this increase was of 13-fold in trf4D
cells (Figure 7). As expected if this specific enhancement of trf4D
was linked to R-loop formation, overexpression of RNase H
reduced the frequency of Ura- mutations to values close to those of
the wild-type (Figure 7). Altogether the data indicate that R-loops
accumulate in the absence of TRF4 and mediate the genomic
instability of trf4D cells.
Here, we show the results of a screening for mutations that
increased homologous recombination between repeated DNA
fragments in yeast using genetic assays based on artificially
constructed DNA repeats. We identified different mRNP
biogenesis and transcription related proteins, as well as other factors,
whose deletions lead to an increase in recombination in plasmid
borne assays. We focused our studies in TRF4, a factor involved in
RNA surveillance, because its absence causes a
transcriptionassociated hyperrecombination phenotype both in chromosome
and plasmid-borne systems. Using different genetic tools we show
that this phenotype is dependent on the presence of the nascent
mRNA and is mediated by R-loops. Our results therefore provide
a new link between RNA quality control and genetic instability
Our screening has permitted to identify a number of
transcription and RNA related factors as suppressors of genome
instability (Figure 1). These include MED2, one subunit of the
transcription Mediator complex; RPL13A, a ribosomal subunit;
LSG1, a GTPase involved in ribosomal biogenesis; and TRF4, a
poly(A) polymerase of the TRAMP complex. Our results provide
new evidence for the link between mRNA biogenesis and genome
instability. Mutants affecting various steps of transcription, from
initiation to termination, and RNA processing have been shown to
lead to an increase of cH2A foci, YAC instability or
hyperrecombination in yeast and human cells [14,17,19,20,31,43,44]. In
addition, our screening identified other non-RNA related factors
whose deletion could have an indirect impact on genome
instability: APC9, encoding a subunit of the Anaphase-Promoting
Complex/Cyclosome (APC/C), and TOS3, considered as the
functional orthologous of LKB1, a mammalian kinase associated
with Peutz-Jeghers cancer-susceptibility syndrome (Figure 1).
Several studies have explored the role of LKB1 as a major actor
of the AMPK/mTOR pathway connecting cellular metabolism,
cell growth and tumorigenesis .
We have focused our interest in deciphering the genetic basis of
genome instability in trf4D mutants. TRF4 is a non-canonical
polyA-polimerase that acts as a cofactor of the exosome complex
for the quality control of different types of RNAs .
Interestingly, it was originally isolated in a synthetic growth screen
with top1 (topoisomerase one-requiring function) . This is a
notable observation because THO mutations also show a synthetic
growth defect with top1  and indeed hpr1D was also recovered
in that screen. In addition, trf4 interacts genetically with mutations
in different components of transcription, histone modification and
histone remodeling complexes, and proteins involved in cohesion
and DNA repair (reviewed in ). Beside its function in RNA
surveillance, Trf4 has been shown to control chromatid cohesion,
mitotic chromosome condensation and mitotic segregation
[50,51,52], rDNA copy number , and telomere length .
Therefore, it seems that Trf4 plays a role in DNA metabolism of
We show that TRF4 prevents genetic instability, as trf4D was
identified as in our screening with recombination systems
containing truncated repeats of the LEU2 gene (Figure 1).
Modulating the transcription levels of the repeats through
constitutive and regulatable promoters, we have demonstrated
that the hyperrecombination phenotype of trf4D is
transcriptiondependent (Figure 2). In addition, the hyperrecombination
phenotype of trf4D cells can be suppressed by the action of a
ribozyme inserted at the nascent mRNA as well as by RNase H
overexpression (Figure 4 and Figure 6). The data indicate that
RNA:DNA hybrids accumulate in the absence of this mRNA
surveillance factor. Interestingly, we have previously reported that
mutation in RRP6, the exonuclease subunit of the nuclear
exosome, has an effect on transcription elongation and genome
integrity . Recently, it has been shown that deletion of TRF4,
and of other mRNA surveillance factors, such as KEM1 (an
exonuclease involved in cytoplasmic mRNA decay), AIR1 (a
RNAbinding protein of the TRAMP complex) and RRP6 lead to
elevated GCRs in the form of terminal deletions and
minichromosome losses using YACs. These events were partially
suppressed by RNAse H overexpresssion , although its
dependency on transcription was not established. Our results
indicate that Trf4 is a factor that prevents different forms of
genome instability, including that associated with transcription
(Figure 1 and Figure 2). Importantly, we provide evidence that
both recombination and mutation were enhanced in trf4D mutants
(Figure 7). Altogether, our data suggest that cotranscriptional
Rloop are responsible for both phenotypes, consistent with the
exacerbated recombination and mutation phenotypes of trf4D cells
upon AID cytidine deaminase overexpression (Figure 6 and
The impact of mutations in the RNA surveillance machinery on
genome integrity reveals the global relevance of RNA metabolism
in genome dynamics. Interestingly, in mammalian cells the core
nuclear exosome subunit Rrp40 has been shown to be recruited to
S regions of Ig genes and to be required for optimal class switching
recombination . It has been proposed that the exosome could
provide the ribonuclease activity for degradation of the RNA
strand of RNA-DNA hybrids exposing the template DNA strand
to AID activity , but this is in principle unrelated with the
AID-independent phenomenon described here. As AID induces
mutation and recombination in different yeasts cells [55,13],
including trf4 cells (this study) we believe that the loss of Trf4 may
cause an accumulation of transcripts at the site of transcription
with the potential to form R-loops, which are highly susceptible of
AID, as previously shown for THO mutants .
R-loops structures accumulate in different mutants of mRNP
biogenesis. mRNA processing and transcription factors could
prevent R-loop formation by facilitating assembly of the nascent
mRNA into a ribonucleoprotein particle, therefore limiting its
ability to rehybridize with the template DNA strand (reviewed in
). Accumulative data indicate that R-loops in THO mutants
hinder transcription elongation and generate recombinogenic
structures that could represent an obstacle for the replication
machinery [11,56,57,58]. However, these properties are not
shared by every mutant impairing mRNA biogenesis . Indeed
trf4D mutants do not show a reduction in the expression level the
GC-rich lacZ gene from E. coli (Figure 3), in contrast to mutants of
THO/TREX and other transcription elongation factors [31,44].
On the other hand, mRNA processing and assembly into an
export-competent mRNP is a tightly regulated process and a
defect in mRNA processing can affect downstream steps
[10,59,60]. TRAMP together with the nuclear exosome have
been proposed to mediate a quality-control checkpoint activated
upon mRNA export blockage . Therefore, it is possible that
aberrant mRNA transcripts that escape degradation in trf4D cells
hybridize with the DNA contributing to R-loop formation and
TRAMP plays a role in polyadenylation and stimulates RNA
degradation mediated by the nuclear exosome [25,26,27].
However, polyadenylation is not essential for active degradation
in vitro  and a polyadenylation-defective Trf4 protein is fully
active, suggesting that mRNA degradation triggered by Trf4 is
independent of its polyadenylation activity . Indeed,
genomewide expression analysis shows that the overexpression of
TRF4DADA restores the levels of most RNAs with an altered expression
in trf4D cells, except a small fraction corresponding to highly
expressed and structured RNAs . The fact that
hyperrecombination phenotype of trf4D cells is suppressed by the
overexpression of the TRF4-DADA mutant allele (Figure 5)
indicates that occurs via a polyadenylation-independent
mechanism. Interestingly, other DNA-related phenotypes, such as the
maintenance of telomere, have also been shown to be
polyadenylation independent . In addition, although Trf4 has been
defined as a non-canonical poly-A-polymerase playing a role in
RNA surveillance, its function role is not restricted to RNA
degradation, but rather contributes to the processing of different
RNAs such as tRNAs, snoRNA, snRNAs and rRNA precursors
[32,62,63]. Indeed, Trf4 has been shown recently to be associated
with introns in vivo, as shown by
crosslinking-RNA-immunoprecipitation, and to regulate degradation of spliced-out introns .
Therefore Trf4 could link RNA processing with the maintenance
of genome integrity.
A number of reports suggest that R-loops are formed at a higher
frequency in the genome than previously anticipated with an
impact in both gene expression and genome integrity . Given
the role of Trf4 in the processing and degradation of different
types of RNAs , it is also possible that the loss of rDNA repeats
observed previously in trf4D cells, could be associated with the
formation of R-loops [53,15]. In summary our work suggests that
trf4D leads to a general transcription-associated genome instability
phenotype that is mediated by the cotranscriptional formation of
R-loops, providing a further connection between genome
dynamics and RNA metabolism.
We thank W. Keller, S. Vanacova and A. Corbett for plasmid gifts, A.G.
Rond on for critical reading of the manuscript, and Diane Haun for style
Conceived and designed the experiments: SG MG RL AA. Performed the
experiments: SG MG RL. Analyzed the data: SG MG RL AA. Wrote the
paper: RL AA.
1. Negrini S , Gorgoulis VG , Halazonetis TD ( 2010 ) Genomic instability-an evolving hallmark of cancer . Nat Rev Mol Cell Biol 11 : 220 - 228 .
2. Kim N , Jinks-Robertson S ( 2012 ) Transcription as a source of genome instability . Nat Rev Genet 13 : 204 - 214 .
3. Aguilera A ( 2002 ) The connection between transcription and genomic instability . EMBO J 21 : 195 - 201 .
4. Garcia-Rubio M , Huertas P , Gonzalez-Barrera S , Aguilera A ( 2003 ) Recombinogenic effects of DNA-damaging agents are synergistically increased by transcription in Saccharomyces cerevisiae. New insights into transcriptionassociated recombination . Genetics 165 : 457 - 466 .
5. Schmidt KH , Reimers JM , Wright BE ( 2006 ) The effect of promoter strength, supercoiling and secondary structure on mutation rates in Escherichia coli . Mol Microbiol 60 : 1251 - 1261 .
6. Aguilera A , Gomez-Gonzalez B ( 2008 ) Genome instability: a mechanistic view of its causes and consequences . Nat Rev Genet 9 : 204 - 217 .
7. Bermejo R , Lai MS , Foiani M ( 2012 ) Preventing replication stress to maintain genome stability: resolving conflicts between replication and transcription . Mol Cell 45 : 710 - 718 .
8. Aguilera A , Garcia-Muse T ( 2012 ) R loops: from transcription byproducts to threats to genome stability . Mol Cell 46 : 115 - 124 .
9. Luna R , Gaillard H , Gonzalez-Aguilera C , Aguilera A ( 2008 ) Biogenesis of mRNPs: integrating different processes in the eukaryotic nucleus . Chromosoma 117 : 319 - 331 .
10. Perales R , Bentley D ( 2009 ) "Cotranscriptionality": the transcription elongation complex as a nexus for nuclear transactions . Mol Cell 36 : 178 - 191 .
11. Huertas P , Aguilera A ( 2003 ) Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination . Mol Cell 12 : 711 - 721 .
12. Dominguez-Sanchez MS , Barroso S , Gomez-Gonzalez B , Luna R , Aguilera A ( 2011 ) Genome instability and transcription elongation impairment in human cells depleted of THO/TREX . PLoS Genet 7 : e1002386 .
13. Gomez-Gonzalez B , Aguilera A ( 2007 ) Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex . Proc Natl Acad Sci U S A 104 : 8409 - 8414 .
14. Li X , Manley JL ( 2005 ) Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability . Cell 122 : 365 - 378 .
15. El Hage A , French SL , Beyer AL , Tollervey D ( 2010 ) Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis . Genes Dev 24 : 1546 - 1558 .
16. Mischo HE , Gomez-Gonzalez B , Grzechnik P , Rondon AG , Wei W , et al ( 2011 ). Yeast Sen1 helicase protects the genome from transcription-associated instability . Mol Cell 41 : 21 - 32 .
17. Wahba L , Amon JD , Koshland D , Vuica-Ross M ( 2011 ) RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability . Mol Cell 44 : 978 - 988 .
18. Tuduri S , Crabbe L , Conti C , Tourriere H , Holtgreve-Grez H , et al. ( 2009 ) Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription . Nat Cell Biol 11 : 1315 - 1324 .
19. Stirling PC , Chan YA , Minaker SW , Aristizabal MJ , Barrett I , et al. ( 2012 ) Rloop-mediated genome instability in mRNA cleavage and polyadenylation mutants . Genes Dev 26 : 163 - 175 .
20. Paulsen RD , Soni DV , Wollman R , Hahn AT , Yee MC , et al. ( 2009 ) A genomewide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability . Mol Cell 35 : 228 - 239 .
21. Chen C , Kolodner RD ( 1999 ) Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants . Nat Genet 23 : 81 - 85 .
22. Yuen KW , Warren CD , Chen O , Kwok T , Hieter P , et al. ( 2007 ) Systematic genome instability screens in yeast and their potential relevance to cancer . Proc Natl Acad Sci U S A 104 : 3925 - 3930 .
23. Aguilera A , Klein HL ( 1988 ) Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae . I. Isolation and genetic characterization of hyper-recombination mutations . Genetics 119 : 779 - 790 .
24. Alvaro D , Lisby M , Rothstein R ( 2007 ) Genome-wide analysis of Rad52 foci reveals diverse mechanisms impacting recombination . PLoS Genet 3 : e228 .
25. Wyers F , Rougemaille M , Badis G , Rousselle JC , Dufour ME , et al. ( 2005 ) Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase . Cell 121 : 725 - 737 .
26. Vanacova S , Wolf J , Martin G , Blank D , Dettwiler S , et al. ( 2005 ) A new yeast poly(A) polymerase complex involved in RNA quality control . PLoS Biol 3 : e189 .
27. LaCava J , Houseley J , Saveanu C , Petfalski E , Thompson E , et al. ( 2005 ) RNA degradation by the exosome is promoted by a nuclear polyadenylation complex . Cell 121 : 713 - 724 .
28. Prado F , Aguilera A ( 1995 ) Role of reciprocal exchange, one-ended invasion crossover and single-strand annealing on inverted and direct repeat recombination in yeast: different requirements for the RAD1, RAD10, and RAD52 genes . Genetics 139 : 109 - 123 .
29. Piruat JI , Aguilera A ( 1998 ) A novel yeast gene, THO2, is involved in RNA pol II transcription and provides new evidence for transcriptional elongationassociated recombination . EMBO J 17 : 4859 - 4872 .
30. Mumberg D , Muller R , Funk M ( 1994 ) Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression . Nucleic Acids Res 22 : 5767 - 5768 .
31. Jimeno S , Rondon AG , Luna R , Aguilera A ( 2002 ) The yeast THO complex and mRNA export factors link RNA metabolism with transcription and genome instability . EMBO J 21 : 3526 - 3535 .
32. San Paolo S , Vanacova S , Schenk L , Scherrer T , Blank D , et al. ( 2009 ) Distinct roles of non-canonical poly(A) polymerases in RNA metabolism . PLoS Genet 5 : e1000555 .
33. Santos-Rosa H , Aguilera A ( 1994 ) Increase in incidence of chromosome instability and non-conservative recombination between repeats in Saccharomyces cerevisiae hpr1 delta strains . Mol Gen Genet 245 : 224 - 236 .
34. Kornberg RD ( 2005 ) Mediator and the mechanism of transcriptional activation . Trends Biochem Sci 30 : 235 - 239 .
35. Planta RJ , Mager WH ( 1998 ) The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae . Yeast 14 : 471 - 477 .
36. Kallstrom G , Hedges J , Johnson A ( 2003 ) The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively. Mol Cell Biol 23 : 4344 - 4355 .
37. Hong SP , Leiper FC , Woods A , Carling D , Carlson M ( 2003 ) Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases . Proc Natl Acad Sci U S A 100 : 8839 - 8843 .
38. Lin CH , MacGurn JA , Chu T , Stefan CJ , Emr SD ( 2008 ) Arrestin-related ubiquitin-ligase adaptors regulate endocytosis and protein turnover at the cell surface . Cell 135 : 714 - 725 .
39. Zachariae W , Nasmyth K ( 1999 ) Whose end is destruction: cell division and the anaphase-promoting complex . Genes Dev 13 : 2039 - 2058 .
40. Rondon AG , Jimeno S , Garcia-Rubio M , Aguilera A ( 2003 ) Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation . J Biol Chem 278 : 39037 - 39043 .
41. Tous C , Rondon AG , Garcia-Rubio M , Gonzalez-Aguilera C , Luna R , et al. ( 2011 ) A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors . EMBO J 30 : 1953 - 1964 .
42. Chavez S , Aguilera A ( 1997 ) The yeast HPR1 gene has a functional role in transcriptional elongation that uncovers a novel source of genome instability . Genes Dev 11 : 3459 - 3470 .
43. Luna R , Jimeno S , Marin M , Huertas P , Garcia-Rubio M , et al. ( 2005 ) Interdependence between Transcription and mRNP Processing and Export, and Its Impact on Genetic Stability . Mol Cell 18 : 711 - 722 .
44. Gonzalez-Aguilera C , Tous C , Gomez-Gonzalez B , Huertas P , Luna R , et al. ( 2008 ) The THP1-SAC3-SUS1-CDC31 complex works in transcription elongation-mRNA export preventing RNA-mediated genome instability . Mol Biol Cell 19 : 4310 - 4318 .
45. Woods A , Johnstone SR , Dickerson K , Leiper FC , Fryer LG , et al. ( 2003 ) LKB1 is the upstream kinase in the AMP-activated protein kinase cascade . Curr Biol 13 : 2004 - 2008 .
46. Houseley J , Tollervey D ( 2009 ) The many pathways of RNA degradation . Cell 136 : 763 - 776 .
47. Sadoff BU , Heath-Pagliuso S , Castano IB , Zhu Y , Kieff FS , et al. ( 1995 ) Isolation of mutants of Saccharomyces cerevisiae requiring DNA topoisomerase I . Genetics 141 : 465 - 479 .
48. Aguilera A , Klein HL ( 1990 ) HPR1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene . Mol Cell Biol 10 : 1439 - 1451 .
49. Houseley J , Tollervey D ( 2008 ) The nuclear RNA surveillance machinery: the link between ncRNAs and genome structure in budding yeast? Biochim Biophys Acta 1779 : 239 - 246 .
50. Wang Z , Castano IB , Adams C , Vu C , Fitzhugh D , et al. ( 2002 ) Structure/ function analysis of the Saccharomyces cerevisiae Trf4/Pol sigma DNA polymerase . Genetics 160 : 381 - 391 .
51. Edwards S , Li CM , Levy DL , Brown J , Snow PM , et al. ( 2003 ) Saccharomyces cerevisiae DNA polymerase epsilon and polymerase sigma interact physically and functionally, suggesting a role for polymerase epsilon in sister chromatid cohesion . Mol Cell Biol 23 : 2733 - 2748 .
52. Castano IB , Brzoska PM , Sadoff BU , Chen H , Christman MF ( 1996 ) Mitotic chromosome condensation in the rDNA requires TRF4 and DNA topoisomerase I in Saccharomyces cerevisiae . Genes Dev 10 : 2564 - 2576 .
53. Houseley J , Kotovic K , El Hage A , Tollervey D ( 2007 ) Trf4 targets ncRNAs from telomeric and rDNA spacer regions and functions in rDNA copy number control . EMBO J 26 : 4996 - 5006 .
54. Basu U , Meng FL , Keim C , Grinstein V , Pefanis E , et al. ( 2011 ) The RNA exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates . Cell 144 : 353 - 363 .
55. Poltoratsky VP , Wilson SH , Kunkel TA , Pavlov YI ( 2004 ) Recombinogenic phenotype of human activation-induced cytosine deaminase . J Immunol 172 : 4308 - 4313 .
56. Wellinger RE , Prado F , Aguilera A ( 2006 ) Replication fork progression is impaired by transcription in hyperrecombinant yeast cells lacking a functional THO complex . Mol Cell Biol 26 : 3327 - 3334 .
57. Gomez-Gonzalez B , Garcia-Rubio M , Bermejo R , Gaillard H , Shirahige K , et al. ( 2011 ) Genome-wide function of THO/TREX in active genes prevents Rloop-dependent replication obstacles . EMBO J 30 : 3106 - 3119 .
58. Gan W , Guan Z , Liu J , Gui T , Shen K , et al. ( 2011 ) R-loop-mediated genomic instability is caused by impairment of replication fork progression . Genes Dev 25 : 2041 - 2056 .
59. Schmid M , Jensen TH ( 2008 ) Quality control of mRNP in the nucleus . Chromosoma 117 : 419 - 429 .
60. Tutucci E , Stutz F ( 2011 ) Keeping mRNPs in check during assembly and nuclear export . Nat Rev Mol Cell Biol 12 : 377 - 384 .
61. Rougemaille M , Gudipati RK , Olesen JR , Thomsen R , Seraphin B , et al. ( 2007 ) Dissecting mechanisms of nuclear mRNA surveillance in THO/sub2 complex mutants . EMBO J 26 : 2317 - 2326 .
62. Kadaba S , Wang X , Anderson JT ( 2006 ) Nuclear RNA surveillance in Saccharomyces cerevisiae: Trf4p-dependent polyadenylation of nascent hypomethylated tRNA and an aberrant form of 5S rRNA . Rna 12 : 508 - 521 .
63. Egecioglu DE , Henras AK , Chanfreau GF ( 2006 ) Contributions of Trf4p- and Trf5p-dependent polyadenylation to the processing and degradative functions of the yeast nuclear exosome . Rna 12 : 26 - 32 .
64. Gallardo M , Aguilera A ( 2001 ) A new hyperrecombination mutation identifies a novel yeast gene, THP1, connecting transcription elongation with mitotic recombination . Genetics 157 : 79 - 89 .