Nucleic Acids Research Advance Access published online on May 18, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp385
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gene Regulation, Chromatin and Epigenetics |
R-loops do not accumulate in transcription-defective hpr1-101 mutants: implications for the functional role of THO/TREX
Belén Gómez-González and
Andrés Aguilera*
Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Av. Américo Vespucio s/n, 41092 SEVILLA, Spain
*To whom correspondence should be addressed. Tel: +34 954 468 372; Fax: +34 954 461 664; Email: aguilo{at}us.es
Received March 23, 2009. Revised April 27, 2009. Accepted April 28, 2009.
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ABSTRACT
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To get further insight into the effect that THO/TREX and R-loops
have in transcription-associated recombination and transcription,
we analyzed the ability to form R-loops of
hpr1-101, a THO mutation
that impairs transcription and mRNP biogenesis without triggering
hyper-recombination. Human AID, a cytidine deaminase that acts
on ssDNA displaced by RNA-DNA hybrids, strongly induced both
hyper-recombination and hyper-mutation in
hpr1-101, similar
to
hpr1
mutants. However, in contrast to
hpr1, AID-induced mutations
in
hpr1-101 occur at similar frequencies in both the transcribed
and non-transcribed strands, implying that the enhanced AID
action in these mutants is not caused by co-transcriptional
R-loops. These results indicate for the first time that THO
has a transcriptional function that is not mediated by R-loops,
providing a new perspective for the understanding of the coupling
of transcription with mRNP biogenesis and export.
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INTRODUCTION
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In eukaryotic RNA polymerase II (RNAPII) transcription, nascent
pre-mRNAs are processed and matured into mRNAs by protein complexes
that are loaded onto transcription sites
via their interaction
with the C-terminal domain of RNAPII. Processing and maturation
events are coupled with the formation of an export-competent
ribonucleoparticle, mRNP, which is actively exported through
the nuclear pore complex (NPC) into the cytoplasm (
1–4).
One key factor in the coupling between transcription and mRNP
processing and export is THO, a conserved eukaryotic nuclear
complex containing Tho2, Hpr1, Mft1 and Thp2, as first isolated
from yeast (
5). THO interacts physically and functionally with
proteins involved in mRNA export, such as the Sub2 RNA-dependent
ATPase, to form a larger complex termed TREX (
6,
7). THO mutations
lead to gene expression defects particularly evident for long
and GC-rich DNA sequences, (
5) as well as for repeat-containing
genes (
8). Such defects reflect impairment in transcription
elongation as determined both
in vivo and
in vitro (
5,
9,
10).
In the present view, it is believed that THO participates during
transcription elongation in the formation of export-competent
mRNPs. Consistent with it, overexpression of the RNA-dependent
ATPase Sub2 or the RNA binding protein Tho1 suppresses the transcription
and RNA-export defects of THO mutants (
6,
11,
12).
It has been shown that hpr1 mutants accumulate co-transcriptional R-loop structures in which the nascent RNA forms an RNA-DNA hybrid with the transcribed (T) DNA strand, the non-transcribed (NT) strand remaining single-stranded behind the RNA polymerase II (RNAPII) (13). Such R-loops are linked to transcription defects and transcription-associated recombination (TAR) in THO mutants. The relevance of R-loops in genetic instability has also been shown in class switch recombination (CSR) as well as in depleted vertebrate cells of the ASF/SF2 splicing factor (14,15). R-loops in THO mutants have been confirmed genetically by showing that human AID, a cytidine deaminase that acts preferentially on ssDNA in the V and S-regions of Immunoglobulin (Ig) genes during somatic hypermutation (SHM) and CSR (16), induces both mutation and recombination by acting at the ssDNA that is displaced at the R-loops (17). However, the mechanisms by which R-loops impair transcription and induce TAR are unknown. It has been hypothesized that R-loops may become an obstacle for the next elongating RNAPII, impairing transcription elongation, as well as for the replicative DNA polymerase machinery, yielding to DNA lesions whose repair would require recombination (18–20). Consistent with this view, artificially constructed RNA–DNA hybrids have been shown to reduce transcription elongation efficiency in vitro (21). Nonetheless, the possibility that THO could play a more direct role in transcription not mediated by R-loops has not been ruled out. In this sense, it is worth noting that R-loops formed in ASF/SF2-depleted DT40 chicken cells do not seem to be linked to transcription impairment (15).
A particularly interesting allele to answer this question is hpr1-101, a point mutation that, in contrast to THO null mutations, confers a gene expression defect not accompanied by a significant increase neither in TAR nor in replication-fork progression slowdown (22). Despite the separation of phenotypes of hpr1-101, whether R-loops may differentially contribute to the transcription or genetic stability functions of THO mutants has not been elucidated. Here we show that human AID induced both hyper-recombination and hyper-mutation in hpr1-101, similar to hpr1 null mutants. However, in contrast to hpr1, AID-induced mutations in hpr1-101 were found at similar frequencies in both the T and NT strands, implying that the enhanced AID action in these mutants is not caused by co-transcriptional R-loops but by the opening of DNA strands during transcription. These results suggest that, in addition to the impact of R-loops on transcription elongation, THO mutations cause a transcriptional impairment that is independent of R-loops. This result provides a novel perspective in the understanding of the coupling between transcription and mRNP processing and export.
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MATERIALS AND METHODS
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Strains and plasmids
We used W303-1A isogenic strains U678-4C (
hpr1::HIS3), WMK-2A
(
mft1::KAN) (
5), WRS52-4B (
rad52::KAN) (
23) and WH101-4A (
hpr1-101)
(
22), described previously. Centromeric plasmids pCM184-LAUR
containing
lacZ::URA3 under the tet promoter (
6), pRS316-LY
NS
containing the
leu2 repeats recombination assay (
24), pGAID
carrying the human AID ORF under the
GAL1 promoter and pGLG
containing the GFP-based recombination system (
17) were described
previously. pRS425GALRNH1 was constructed by subcloning the
SalI-SpeI GALRNH1 fragment from pRS416GALRNH1 (
13) into SalI-SpeI
digested pRS425.
Recombination and mutation analysis
GFP fluorescence was determined in a FACScalibur (Becton-Dickinson, USA) from 106 cells grown in SC overnight and re-suspended in 1 ml H2O as described (17). For the LYNS recombination and LAUR mutation assay, cells were cultured in SC plates, from which six independent colonies were analyzed. Leu+ recombinants were selected on SC-leu and Ura- mutants were selected on SC containing SC + 700 mg/l FOA as previously described (17). lacZ+ and lacZ– were distinguished by color on SC + X-gal medium. Median mutation and recombination frequencies were obtained by fluctuation tests as the median value of six independent colonies isolated from SC plates. The final frequency given for each strain and condition is the mean and standard deviation of three to four median values.
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RESULTS
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Increased mutagenic and recombinogenic activity of AID in hpr1-101 cells
In order to establish whether the transcription–elongation
impairment of THO null mutants is linked to the accumulation
of R-loops we determined whether
hpr1-101, a THO point mutation
that impairs transcription but does not trigger hyper-recombination
(
22), forms R-loops. For this, we analyzed the effect of AID
in the direct-repeat recombination assay LY
NS based on 0.6-kb
leu2 repeats in which transcription has to proceed through a
3.7-kb intervening sequence (
24) (
Figure 1). Due to the strong
hyper-recombination of THO mutants in direct-repeat systems,
we did not expect AID to cause a strong increase in recombination,
but a significant 4.5-fold AID effect was observed in the
hpr1 allele reaching 98% of recombination frequency. Unexpectedly,
although the recombination frequency in
hpr1-101 was lower,
AID overexpression also increased this frequency to 79%.
To confirm this result, we used the GFP repeat recombination
assay GLG, in which GFP
+ recombinants can be directly scored
by FACS analysis (
17). Strikingly, AID led to a strong synergistic
increase in the formation of GFP
+ recombinants in
hpr1-101 (
Figure 2),
similar to what was previously reported for R-loop-forming
mft1 cells (
17). These results indicate that, in contrast to wild-type
cells, AID can access the DNA very efficiently in
hpr1-101 as
well as in THO null mutants inducing a strong hyper-recombination.
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Figure 2. Effect of AID on direct-repeat recombination as detected by FACS. Recombination frequencies were obtained for the GLG direct-repeat recombination system in wild-type and hpr1-101 strains with or without AID overexpression. y-axis, green fluorescence (FL1H); x-axis, unspecific fluorescence (FL2H). Mean recombination frequency and standard deviation of three to four different experiments are plotted. A diagram of the system is shown at the top.
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To further test the ability of AID to access transcribed DNA
in
hpr1-101 we looked at AID induced mutation in
hpr1-101 in
the LAUR mutation assay, a
lacZ::URA3 translational fusion under
control of the regulated
Tet promoter in which Ura
– mutants
can be selected on synthetic complete (SC) medium supplemented
with 5-fluoroorotic acid (
17). In
Figure 3A, we can observe
that AID increased the mutation frequency 136-fold in
hpr1,
consistently with what was reported for
mft1 (
17). As expected,
AID also led to an 89-fold enhanced hyper-mutation in
hpr1-101 (
Figure 3A). Therefore, AID action is enhanced in
hpr1-101 mutant,
similarly to what occurred in R-loop forming THO null mutants.
Nevertheless, we observed a clear difference analyzing the pattern
of distribution of the mutations obtained in each mutant. While
all mutations obtained in
hpr1 were in
lacZ, mutations obtained
in
hpr1-101 appeared both in
lacZ and
URA3 (
Figure 3B). The
abundance of mutations in
lacZ in THO null mutants was previously
proved to be due to the strong hyper-recombination (
17). Therefore,
the mutation distribution in
hpr1-101 was similar to the wild
type rather than to the hyper-recombinant THO null mutants.
AID accesses with similar frequency both the transcribed and non-transcribed DNA strands of a transcribed reporter
We have previously shown that in THO null mutants AID acts preferentially
on the NT strand in a 3:1 ratio versus the T strand, consistent
with the displacement of the NT strand as ssDNA in R-loops (
17).
To assess whether R-loops form in
hpr1-101, we sequenced the
mutations that had occurred in
hpr1-101 with or without AID
overexpression (
Figure 4 and
Supplementary Table 1) and analyzed
the ratio of mutations in C or G as a measurement of AID targeting
to the non-transcribed versus the transcribed strand (NT:T)
(
Table 1). In the absence of AID,
hpr1-101 yielded a NT:T ratio
of mutations of 1:1 (four mutations at C and four mutations
at G). Unexpectedly, this ratio was very similar in the presence
of AID (0.6:1, 14 at C and 23 at G). The WRC AID-target motif
was also mutated at an NT:T ratio 1:1 in the wild type (15 at
WRC and 15 at GYW) and 0.6:1 in
hpr1-101 (12 at WRC and 18 at
GYW) while it was 5:1 in
mft1 (10 at WRC and 2 at GYW) [
Table 1 and (
17)]. Therefore, both strands are equally accessible to
AID in
hpr1-101. This is indicative that R-loops are either
not formed or formed at a reduced rate in
hpr1-101. The major
accessibility of AID must be explained by the transient opening
of the DNA strands produced by the local negative supercoiling
generated during transcription. Such a negatively supercoiled
region may be larger or last longer in
hpr1-101 due to the transcription
impairment.
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Figure 4. Spectra and distribution of mutations obtained in the LAUR system. DNA sequence of 39 and 45 lacZ+ Ura– spontaneous (–AID; upper part of the sequence) and AID-induced (+AID; lower part of the sequence) mutations, respectively, in hpr1-101 cells. Mutations are shown in bold. Symbols: ‘plus sign’, insertion; ‘triangle’, deletion. Grey highlighting identifies sites where mutations occurred in the WRC/GYW AID target motif, in which W is A or T, R is A or G and Y is C or T.
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Table 1. Spontaneous and AID-induced base substitutions in wild-type (WT) and hpr1-101 mutant classified according to different sequence features
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RNaseH1 overexpression in R-loop-forming THO null mutants mimics the hpr1-101 mutation
Our results indicate that impairment of mRNP formation and transcription
elongation caused by
hpr1-101 is not accompanied by R-loop formation.
Since
hpr1-101 is not hyper-recombinant and does not show replication-fork
slowdown, it is likely that R-loops are linked to the hyper-recombination
and replication fork slowdown that occurs in THO null mutants
(
13). However, the fact that AID efficiently acts in
hpr1-101 mutants, which do not form co-transcriptional RNA-DNA hybrids,
opens the possibility that R-loop removal in
hpr1 cells may
not affect the action of AID either. We therefore reasoned that
overexpression of RNase H1, which specifically remove RNA-DNA
hybrids and has been shown to partially suppress
hpr1 hyper-recombination
(
13), might not suppress AID-induced recombination in THO null
mutants. To test this, we studied the effect of RNase H1 overexpression
in
mft1 in the GLG recombination and LAUR mutation system in
the presence of AID. As shown in
Figure 5, overexpression of
RNH1 did not suppress AID-induced recombination or AID-induced
mutation in
mft1 or
hpr1-101. Altogether, these results imply
that the transcription defect of
hpr1, even though it can be
exacerbated by R-loop formation (
13), is a direct consequence
of the lack of THO and is independent on R-loop formation, consistent
with the fact that
hpr1-101 impairs transcription without forming
R-loops.
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DISCUSSION
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We provide genetic evidence that co-transcriptional R-loops
are either not formed or formed at a reduced rate in the
hpr1-101 point mutant, which, in contrast to
hpr1, is impaired in transcription
but does not lead to a strong increase in TAR or to a defect
in replication fork progression. AID is able to act on both
DNA strands of a transcribed sequence in
hpr1-101 mutant, indicating
that R-loops are not stably formed in this THO point mutant.
The fact that
hpr1-101 mutants show a transcription defect but
no hyper-recombination nor replication slowdown suggests that
whereas the two latter phenotypes of THO mutants are linked
to R-loop formation, as previously reported (
13,
22), the transcription
impairment also occurs in the absence of R-loop formation.
In mRNP biogenesis mutants, such as THO null mutants, a co-transcriptional R-loop is formed, in which the mRNA hybridizes with the complementary transcribed strand, displacing the other strand that remains as ssDNA (13) (Figure 6). Short and unstable, transient R-loops may sporadically occur during transcription in wild-type cells but R-loops are accumulated in THO null mutants. This is presumably favored by the accumulation of negative supercoiling associated with transcription. Indeed, in vitro transcription experiments have revealed that R-loop formation is more extensive on hypernegatively supercoiled templates (25). One possibility could be that THO deficiency in hpr1-101 causes retention of the elongating RNA polymerase favoring the persistence of negative supercoiling. In this sense it is interesting to note that THO mutants are extremely sick in combination with topoisomerase mutations (26). As it can be seen in Figure 6, the NT strand in the form of ssDNA is more susceptible to be deaminated by AID leading to the strong hyper-mutation and hyper-recombination (17). Interestingly, recent in vitro transcription experiments have shown that AID treatment of R-loops, caused by arresting the elongation complexes by a quick removal of NTPs, results in an increase in revertants with multiple clustered mutations (27). Long and unusually stable R-loops are involved in the priming of DNA replication in certain bacterial plasmids as well as in the mitochondrial genome of eukaryotic cells from yeasts to humans (28,29). These bacterial RNA–DNA hybrids involved in replication initiation have been suggested as interfering with replication fork progression (30). Similarly, in THO null-mutants, the formation of R-loops correlates with replication fork progression impairment as seen by 2D-gel analysis (18). Replication disturbance would thus yield recombination-mediated repair, consistent with the strong TAR phenotype of THO null mutants.
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Figure 6. Transcription-elongation impairment in THO mutants. In THO null mutants, a co-transcriptional R-loop can be formed. The R-loop allows AID to act on the non-transcribed ssDNA causing strong AID-induced TAM and TAR. In hpr1-101, the nascent mRNA is packed into an mRNP, although not properly, causing transcription impairment. Negative supercoiling is accumulated behind the RNAPII allowing the accumulation of ssDNA in both DNA strands, which enhances AID action. A putative mRNP-biogenesis checkpoint would sense the export-incompetent sub-optimal mRNPs and in response would slowdown or halt transcription.
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In
hpr1-101, co-transcriptional R-loops are not stably formed.
The THO complex is present, stable and recruited to chromatin
in
hpr1-101 cells (
22) (
Figure 6). Nevertheless,
hpr1-101 may
still cause retention of the elongating RNA polymerase and an
upstream accumulation of negative supercoiling, thus enhancing
the accessibility of both DNA strands to AID and leading to
the strong AID-induced hyper-mutation and hyper-recombination
(
Figure 6). The conclusions from this result might be extrapolated
to the molecular mechanisms leading to CSR and SHM. We previously
proposed a role of defective mRNP biogenesis formation in the
initiation of CSR and SHM, in which sub-optimal mRNP biogenesis
could be leading to a better accessibility of AID to the transcribed
S or V-regions, respectively (
17). This could be due to the
formation of R-loops, as it occurs in yeast THO null mutants,
in which the NT strand is maintained single-stranded thus favoring
AID action to initiate both SHM and CSR. In fact, R-loops are
formed in S-regions (
14,
31). However, the analysis of the products
of SHM reveals that both DNA strands must be mutated
in vivo (
32,
33). Here we present a yeast model in which defective mRNP
biogenesis enhances the action of AID independent of R-loops.
AID mutates both strands in this yeast model, which is consistent
with the pattern of AID action in B-cells (
Figure 6). We found
that AID induced a high number of mutations not explained by
direct replication of an U:G mismatch, which would lead to C
to T and G to A transitions (82% in wt and 72% in
hpr1-101;
Supplementary Table 1). Interestingly, this is similar to the
pattern observed in SHM, in which 60% and 75% of mutations are
neither C to T nor G to A in human and mice, respectively (see
(
34) and references therein). This suggests that AID-induced
mutations in yeast would occur by an initial cytosine deamination
leading to a U:G mismatch that is later processed by aberrant/error
prone repair processes involving Ung and Msh proteins, as it
is believed for SHM in B cells (
34).
Notably, transcription is impaired in hpr1-101 as it is in THO null mutants (22), possibly due to the negative supercoiling transiently accumulated upstream of the elongating RNA polymerase II, which enhances the accessibility of both DNA strands to AID, leading to strong AID-induced hyper-mutation and hyper-recombination (Figure 3). Several hypotheses have been suggested as explanations of the effect of R-loop formation in transcription. The most extended one is that an R-loop could constitute a roadblock for the next RNA polymerase, so that the overall levels of transcription elongation would be reduced (3,19,20). Consistent with this, we have shown that artificially constructed RNA–DNA hybrids reduce the efficiency of transcription elongation in vitro (21). It seems, therefore, that R-loops by themselves can impair the progression of RNA polymerases, as it also seems to be the case for DNA polymerases (35). In the case of THO null mutants, the transcription-elongation impairment can be due in part to R-loop formation (13), but the extent of the impact of the R-loop, or a more direct role of THO in transcription, was unclear. The results of this study imply that Hpr1 also has a role in the control of transcription that is not mediated by R-loops. This, indeed, may explain why RNase HI overexpression does not apparently suppress the transcription defect of THO null mutants despite reducing the amount of R-loops (13). It is likely that an mRNP-biogenesis checkpoint might exist that senses export-incompetent sub-optimal mRNPs to impair transcription and to facilitate its degradation by the nuclear exosome. These results provide a new and intriguing perspective on the functional role of THO in transcription elongation and the coupling between transcription and mRNA processing. Further analysis of the effect of THO and related mRNP biogenesis and export factors on transcription elongation should, therefore, contribute to clarify the different mechanisms by which transcription and mRNP biogenesis and export are coupled.
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SUPPLEMENTARY DATA
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Supplementary Data are available at NAR Online.
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FUNDING
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Spanish Ministry of Science and Innovation (BFU2006-05260, CSD2007-015);
Junta de Andalucía (CVI-2549, BIO-102). B.G.-G. was a
recipient of a predoctoral training grant from the Spanish Ministry
of Science and Innovation. Funding for open access charge: grant
BIO-102 from Junta de Andalucía.
Conflict of interest statement. None declared.
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ACKNOWLEDGEMENTS
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We thank R. Luna and S. Jimeno for critical reading of the manuscript,
and D. Haun for style supervision.
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