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3 - Introns Protect Eukaryotic Genomes From Transcription Associated Genetic Instability
3 - Introns Protect Eukaryotic Genomes From Transcription Associated Genetic Instability
Correspondence
benoit.palancade@ijm.fr
In Brief
By combining the genetic manipulation of
intron content with genome-wide
analyses in both yeasts and human cells,
Bonnet et al. reveal a function for introns
in counteracting DNA:RNA hybrid
(R-loop) formation and its deleterious
impact on genetic stability.
Highlights
d Introns prevent R-loop and DNA damage accumulation on
highly expressed yeast genes
Article
*Correspondence: benoit.palancade@ijm.fr
http://dx.doi.org/10.1016/j.molcel.2017.07.002
608 Molecular Cell 67, 608–621, August 17, 2017 ª 2017 Elsevier Inc.
factors contributing to proper formation of export-competent occurrence of hybrids was significantly reduced among intron-
mRNPs, notably the conserved THO/TREX and THSC/TREX-2 containing as compared to intronless genes in the highest tran-
complexes (Huertas and Aguilera, 2003; González-Aguilera scriptional class (>50 mRNAs/hr; Figure 1A). Furthermore, for
et al., 2008). In addition, previous studies, including systematic R-loop-positive loci of this category, hybrid densities calculated
screens, uncovered elevated levels of R-loops or DNA damage from DRIP sequencing (DRIP-seq) data were strongly reduced
upon inactivation of splicing factors in both budding yeast and for intron-containing genes (Figure 1B; Table S1). Differences
mammals (Li and Manley, 2005; Paulsen et al., 2009; Chan in transcription levels, gene length, G-C content, GC skew, or
et al., 2014a). These observations could reflect an indirect contri- AT skew did not appear to bias the formation of R-loops on highly
bution of splicing to the expression of factors targeting R-loops. expressed intron-containing genes, since these parameters
However, the fact that the artificial insertion of an intron in a re- were not distinguishable between R-loop-positive and R-loop-
porter gene alleviates the transcriptional defects arising in a negative loci (Figures S1C–S1G). In addition, similar conclusions
yeast mRNP biogenesis mutant (tho) previously argued in favor could be reached when re-analyzing an alternative dataset of
of a protective effect of introns in cis (Bonnet et al., 2015). R-loop-prone sites (Figure S1H), which could be detected in
Spliceosomal introns are intervening sequences that are wild-type (WT) cells using a less stringent DRIP protocol (Chan
removed from the pre-mRNA molecule by two sequential trans- et al., 2014a), and further associated with hotspots of genetic
esterification reactions requiring the recognition of cis-acting instability (Chen et al., 2016).
elements by the spliceosome complex. Since their identification To strengthen the relevance of these observations, we then
(Berget et al., 1977; Chow et al., 1977), these sequences have sought a natural situation where intron-containing and intronless
emerged as a distinctive feature of eukaryotic genomes, even versions of the same gene could be directly compared for R-loop
though their frequency and length greatly vary between organ- formation. Among the numerous gene pairs arising from the
isms. However, their functions, as well as the evolutionary con- whole-genome duplication event that has occurred in an
straints that drive their maintenance in genomes, have remained ancestor of S. cerevisiae (Byrne and Wolfe, 2005), only one
debated. On the one hand, the presence of introns increases the (RPP1A and RPP1B) corresponded to two highly expressed
regulatability and the coding potential of the genome: introns can loci (>50 mRNAs/hr) with a distinct intron content. Strikingly,
modulate mRNA synthesis rates and stability (Heyn et al., 2015), although being more transcribed, the intron-containing RPP1B/
in particular through developmentally regulated intron retention YDL130w gene displayed a lower R-loop density than its intron-
(Braunschweig et al., 2014), and allow alternative splicing events less paralog RPP1A/YDL081c (Figure 1B; Table S1). Intron-con-
that contribute to proteome diversity (Nilsen and Graveley, taining genes are thereby less prone to accumulate DNA:RNA
2010). On the other hand, recent advances in transcriptome hybrids than their intronless counterparts.
profiling have revealed that a meaningful fraction of exon-intron To further determine whether the presence of introns is the
junctions is constitutively spliced in mammals and thereby un- direct cause of decreased R-loop formation, we performed
likely to contribute to regulatory events (Ryu et al., 2015). DRIP experiments on a yeast strain (Di) in which introns have
Furthermore, a large proportion of introns can be removed been removed from RPL7A and RPL7B, two intron-containing
from the yeast genome without altering gene expression or cell genes of the highest transcriptional class (Parenteau et al.,
fitness (Parenteau et al., 2008, 2011). This paradox prompted 2011; Figure 1C). In WT cells, our DRIP assay readily detected
us to question whether introns could have been selected and/ RNH-sensitive hybrids on YEF3, one of the intronless genes of
or maintained in eukaryotic genes to counteract R-loop forma- this transcriptional class identified in the DRIP-seq analysis,
tion through spliceosome recruitment, further reducing tran- but it failed to score any RNH-sensitive signal on both intron-
scription-associated genetic instability. containing RPL7A and RPL7B loci as well as on an untranscribed
region (Figures 1D–1G, top panels). However, intron deletion (Di),
RESULTS while not causing an increased expression of RPL7A/B (Fig-
ure S1I), was sufficient to trigger a specific appearance of
Intron-Containing Genes Display Decreased R-Loop detectable RNH-sensitive R-loops at these two loci (Figures 1D
and DNA Damage Levels in Yeast and 1E, bottom panels).
We first analyzed a dataset of budding yeast R-loop-prone sites, We then wondered whether the decreased R-loop accumula-
mapped by DNA:RNA hybrid immunoprecipitation (DRIP) using tion detected on intron-containing genes was associated with a
the S9.6 hybrid-specific antibody in cells mutated for RNases lowered genetic instability. For this purpose, we intersected the
H (RNH; Wahba et al., 2016). Since R-loops mainly stem dataset of R-loop-positive loci with the previously reported
from high levels of transcription (Wahba et al., 2016) and as genome-wide map of H2A-Ser129 phosphorylation (g-sites; Stir-
intron-containing genes, although representing a minor fraction ling et al., 2012). This histone modification is a landmark of DNA
(4.4%) of protein-coding loci, are heavily transcribed in yeast damage, including, but not restricted to, R-loop- and transcrip-
(Ares et al., 1999), we focused our study on highly transcribed tion-dependent DSBs (Stirling et al., 2012). Although H2A-
genes (>50 mRNAs/hr). Analysis of this category, which ac- Ser129 phosphorylation was not systematically detected on
counts for 50% of the transcriptome, allowed comparison of R-loop-prone loci in WT cells, which are likely to maintain non-gen-
equivalent numbers of intronless and intron-containing genes otoxic levels of hybrids, g-sites were found to form with a lower
of similar expression levels (Figure S1A). While the propensity occurrence (Figure 1H) and at a lower density (Figure 1I) on
to form R-loops increased gradually as a function of transcription intron-containing as compared to intronless genes. This observa-
for both intronless and intron-containing loci (Figure S1B), the tion was not merely caused by a difference in the number of
intronless and intron-containing loci, as proved by data bootstrap- ChIP dataset (Capra et al., 2010; Figures S1J and S1K). Collec-
ping (Figure 1I), and it was further confirmed upon re-analysis of an tively, our results thereby demonstrate that intron-containing loci
alternative phospho-H2A chromatin immunoprecipitation (ChIP)- are specifically protected against R-loop and DNA damage
(E) Detection of DNA:RNA hybrids on the GAL-YAT1 gene expressed in WT yeast cells by DRIP-qPCR (percentage of IP; n = 3). Values from RNH-treated im-
munoprecipitations appear as hatched. DRIP signals obtained on two control loci, either highly transcribed (YEF3) or untranscribed (intergenic), are indicated.
(F) Nascent mRNA levels detected in WT or tho (mft1D) yeast cells expressing the GAL-YAT1 gene (qRT-PCR, normalized to ACT1 mRNA values; n = 3). WTS, the
induction of the GAL-YAT1 gene was performed for 30 min instead of 5 hr.
(G) DNA:RNA hybrid detection by DRIP-qPCR in WT or tho yeast cells expressing the GAL-YAT1 gene (percentage of IP; n = 3).
(H) DNA:RNA hybrid formation per nascent mRNA in WT or tho yeast cells expressing the GAL-YAT1 gene. DRIP values (from G) were normalized to the amount of
nascent mRNAs expressed from the corresponding strains (from F).
(I) RNA polymerase II (Pol II) distribution on the GAL-YAT1 gene as determined by chromatin immunoprecipitation (ChIP) in WT or tho yeast cells (percentage of IP;
n = 4).
(J) Recombination frequencies (n = 3) for WT or tho yeast strains expressing the GAL-YAT1 reporter and either an empty vector or an RNH1-overexpressing
construct. The frequency of Leu+ prototrophs arising from recombination upon transcriptional induction (Gal) or repression (Glu, hatched) is indicated.
Means and SD are plotted (*p % 0.05 and **p % 0.01; ns, not significant; Welch’s t test). See also Figure S2.
in tho mutants, as scored by nascent mRNA quantification (Fig- on the YAT1 intronless gene (Figure 3F), in agreement with earlier
ure 3C) and Pol II ChIP (Figure 3D). Finally, the intron virtually studies using alternative reporter systems (Mischo et al., 2011;
suppressed the unwanted recombination induced on the YAT1 Stirling et al., 2012), and this phenotype was partially alleviated
gene by THO inactivation (Figure 3E). on the intron-containing construct (Figure 3F).
Importantly, the presence of the RPL51A* intron did not affect In addition, the effect of the intron was not restricted to the
R-loop formation in the in vitro transcription assay (Figures S3D case of the YAT1 gene. LYS2 is a long gene similarly reported
and S3E). In contrast, its protective effect was observed in to display transcription defects and transcription-associated
distinct in vivo mutant situations associated with R-loop accu- recombination in tho cells (Chávez et al., 2001), and insertion
mulation. Indeed, loss of function of either Hpr1, another THO of the RPL51A* intron at its 50 end partially rescued these
complex subunit (Figures S4A and S4B), or Sus1, a subunit of R-loop-associated phenotypes (Figures S4E–S4G). Finally, other
the THSC/TREX-2 that functions downstream of the THO com- intronic sequences were also tested for their ability to protect
plex in the mRNP biogenesis and export pathway (Figures 3F against the consequences of R-loop accumulation. Strikingly,
and S4C), similarly triggered transcription defects and hyper- insertion of the RPL35A or SEC27 natural introns at the 50 end
recombination phenotypes that were reduced in the presence of the YAT1 reporter (Figure 4A) similarly supported splicing
of the intron. Similarly, inactivation of the R-loop-resolving (Figure S3B), and it further alleviated the deleterious effect of
Sen1 helicase triggered transcription-dependent recombination THO inactivation on transcription (Figure 4B) and recombination
(Figure 4C). In contrast, insertion of an exonic sequence origi- small nuclear (sn)RNPs was indeed defective for both 50 SS
nating from the intronless RPL4A gene and of similar length as mutants (D5 and 5II3I) but virtually unaffected for the 30 SS
the RPL51A* intron failed to rescue the decreased transcription mutant intron (Figure S3A). In addition, RT-PCR analyses
and the hyper-recombination phenotype of tho cells (Figures confirmed that splicing was strongly reduced for the three
4B and 4C). This set of results therefore demonstrates that de mutants (Figure S3B). Strikingly, both 50 SS mutants rescued
novo insertion of introns in R-loop-prone genes can suppress neither the transcriptional defect (Figure 5B) nor the hyper-
the pathological formation of R-loops and their impact on tran- recombination (Figure 5C) arising at the intronless YAT1 gene
scription and genetic stability in vivo. in R-loop-forming tho cells. In contrast, the 30 SS mutant intron
fully rescued both R-loop-associated cellular phenotypes (Fig-
RNP Formation, but Not Splicing Per Se, Attenuates ures 5B and 5C), but it failed to suppress hybrid formation in
R-Loop Formation and Genetic Instability the in vitro transcription system (Figures S3D and S3E), demon-
Based on these findings, we used transcription and hyper- strating that spliceosome recruitment in vivo, but not splicing per
recombination of YAT1 reporters in R-loop-accumulating tho se, is the main cause of R-loop suppression.
mutants as a readout of hybrid formation to further decipher To further confirm this finding, we next inserted within the
the mechanisms by which introns attenuate R-loop accumula- YAT1 reporter a Tetrahymena group I self-splicing intron, which
tion (Figure 5A). On the one hand, introns could prevent R-loop readily excises in the context of the YAT1 sequence (Figure S3C)
formation by recruiting the spliceosome that would directly without recruiting any protein machineries (Chalamcharla et al.,
antagonize invasion of the pre-mRNA into its DNA template or 2010). Self-splicing occurred co-transcriptionally (i.e., on
further contribute to alternative mRNP assembly pathways nascent mRNAs; Figure S3C), but it failed to rescue the impaired
specifically taking over upon impaired mRNP biogenesis (tho transcription and the hyper-recombination phenotype triggered
mutants). On the other hand, intron splicing could itself attenuate by R-loop accumulation (Figures 5B and 5C), confirming that
R-loop formation by decreasing the sequence homology be- splicing per se was not sufficient to reduce R-loop formation.
tween the mRNA and its template. To discriminate between Conversely, artificial tethering of MS2-coat proteins (MS2-CPs)
these two hypotheses, we inserted within the YAT1 reporter onto the intronless YAT1 mRNA through two or six MS2-loops
different splice site (SS) mutants of the RPL51A* intron as fol- (MS2L) inserted in place of the intron partially rescued the tran-
lows: (1) a combined mutation of the 50 SS and of the branchpoint scriptional defects of tho mutants, as probed by measurement
(5II3I), which fully suppresses spliceosome recruitment and im- of mRNA levels (Figure 5D) and Pol II recruitment (Figures S5F
pairs splicing (Lacadie and Rosbash, 2005); (2) a deletion of and S5G). The observation of an increased Pol II occupancy
the 50 SS (D5), which affects early spliceosome assembly and, upon MS2-CP binding to MS2-loops rules out the possibility
subsequently, inhibits the first step of splicing (Lacadie and that the rescue of YAT1 mRNA levels would solely reflect the re-
Rosbash, 2005); and (3) a mutation of the 30 SS (30 ss*), which ported stabilization of MS2-CP-bound RNA species (Garcia and
supports early stages of spliceosome formation but prevents Parker, 2015). Furthermore, MS2-CP recruitment onto the YAT1
the second step of splicing (Alexander et al., 2010). ChIP exper- mRNA suppressed the hyper-recombination phenotype caused
iments revealed that the recruitment of spliceosomal U1 and U2 by THO inactivation (Figure 5E). In agreement with a direct
suppression of R-loop formation by the presence of MS2-CP rich (S. pombe and C. neoformans) species (Figure 6A). As pre-
bound to the mRNA, our DRIP assay detected decreased hybrid viously observed in S. cerevisiae (Garcı́a-Rubio et al., 2008), loss
levels at the MS2L-YAT1 reporter upon MS2-CP expression (Fig- of HPR1 function triggered a noticeable growth defect in
ure 5F). Taken together, these experiments support a model in C. glabrata, S. pombe, or C. neoformans cells (Figures S6A–
which introns prevent R-loop formation by recruiting protein fac- S6C). Using genomic DNA from cells of these distinct species,
tors that directly antagonize hybridization of the nascent mRNA we then detected RNH-sensitive hybrids on dot blots (Figures
onto its template (Figure S5H). S6D–S6G), as previously performed for templates forming
R-loops in vitro (as shown in Figure 2C). This analysis readily
Intron-Rich Genomes Do Not Accumulate R-Loops upon detected DNA:RNA hybrids on genomic DNA from WT cells of
Impaired mRNP Biogenesis these different species, possibly originating from transcription
We then sought to determine whether this function of introns had by all RNA polymerases. Strikingly, THO inactivation, which is
been maintained throughout evolution. Since the function of likely to specifically increase Pol II-dependent hybrids, triggered
the THO complex in preventing the accumulation of genotoxic a clear accumulation of R-loops in intron-poor genomes, e.g., in
R-loops on Pol II genes has been conserved from yeast to human C. glabrata (3.3% of intron-containing genes) and S. cerevisiae
(Huertas and Aguilera, 2003; Domı́nguez-Sánchez et al., 2011), (4.4%) (Figures 6B and 6C). However, loss of the THO complex
we systematically triggered hybrid formation by inactivating its had little or no effect in yeasts with an elevated proportion of
Hpr1 subunit in a panel of yeasts differing for their intron content, intron-containing genes, S. pombe (47%) and C. neoformans
including intron-poor (C. glabrata and S. cerevisiae) and intron- (99.5%) (Figures 6D and 6E), revealing an inverse correlation
between hybrid formation and intron content (Figure 6F; Pearson loci (Figures 7A and S7A). This observation was not merely
correlation coefficient = 0.89). Intron-richest genomes are due to the difference in the number of intronless and intron-con-
thereby less prone to accumulate R-loops in a context of defec- taining loci, as proved by data bootstrapping (Figure 7A), and
tive mRNP biogenesis. it was also confirmed by examining independent datasets
obtained from distinct cell types (IMR90, NTERA-2, and human
Intron-Containing Human Genes Display Decreased fibroblasts; Figures S7C, S7E, and S7G). Furthermore, analysis
Levels of R-Loops and DNA Damage of potential confounding effects (transcription levels, gene
We then examined various datasets of genome-wide R-loop dis- length, G-C content, and GC skew) for these distinct datasets
tribution obtained from human cells (Ginno et al., 2013; Lim et al., did not identify any genomic feature susceptible to bias the for-
2015; Nadel et al., 2015), and we calculated R-loop densities for mation of R-loops, as shown for highly expressed intron-con-
intronless and intron-containing genes. The analysis of hybrid- taining genes (Figures S7B, S7D, S7F, and S7H). In particular,
positive regions in HEK293 cells revealed that R-loop densities increased R-loop levels on intronless genes do not seem to be
do not vary much among transcribed genes in these cells, as caused by their reduced length, since R-loop-forming intronless
shown previously (Nadel et al., 2015), but that they are system- genes are frequently longer that intronless genes with no detect-
atically lower on intron-containing as compared to intronless able R-loops (see, for example, Figures S7D, S7F, and S7H,
‘‘length’’ panels). Human intron-containing genes are thereby lowered R-loop accumulation in intron-rich genomes. Finally,
less prone to accumulate DNA:RNA hybrids as compared to their genome-wide analyses revealed that intron-containing genes
intronless counterparts. display decreased R-loop levels and DNA damage as compared
We then compared DNA damage accumulation on intronless to intronless genes of similar expression, in both yeast and
and intron-containing R-loop-forming loci using a genome- human cells.
wide g-H2AX map also obtained in the HEK293 cell line (Bunch It is noteworthy that the presence of introns is not the only
et al., 2015). Although g-H2AX accumulation was not system- determinant of R-loop formation in our genome-wide analyses,
atic on R-loop-prone loci, we scored a significantly decreased as evidenced by the detection of both hybrid-positive intron-
signal on intron-containing genes of the highest transcrip- containing genes and hybrid-negative intronless genes (Figures
tion class (Figure 7B), a result that was still significant after 1A and 7A). However, when R-loops form on intron-containing
correcting for multiple hypothesis testing (p = 0.0004). These loci, e.g., for highly expressed yeast genes or in hybrid-prone
observations support that, among other features, R-loop accu- mutants, their levels are strongly attenuated as compared to
mulation can be a cause of damage accumulation and that those scored on their intronless counterparts (Figures 1B, 3B,
reduced R-loop formation on intron-containing genes also and 7A). The genomic R-loop distribution is thereby likely to be
dampens transcription-associated genetic instability in the modulated by the occurrence of introns, together with other
human genome. cis-acting determinants previously reported to impact on hybrid
formation. Among them, high levels of transcription from strong
DISCUSSION promoters have been shown to drive R-loop accumulation in
yeast (Chan et al., 2014a; Wahba et al., 2016). In addition, the
In this study, we provide multiple lines of evidence supporting influence of sequence properties on the propensity to form
our initial hypothesis that introns protect eukaryotic genomes R-loops has been pinpointed by different studies: while GC rich-
from R-loop accumulation and subsequent transcription-associ- ness was shown to positively influence R-loop formation both
ated genetic instability. First, by modifying the intron content of in vitro on transcribed reporters (Roy and Lieber, 2009) and
budding yeast genes, we have demonstrated that removing in vivo on human promoters (Ginno et al., 2013), A:T tracts
the introns from intron-containing genes is sufficient to trigger were recently shown to facilitate hybrid accumulation (Wahba
hybrid formation; conversely, inserting an intron within an et al., 2016). However, these features are unlikely to account
intronless R-loop-prone gene suppresses R-loop formation for the lowered R-loop formation scored on intron-containing
and hyper-recombination in R-loop-forming mutants. Second, genes in our study. Indeed, (1) intronless genes harbor higher
by comparing distinct yeast species differing for their intron con- R-loop densities than intron-containing genes of similar expres-
tent in situations of impaired mRNP biogenesis, we have scored sion (Figures 1B, 7A, S1C, and S7A), (2) hybrid-positive and
Further information and requests for reagents may be directed to and will be fulfilled by the Lead Contact, Benoit Palancade (benoit.
palancade@ijm.fr).
Plasmids
Plasmids used in this study are listed in the Key Resources Table and were constructed using PCR-based molecular cloning tech-
niques. Introns from RPL35A and SEC27, as well as an exonic sequence from RPL4A (1-70), were inserted downstream the ATG
within p-GAL-YAT1. The complete LYS2 CDS was subcloned downstream the GAL1 promoter to generate p-GAL-LYS2. Plasmids
of the p-L-GAL serie (used for hyper-recombination assays) were obtained by subcloning the different GAL1-promoter(+ATG)-YAT1
cassettes from plasmids of the p-GAL serie between the two leu2 repeats of pRS316-L, which share 600pb of homology. Previously
described intron mutations (5II3I and D5; Lacadie and Rosbash, 2005) were integrated in p-GAL-[RPL51A*intron]-YAT1 by PCR-
based mutagenesis. The 30 SS was mutated by introducing a AG- > Ac substitution (Alexander et al., 2010) at the end of the
RPL51A*intron, together with a G- > c substitution at a secondary 30 SS mapped 9 nt downstream. The Tetrahymena GroupI self-
splicing intron sequence was amplified from ptGpI-CUP1 (Chalamcharla et al., 2010) and subcloned between the ATG and the
YAT1 CDS within p-GAL-YAT1. MS2-loop coding sequences were amplified from pIIIA/MS2-1 (a gift from M. Wickens) and inserted
between the ATG and the YAT1 CDS within p-GAL-YAT1. For in vitro transcription, the YAT1 CDS, eventually flanked by wt or mutant
RPL51A* introns, was subcloned downstream the T7 promoter within the pCR4-TOPO vector (Lifetech). For RNH1 overexpression, a
GPDpromoter-hsRNH1 cassette from p425-GPD-hsRNH1 (Wahba et al., 2011) was subcloned in pRS423.
METHOD DETAILS
Hyper-recombination rates were defined as the mean of 3 experiments, each one performed with six independent colonies: cells
were plated on SC medium lacking leucine to estimate the number of Leu+ recombinants, while cell survival was estimated following
plating on SC medium. For statistics, n values correspond to the number of biological replicates (e.g., independent yeast cultures).
The following statistical tests were used: Fisher exact test (to compare R-loop/g-sites occurrences between intronless and intron-
containing gene populations; Figures 1 and S1); Bootstrapping Method with 100000 replications of the samples mean to compare
R-loop/g-sites densities of intronless and intron-containing dataset; Figures 1 and 7); Mann-Whitney-Wilcoxon with FDR adjusted
p values (to compare R-loop densities and genomic features of intronless and intron-containing datasets; Figures 1, S1, and S7);
two-sided Welch’s t test, allowing unequal variance (other figures). *p % 0.05; **p % 0.01; ***p % 0.001; ns, not significant.
The raw images have been deposited in Mendeley Data and are available at http://dx.doi.org/10.17632/b3f8k56vjs.1.