Volume 67, Issue 4, Pages 608-621.e6 (August 2017) Introns Protect Eukaryotic Genomes from Transcription-Associated Genetic Instability  Amandine Bonnet, Ana R. Grosso, Abdessamad Elkaoutari, Emeline Coleno, Adrien Presle, Sreerama C. Sridhara, Guilhem Janbon, Vincent Géli, Sérgio F. de Almeida, Benoit Palancade  Molecular Cell  Volume 67, Issue 4, Pages 608-621.e6 (August 2017) DOI: 10.1016/j.molcel.2017.07.002 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 67, 608-621.e6DOI: (10.1016/j.molcel.2017.07.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Introns Prevent R-Loop and DNA Damage Accumulation on Highly Transcribed Yeast Genes (A) Occurrence of DNA:RNA hybrids in intronless and intron-containing genes of the highest transcriptional category (>50 mRNAs/hr), evaluated according to a dataset of hybrid-prone sites detected in the rnh1Δ rnh201Δ mutant (Wahba et al., 2016). (B) DNA:RNA hybrid densities on intronless and intron-containing hybrid-positive loci from (A). The positions of the pair of paralogous genes RPP1A and RPP1B are indicated. (C) Organization of RPL7A and RPL7B loci in WT and Δi strains. The positions of the deleted introns (dark orange) are indicated. (D–G) DNA:RNA hybrid detection by DRIP-qPCR (percentage of IP; means and SD are plotted; n = 4) at the indicated loci (D, RPL7A; E, RPL7B; F, intergenic; G, YEF3) in either WT or Δi yeast cells. Values from RNH-treated immunoprecipitations appear as hatched. (H) Occurrence of γ-sites in intronless and intron-containing hybrid-positive loci, evaluated according to a ChIP-ChIP dataset of phosphorylated H2A (Stirling et al., 2012). (I) γ-site densities on intronless and intron-containing hybrid-positive loci. ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001; Fisher’s exact test (A and H), Mann-Whitney-Wilcoxon test (B), Welch’s t test (D and E), and Bootstrapping Method (I). See also Figure S1. Molecular Cell 2017 67, 608-621.e6DOI: (10.1016/j.molcel.2017.07.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 R-Loop-Forming Reporters Allow Scoring of R-Loop-Associated Phenotypes (A) Principle of the YAT1 reporter genes. The YAT1 gene was expressed either in vitro from the T7 promoter (pT7) or in yeast cells under the control of the GAL1-inducible promoter (pGAL1). The GAL-YAT1 reporter was integrated between two direct leu2 repeats (dark gray) to score transcription-associated recombination (J). The position of the 5′ and 3′ qPCR amplicons used in (I) is indicated. (B) Electrophoretic mobility of the T7-YAT1 plasmid, either untranscribed or transcribed by the T7 RNA polymerase (T7 RNA pol) and further treated or not with RNase H (RNH). Nucleic acids were stained with ethidium bromide. The positions of the distinct T7-YAT1 plasmid species, as well as molecular weight markers (kb), are indicated. (C) R-loop-forming YAT1 plasmids (from B) were extracted following electrophoresis and further used for dot blotting with antibodies against DNA:RNA hybrids (S9.6) or double-stranded DNA (dsDNA). (D) Detection of DNA:RNA hybrids on in-vitro-transcribed YAT1 by DRIP-qPCR (percentage of IP; n = 3). (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 immunoprecipitations 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 (mft1Δ) 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. Molecular Cell 2017 67, 608-621.e6DOI: (10.1016/j.molcel.2017.07.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Insertion of an Intron within an R-Loop-Prone Gene Prevents R-Loop Accumulation and Transcription-Associated Genetic Instability (A) Principle of the yeast intronless and intron-containing YAT1 reporter genes. Both reporters were integrated between leu2 repeats to score transcription-associated recombination. (B) DNA:RNA hybrid formation per nascent mRNA in WT or tho (mft1Δ) yeast cells expressing the indicated reporter constructs. DRIP/nascent mRNA values were calculated as in Figure 2H (percentage of IP/mRNA; n = 3), and RNH-treated immunoprecipitations appear as hatched. (C) Nascent mRNA levels detected in WT or tho yeast cells expressing the indicated reporter constructs (qRT-PCR, normalized to ACT1 mRNA values; n = 3). (D) Pol II distribution on the reporter genes as determined by ChIP in WT or tho yeast cells (percentage of IP; n = 4). Values for the intronless reporter are the same as used in Figure 2I. (E and F) Recombination frequencies (n = 3) for WT, tho (mft1Δ), sus1Δ, or sen1-1 yeast strains expressing the indicated constructs. The frequency of Leu+ prototrophs arising from recombination upon transcriptional induction (Gal) or repression (Glu, hatched) of each reporter is indicated. Note that SEN1 inactivation triggers hyper-recombination in both a transcription-independent and a transcription-dependent manner but that the intron mainly decreases the transcription-dependent phenotype. Means and SD are plotted (∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001; Welch’s t test). See also Figures S3 and S4. Molecular Cell 2017 67, 608-621.e6DOI: (10.1016/j.molcel.2017.07.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Distinct Intronic Sequences Alleviate R-Loop-Associated Phenotypes (A) Principle of the reporter constructs used in the different panels. All reporters were further integrated between leu2 repeats to score transcription-associated recombination. (B) mRNA levels in tho (mft1Δ) yeast cells expressing the indicated reporter constructs (qRT-PCR, normalized to ACT1 mRNA values; n = 3). (C) Recombination frequencies scored upon transcriptional induction for tho yeast strains expressing the indicated constructs (n = 3). Means and SD are plotted (∗p ≤ 0.05 and ∗∗p ≤ 0.01; ns, not significant; Welch’s t test). See also Figure S3. Molecular Cell 2017 67, 608-621.e6DOI: (10.1016/j.molcel.2017.07.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Spliceosome-Dependent mRNP Assembly, but Not Splicing Per Se, Is Required to Prevent R-Loop Formation (A) Principle of the reporter constructs used in the different panels. All reporters were further integrated between leu2 repeats to score transcription-associated recombination. The inserted sequences do not disturb transcription or recombination at the YAT1 locus in WT cells (see Figures S5B–S5E). (B) mRNA levels in tho (mft1Δ) yeast cells expressing the indicated reporter constructs (qRT-PCR, normalized to ACT1 mRNA values; n = 3). (C) Recombination frequencies for tho yeast strains expressing the indicated constructs (n = 3). The frequency of recombination events arising upon transcriptional induction (Gal) or repression (Glu, hatched) is scored for each reporter. The ability to properly recruit the spliceosome and to get spliced is indicated for each intron tested (see also Figures S3A–S3C). 5II3I, Δ5, and 3′ss∗ are splice site mutations within the RPL51A∗ intron (Lacadie and Rosbash, 2005; Alexander et al., 2010). (D) mRNA levels in WT or tho yeast cells expressing the indicated reporter constructs (as in B, n = 3). (E) Recombination frequencies for WT or tho yeast strains expressing the indicated constructs (as in C, n = 3). (F) DNA:RNA hybrid formation per mRNA in WT or tho yeast cells expressing the indicated reporter constructs (percentage of IP/mRNA; n = 3). The expression of MS2-coat proteins (MS2-CPs) artificially tethered onto the mRNA through two or six MS2-loops is indicated. Means and SD are plotted (∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001; ns, not significant; Welch’s t test). See also Figure S5. Molecular Cell 2017 67, 608-621.e6DOI: (10.1016/j.molcel.2017.07.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Intron-Rich Yeast Genomes Do Not Accumulate R-Loops upon Improper mRNP Biogenesis (A) Occurrence of introns in the genomes of the indicated yeast species. The following metrics are indicated: percentage of intron-containing genes (among protein-coding genes), mean number of introns (per intron-containing gene), positional bias of the introns (5′, introns preferentially located at the 5′ end of the mRNA). aLinde et al., 2015; bSaccharomyces Genome Database (http://www.yeastgenome.org); cPomBase (http://www.pombase.org); dJanbon et al., 2014; eLin and Zhang, 2005. (B–E) DNA:RNA hybrid detection by dot blot on genomic DNA from either WT or hpr1 (tho) cells in the indicated species (B, C. glabrata; C, S. cerevisiae; D, S. pombe; E, C. neoformans). Decreasing amounts of DNA extracts from the indicated cells were probed using antibodies directed against DNA:RNA hybrids (left panels) or dsDNA (right panels). (F) DNA:RNA hybrid levels were quantified from (B)–(E) using serial dilutions of a reference sample as a standard and normalized to the amount of DNA in each sample (for each species, n = 3). The accumulation of DNA:RNA hybrids upon THO inactivation (HPR1 knockdown, relative to WT) was plotted as a function of the fraction of intron-containing genes (among protein-coding genes) in the genomes of the different species. Means and SD are plotted. The Pearson correlation coefficient is indicated. See also Figure S6. Molecular Cell 2017 67, 608-621.e6DOI: (10.1016/j.molcel.2017.07.002) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Intron-Containing Genes Are Protected from R-Loop and DNA Damage Accumulation in Humans (A) DNA:RNA hybrid densities on intronless and intron-containing hybrid-positive loci, evaluated according to a dataset of hybrid-prone sites detected in HEK293 cells (Nadel et al., 2015) and ranked according to nascent mRNA levels (see also Figure S7A). L, low expression; M, medium expression; H, high expression; VH, very high expression. (B) γ-H2AX association to intronless and intron-containing hybrid-positive loci from (A), evaluated according to a γ-H2AX ChIP-seq dataset obtained from HEK293 cells (Bunch et al., 2015) and displayed according to expression levels. (C) Model for the role of introns in R-loop prevention. Intron-mediated recruitment of the spliceosome, and possibly of other factors, would favor RNP formation and antagonize hybridization of the mRNA onto its DNA template. ∗∗∗p ≤ 0.001, Bootstrapping Method. See also Figure S7. Molecular Cell 2017 67, 608-621.e6DOI: (10.1016/j.molcel.2017.07.002) Copyright © 2017 Elsevier Inc. Terms and Conditions