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. 2013 Nov 15;27(22):2445-58.
doi: 10.1101/gad.229880.113.

The Npl3 hnRNP Prevents R-loop-mediated Transcription-Replication Conflicts and Genome Instability

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Free PMC article

The Npl3 hnRNP Prevents R-loop-mediated Transcription-Replication Conflicts and Genome Instability

José M Santos-Pereira et al. Genes Dev. .
Free PMC article

Abstract

Transcription is a major obstacle for replication fork (RF) progression and a cause of genome instability. Part of this instability is mediated by cotranscriptional R loops, which are believed to increase by suboptimal assembly of the nascent messenger ribonucleoprotein particle (mRNP). However, no clear evidence exists that heterogeneous nuclear RNPs (hnRNPs), the basic mRNP components, prevent R-loop stabilization. Here we show that yeast Npl3, the most abundant RNA-binding hnRNP, prevents R-loop-mediated genome instability. npl3Δ cells show transcription-dependent and R-loop-dependent hyperrecombination and genome-wide replication obstacles as determined by accumulation of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Npl3 is preferentially bound in wild-type cells, and are reduced by RNase H1 overexpression. The resulting replication stress confers hypersensitivity to double-strand break-inducing agents. Therefore, our work demonstrates that mRNP factors are critical for genome integrity and opens the option of using them as therapeutic targets in anti-cancer treatment.

Keywords: DNA damage response; Npl3; R loops; hnRNPs; transcription-associated genome instability; transcription–replication conflicts.

Figures

Figure 1.
Figure 1.
DNA repair in npl3Δ cells. (A) Sensitivity of BY4741 (WT) and SC086 (npl3Δ) strains to trabectedin (ET-743), MMS, Phl, UV light, and HU. (B) Sensitivity of BY4741 (WT), SC086 (npl3Δ), SC063 (rad52Δ), SC087 (rad52Δ npl3Δ), SC064 (yku80Δ), and SC088 (yku80Δ npl3Δ) strains to MMS. (C) Plasmid relegation assay. (Top) Scheme of pBTM116 and enzymes used in the assay. (Bottom) Percentage of plasmid repair in SC071 (WT), SC073 (yku80Δ), and SC089 (npl3Δ) strains. (*) P < 0.05; (***) P < 0.001 (Student's t-test). (D) Repair of replication-borne HO-induced DSBs by SCR. (Top) Scheme of pRS316TINV plasmid carrying two inverted leu2 repeats. Fragments generated by HO cleavage and XhoI SpeI digestion, as detected by the LEU2 probe (represented as a black line), are indicated with their corresponding sizes. (Left) Physical analysis of HO-induced DSB formation and its repair kinetics in WS (WT) and WSNPL3-01 (npl3Δ) strains. (Right) Quantification of DSBs (1.4-kb plus 2.4-kb bands) and SCR (4.7-kb band) related to total plasmid DNA. Average and standard deviation of three independent experiments are shown.
Figure 2.
Figure 2.
Replication impairment and persistent checkpoint activation in response to MMS in npl3Δ cells. (A) S-phase progression of BY4741 (WT) and SC086 (npl3Δ) cells synchronized in G1 with α-factor and released in the absence (left) or presence (right) of 0.033% MMS. (B) Analysis of completion of replication by PFGE in BY4741 (WT) and SC086 (npl3Δ) strains at different time points after G1 synchronization, treatment with 0.033% MMS, and release into fresh medium. (C) Western against the Rad53 checkpoint kinase in BY4741 (WT) and SC086 (npl3Δ) strains. Cells were treated with MMS as in B. (D) Western against Rad53 in which G1-arrested cells were treated with 200 mM HU for 3 h, washed, and released into fresh medium. A diagram of each experiment is shown at the top of each panel.
Figure 3.
Figure 3.
Genetic instability and TAR in npl3Δ cells. (A) Spontaneous Rad52-YFP foci formation in W303-1A (WT) and WNPL3-1D (npl3Δ) strains and representative microscope images. Average and standard deviation of three to four independent experiments are shown. (B) Percentage of centromeric plasmid pRS316 stability in W303-1A (WT) and WNPL3-1D (npl3Δ) strains. (C) Recombination analysis of AYW3-1B (WT) and AYNPL3-1D (npl3Δ) strains carrying the chromosomal direct repeat system leu2-k∷ADE2-URA3∷leu2-k. (D) Recombination analysis of W303-1A (WT) and WNPL3-1D (npl3Δ) strains carrying L and LYΔNS plasmid systems. (E) Recombination analysis in the TL-lacZ plasmid system, whose transcription is regulated by the tet promoter, in the presence (Low transcription) or absence (High transcription) of 5 μg/mL doxycycline in W303-1A (WT), WNPL3-1D (npl3Δ), and U678-1C (hpr1Δ) strains. For B–E, average and standard deviation of three to four fluctuation tests consisting of the median value of six independent colonies for each one are shown. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 (Student's t-test). For recombination assays, a scheme of the system is shown at the top of each panel.
Figure 4.
Figure 4.
Suppression of Rad52 foci and hyperrecombination by overexpression of RNH1. (A) Rad52-YFP foci formation in W303-1A (WT), WNPL3-1D (npl3Δ), and U678-1C (hpr1Δ) strains carrying pCM189 (−RNH1) or pCM189RNH1 (+RNH1) in the absence of doxycycline. Representative microscope images are shown. Average and standard deviation of six independent experiments are shown normalized to wild-type levels. (B) Recombination analysis in the TL-lacZ system in W303-1A (WT), WNPL3-1D (npl3Δ), and U678-1C (hpr1Δ) strains under the same conditions as in A. Average and standard deviation of three to four fluctuation tests from six independent colonies for each one are shown normalized to wild-type levels. (*) P < 0.05; (**) P < 0.01 (Student's t-test).
Figure 5.
Figure 5.
Effect of multicopy SUB2, THO1, NAB2, and NPL3 overexpression over different strains. (A) Recombination analysis of AYW3-1B (WT) and AYNPL3-1D (npl3Δ) strains carrying the chromosomal direct repeat system leu2-k∷ADE2-URA3∷leu2-k and multicopy plasmids YEp351 (empty vector, YEp), YEpSUB2 (SUB2), YEpTHO1 (THO1), and YEpNAB2 (NAB2). Average and standard deviation of three to four fluctuation tests from six independent colonies for each one is shown. (B) Suppression of temperature, HU, and MMS sensitivity phenotypes by YEpSUB2, YEpTHO1, and YEpNAB2 in serial dilutions of AYW3-1B (WT) and AYNPL3-1D (npl3Δ) cells. (C) Recombination analysis of AYW3-1B (WT), AYNPL3-1D (npl3Δ), and AYW3-3C (hpr1Δ) strains carrying the chromosomal direct repeat system leu2-k∷ADE2-URA3∷leu2-k and multicopy plasmids YEp351 (empty vector, YEp) or YEpNPL3 (NPL3). Average and standard deviation of six fluctuation tests from six independent colonies for each one are shown. (*) P < 0.05; (**) P < 0.01 (Student's t-test).
Figure 6.
Figure 6.
Npl3 and Hpr1 recruitment to highly transcribed ORFs. (A) Genomic view of Npl3-MYC (YAM535) and Hpr1-Flag (SYHPR1) recruitment. A fragment of chromosome XVI is plotted with the signal log2 ratio values. Green (Npl3-IP) and orange (Hpr1-IP) histograms represent the significant clusters. SGD features are represented below as blue bars with white arrows according to the direction of transcription. (B) Table showing the statistical analysis of length, G+C content, and expression levels of the genes mapped by Npl3 clusters. P-value was calculated by Mann-Whitney's U-test. (C) Composite profile of Npl3 and Hpr1 occupancy detected by ChIP–chip across the average ORF plotted as Npl3 or Hpr1 percentage of ChIP clusters per segment.
Figure 7.
Figure 7.
Rrm3 genome-wide recruitment in wild-type and npl3Δ cells. (A) Genomic view of Rrm3 recruitment in WRBb-9B (WT) and N3RBb-4B (npl3Δ) cells. A fragment of chromosome II is plotted with the signal log2 ratio values. Red histograms represent the significant clusters. SGD features are represented below as blue bars with white arrows according to the direction of transcription. (B) Statistical analysis of length, G+C content, and expression values of the top 500 genes showing significant Rrm3 recruitment in wild-type and npl3Δ cells. (***) P < 0.001 (Mann-Whitney's U-test). (C) Composite profile of Rrm3 occupancy detected by ChIP–chip across the average ORF plotted as signal log2 ratio average per each segment. (D) Detailed analysis of ChIP–chip data of Rrm3-Flag at the GCN4 region. (E) Specific Rrm3-Flag ChIP analysis using RT-qPCR of three regions (depicted as black lines in D with numbers 1–3) of GCN4 in WRBb-9B (WT) and N3RBb-4B (npl3Δ) cells carrying pCM184 (−RNH1) or pCM184RNH1 (+RNH1) without doxycycline. Data were normalized to wild-type levels without RNH1 overexpression. Average and standard deviation of three independent experiments are shown. (*) P < 0.05 (Student's t-test).

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