ALC1/eIF4A1-mediated regulation of CtIP mRNA stability controls DNA end resection

RESEARCH ARTICLE ALC1/eIF4A1-mediated regulation of CtIP mRNA stability controls DNA end resection Fernando Mejı ´as-NavarroID 1,2☯ , Guillermo Rodrı ´guez-RealID 1,2☯ , Javier Ramo ´n 1,2¤ , Rosa CamarilloID 1,2 , Pablo HuertasID 1,2 * 1Department of Genetics, University of Seville, Sevilla, Spain, 2Centro Andaluz de Biologı ´a Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Sevilla, Spain ☯These authors contributed equally to this work. ¤Current address: Mitochondrial Disorders Unit, Vall d’Hebron Institut de Recerca, Barcelona, Catalonia, Spain *[email protected] Abstract During repair of DNA double-strand breaks, resection of DNA ends influences how these lesions will be repaired. If resection is activated, the break will be channeled through homologous recombination; if not, it will be simply ligated using the non-homologous end-joining machinery. Regulation of resection relies greatly on modulating CtIP, which can be done by modifying: i) its interaction partners, ii) its post-translational modifications, or iii) its cellular levels, by regulating transcription, splicing and/or protein stability/degradation. Here, we have analyzed the role of ALC1, a chromatin remodeler previously described as an integral part of the DNA damage response, in resection. Strikingly, we found that ALC1 affects resection independently of chromatin remodeling activity or its ability to bind damaged chromatin. In fact, it cooperates with the RNA-helicase eIF4A1 to help stabilize the most abundant splicing form of CtIP mRNA. This function relies on the presence of a specific RNA sequence in the 50UTR of CtIP. Therefore, we describe an additional layer of regulation of CtIP—at the level of mRNA stability through ALC1 and eIF4A1. Author summary The DNA molecule is constantly threatened by the appearance of physical or chemical modifications that endanger the integrity of the genetic information. Among them, the breakage of the DNA molecule is the most challenging to deal with. In order to minimize such risks, cells have developed multiple DNA repair mechanisms that take care of broken chromosomes. The regulation of the usage of each different mechanism is extremely important for the safekeeping of the genetic material. CtIP is a key protein of pivotal importance in the decision between different broken DNA repair pathways. Here we show a novel regulatory mechanism that controls the abundance of this protein by controlling the stability of the different mRNA molecules produced by the CtIP gene. In brief, we show that the factors ALC1 and eIF4A1 affect the stability of some, but not all, CtIP PLOS GENETICS PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 1 / 22 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Mejı ´as-Navarro F, Rodrı ´guez-Real G, Ramo ´n J, Camarillo R, Huertas P (2020) ALC1/ eIF4A1-mediated regulation of CtIP mRNA stability controls DNA end resection. PLoS Genet 16(5): e1008787. https://doi.org/10.1371/journal. pgen.1008787 Editor: Nancy Maizels, University of Washington School of Medicine, UNITED STATES Received: September 23, 2019 Accepted: April 22, 2020 Published: May 11, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pgen.1008787 Copyright: ©2020 Mejı ´as-Navarro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. mRNA variants. This effect is dependent on the presence or absence of a specific RNA structure, called a G-quartet or G-quadruplex, in a non-coding portion of the mRNA. Introduction In order to maintain genomic integrity, cells have to successfully deal with thousands of threats on a daily basis that could potentially compromise the structure or sequence of DNA [1]. While DNA double-strand breaks (DSBs) occur at a low incidence, they nonetheless are very challenging to repair faithfully [1]. The appearance of one or more DSBs triggers a very complex response that is required to minimize genomic instability. This includes the activation of specific DSB repair pathways as well as of a cellular response that will affect virtually every aspect of metabolism, from cell cycle progression, to gene and protein expression [1]. In terms of pure repair, broken DNA molecules can be repaired by the error-prone, but relatively fast and simple, non-homologous end-joining (NHEJ) mechanism or by the more accurate but complex homologous recombination (HR) mechanism [2,3]. HR is considered the most errorfree way to deal with a broken chromosome, at least when the sister chromatid is present [3]. HR is a multi-step and complex pathway, but it is always initiated by processing the DNA ends in an event known as DNA end resection [4,5]. Resection consists of a 50–30degradation of one DNA strand at each side of the break, forming long tails of 30-OH single-strand DNA (ssDNA). ssDNA must be protected from nucleases by the binding of protective protein complexes, such as RPA [4,5]. Importantly, ssDNA is an obligatory intermediate of all HR pathways and is also a potent inhibitor of NHEJ. Resection itself is a two-step mechanism. Its initiation is catalyzed by the action of the Mre11-Rad50-Nbs1 (MRN) complex [4,5]. However, several cellular and local criteria must be met for resection to be activated; therefore, resection is only started at specific breaks under specific circumstances. Resection licensing depends greatly on a single factor, CtIP, which receives and integrates multiple cellular signals and is responsible for activating the MRN-role in resection [4,6]. Not surprisingly, CtIP is heavily regulated at several levels. First, the overall amount of CtIP is strictly controlled, both at the level of CtIP mRNA transcription [7–9] and by protein stability [10–12]. Second, several post-translational modifications (both constitutive and induced) of CtIP are required for it to be able to help activate resection [11–17]. Once resection is initiated by the combined actions of MRN and CtIP, it is extended by other nucleases [5,18], and the break can be repaired by recombination. Finally, a plethora of anti- and pro-resection factors can exert a regulatory role at this level [5]. In addition to the activation of the repair mechanisms, the presence of DSBs triggers a complex, massive cellular response known as the DNA damage response (DDR) [1]. This consists of a signaling cascade that can alter all cellular metabolism (including cell cycle progression, mRNA expression and splicing, and the chromatin environment) to create the perfect environment for repair to take place [19]. In many cases, there is a crosstalk between the DNA repair pathways and DDR. Indeed, many repair factors are controlled at different levels by this global response, either through changes in their transcription or protein stability, or through post-translational modifications. Thousands of proteins are involved in DDR, many of which can be recruited to regions of chromatin flanking a DSB. One such protein is ALC1, a chromatin remodeling factor that is recruited very rapidly to damaged DNA and is involved in local decondensation of chromatin, which is known to facilitate DDR [20]. Also known as CHD1L (for CHD1-like), ALC1 lacks the chromodomains typical of the members of the CHD family but maintains the helicase PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 2 / 22 Funding: This work was financed by an R+D+I grant from the Spanish Ministry of Economy and Competitivity (SAF2016-74855-P) and by the European Union Regional Funds (FEDER). FM-N was funded with an FPU fellowship from the Spanish Ministry of Education, and JR and GR-R were supported by the Regional Government of Andalucı ´a (Junta de Andalucı ´a) with a contract of the program “GARANTI ´A JUVENIL EN LA UNIVERSIDAD DE SEVILLA”. CABIMER is supported by the regional government of Andalucı ´a (Junta de Andalucı ´a). 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. domain [20]. Additionally, ALC1 has a macrodomain that is involved in PAR-dependent recruitment to DNA damage [20]. Further, ALC1 can locally relax the chromatin surrounding DSBs [21]. In addition to its role in DDR, ALC1 has also been implicated in transcription of specific genes [22,23]. Not surprisingly, ALC1 is considered an oncogene with effects in promoting proliferation, cell migration, invasion, and metastasis, and in inhibiting apoptosis [24]. Despite its influence on DDR and oncogenesis, ALC1 has not yet been shown to be involved directly in DNA repair. Here we show that ALC1 indeed facilitates DNA end resection and homologous recombination and, as a consequence, impairs NHEJ. Strikingly, however, it does so in a fashion that is completely independent of its DDR function, its helicase activity, and its PAR-dependent binding to damaged chromatin. Rather, this role is exerted by controlling CtIP expression and relies on the presence of a G-quadruplex (G4) structure in the 50-UTR of the CtIP major mRNA isoform. Remarkably, whereas the G4-containing (G4) CtIP mRNA is less stable in the absence of ALC1, a second mRNA species that lacks such a structure in the 50-UTR (G4less) is not. The mRNA helicase eIF4A1, which is involved in unwinding G4 in the 50-UTR of mRNAs, cooperates with ALC1 in this role. In fact, the G4 50-UTR sequence of CtIP is sufficient to affect expression of the non-related control GFP gene in a way that depends on both eIF4A1 and ALC1. Thus, we have revealed an additional way in which ALC1 and eIF4A1 regulate the responses to DSBs—by directly controlling CtIP mRNA stability. Materials and methods Cell lines and growth conditions U2OS, HeLa and RPE1 human cell lines were grown in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich) and 100 units/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich). U2OS expressing YFP-ALC1 variants [20] were grown in standard U2OS medium supplemented with 0.5 mg/ ml G418 (Gibco, Invitrogen). U2OS cells stably expressing a control shRNA or an shRNA against CCAR2 or 53BP1, and those cells bearing a copy of the DR-GFP, SA-GFP or EJ5-GFP reporter system, were grown in standard DMEM described above supplemented with 1 μg/ml puromycin (Sigma-Aldrich). U2OS cells stably expressing the GFP or GFP-CtIP plasmid were grown in standard DMEM supplemented with 0.5 mg/ml G418 (Sigma-Aldrich). For serum starvation and release, RPE1 cells were cultured for 24h in medium containing 0.1% FBS and then released in fresh medium complemented with 10% FBS. siRNAs, plasmids and transfections siRNA duplexes were obtained from Sigma-Aldrich, Dharmacon or Qiagen (S1 Table) and were transfected using RNAiMax Lipofectamine Reagent Mix (Life Technologies), according to the manufacturer’s instructions. Plasmid transfection of U2OS cells with YFP-ALC1 variants [20] and GFP plasmids containing the 50UTR regions of CtIP was carried out using FuGENE 6 Transfection Reagent (Promega) according to the manufacturer’s protocol. HR and NHEJ analysis U2OS cells bearing a single copy integration of the reporters DR-GFP (Gene conversion), SA-GFP (SSA) or EJ5-GFP (NHEJ) [25,26] were used to analyze the different DSB repair pathways. In all cases, 35,000 cells were plated in 6-well plates in duplicate. One day after seeding, cells were transfected with the indicated siRNA, and medium was replaced with fresh one without siRNAs 6–8 h later. Two days after the start of siRNA transfection, each duplicate PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 3 / 22 culture was infected, one well with lentiviral particles containing I-SceI–BFP expression construct at MOI 10 using 4 μg/ml polybrene in 1 ml of DMEM, and the other well with DMEM including polybrene but without lentivirus (as a control for basal fluorescence). Cells were then left to grow for an additional 24 h before changing the medium for fresh DMEM. One day later, cells were washed with PBS, trypsinized, neutralized with DMEM, centrifuged for 5 min at 700 g, fixed with 4% paraformaldehyde for 20 min and collected by centrifugation. Cell pellets were washed once with PBS before resuspension in 150 μl of PBS. Samples were analyzed with a BD FACSAria with the BD FACSDiva Software v5.0.3. Four different parameters were considered: side scatter (SSC), forward scatter (FSC), blue fluorescence (407 nm violet laser BP, filter 450/40), green fluorescence (488 nm blue laser BP, filter 530/30). Finally, the number of green cells from at least 10,000 events positives for blue fluorescence (infected with the I-SceI–BFP construct) was scored, considering the background of green fluorescence obtained in the samples without infection with lentivirus harboring pBFP-ISceI plasmid as previously described [26–28]. To facilitate comparison between experiments, this ratio was normalized with siRNA control. At least three independent experiments were carried out for each condition; the average and standard deviations are given. SDS-PAGE and Western blot analysis Protein extracts were prepared in 2×Laemmli buffer (4% SDS, 20% glycerol, 125 mM Tris- HCl, pH 6.8) and passed 10 times through a 0.5 mm needle–mounted syringe to reduce viscosity. Proteins were resolved by SDS-PAGE and transferred to low fluorescence PVDF membranes (Immobilon-FL, Millipore). Membranes were blocked with Odyssey Blocking Buffer (LI-COR) and blotted with the appropriate primary antibody (S2 Table) and infra-red dyed secondary antibodies (LI-COR) (S3 Table). Antibodies were prepared in blocking buffer supplemented with 0.1% Tween-20. Membranes were air-dried in the dark and scanned in an Odyssey Infrared Imaging System (LI-COR), and images were analyzed with ImageStudio software (LI-COR). YFP-ALC1 immunoprecipitation To immunoprecipitate YFP-ALC1 protein, U2OS cells were scrapped in lysis buffer containing proteases and phosphatases inhibitors (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.5% NP-40, protease inhibitors [11873580001, Roche] and phosphatase inhibitor cocktail 3 [P0044, Sigma]). To degrade DNA, 100 U/ml Benzonase (70746–4, VWR) was added to protein extracts and incubated 30 min on ice. During that time, chromatin was sheared by passing the sample three times through a syringe with a 0.5x16 mm needle. In parallel, GFP-Trap beads (gtm-20, chromotek) were equilibrated once in lysis buffer without inhibitors for 5 min on ice with rocking, followed by two washes with lysis buffer containing inhibitors. Then, 1 mg of protein extracts was diluted in lysis buffer containing inhibitors to 0.2% NP-40 concentration. GFP-Trap beads were added and incubated under gentle agitation at 4˚C for 2 hours. Beads were then washed twice with lysis buffer containing inhibitors, and the precipitate was eluted in Laemmli buffer, boiled, and resolved in SDS-PAGE as described. Immunofluorescence and microscopy For RPA foci visualization, U2OS cells knocked-down for different proteins were seeded on coverslips. At 1 h after either irradiation (10 Gy) or treatment with 10 μM etoposide, coverslips were washed once with PBS followed by treatment with pre-extraction buffer (25 mM Tris- HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl 2 , 300 mM sucrose and 0.2% Triton X- 100) for 5 min on ice. Cells were fixed with 4% paraformaldehyde (w/v) in PBS for 15 min. PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 4 / 22 Following two washes with PBS, cells were blocked for 1 h with 5% FBS in PBS, co-stained with the appropriate primary antibodies (S2 Table) in blocking solution overnight at 4˚C or for 2 h at room temperature, washed again with PBS and then co-immunostained with the appropriate secondary antibodies for 1 h (S3 Table) in blocking buffer. After washing with PBS and drying with ethanol 70% and 100% washes, coverslips were mounted into glass slides using Vectashield mounting medium with DAPI (Vector Laboratories). RPA foci immunofluorescences were analyzed using a Leica Fluorescence microscope. For 53BP1 visualization, U2OS cells were seeded and transfected as previous described. Once collected, cells were fixed with methanol (VWR) for 10 min on ice, followed by treatment with acetone (Sigma) for 30 sec on ice. For RIF1 foci visualization, cells were fixed with 4% PFA for 15 min, washed twice with 1×PBS and then permeabilized for 15 min with 0.25% Triton diluted in 1×PBS. Finally, CCAR2 foci were observed in cells permeabilized for 10 min on ice with 0.2% Triton diluted in 1×PBS. Samples were immunostained as described above with the appropriate primary and secondary antibodies (S2 and S3 Tables). Images obtained with a Leica Fluorescence microscope were then analyzed using Metamorph to count the number, intensity and size of the foci. SMART (single-molecule analysis of resection tracks) SMART was performed as described [29]. Briefly, cells were grown in the presence of 10 μM BrdU for 22–24 h. Cultures were then irradiated (10 Gy) and harvested after 1 h. Cells were embedded in low-melting agarose (Bio-Rad), followed by DNA extraction. DNA fibers were stretched on silanized coverslips, and immunofluorescence was carried out to detect BrdU (S2 and S3 Tables). Samples were observed with a Nikon NI-E microscope, and images taken and processed with the NIS ELEMENTS Nikon Software. For each experiment, at least 200 DNA fibers were analyzed, and the length of the fibers was measured with Adobe Photoshop CS4. Cell cycle analysis Cells were fixed with cold 70% ethanol overnight, incubated with 250 μg/ml RNase A (Sigma) and 10 μg/ml propidium iodide (Fluka) at 37˚C for 30 min and analyzed with a FACSCalibur (BD). Cell cycle distribution data were further analyzed using ModFit LT 3.0 software (Verity Software House Inc). RNA extraction, reverse transcription and quantitative PCR RNA extracts were obtained from cells using NZY Total RNA Isolation kit (Nzytech) according to manufacturer’s instructions. To obtain complementary DNA (cDNA), 1 μg RNA was subjected to RQ1 DNase treatment (Promega) prior to reverse transcription reaction using Maxima H Minus First Strand cDNA Synthesis kit (Thermo Scientific) according to manufacturer’s instructions. Quantitative PCR from cDNA was performed to check siRNA-mediated knock-down of several proteins. For this, iTaq Universal SYBR Green Supermix (Bio-Rad) was used following manufacturer’s instructions. DNA primers used for qPCR are listed in S4 Table. Q-PCR was performed in an Applied Biosystem 7500 FAST Real-Time PCR system. The comparative threshold cycle (Ct) method was used to determine relative transcripts levels (Bulletin 5279, Real-Time PCR Applications Guide, Bio-Rad), using β-actin expression as internal control. Expression levels relative to β-actin were determined with the formula 2 -ΔΔCt [30]. PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 5 / 22 Cycloheximide chase assay U2OS cells depleted for the indicated siRNA were treated with fresh DMEM containing 150 μg/ml cycloheximide (CHX, Sigma-Aldrich). Cells were collected 0, 4 and 8 h after CHX addition, and protein extracts were prepared as described before. Samples were resolved by SDS-PAGE, analyzed by immunoblotting (S2 and S3 Tables) and quantified with ImageStudio software (LI-COR). The siNT condition at time 0 was normalized as 100%, and the other conditions were relativized to this point. CtIP expression was calculated as the amount of CtIP divided by α-tubulin signal. Cloning of CtIP 50-UTR sequences Total RNA from U2OS cells was extracted and reverse-transcribed into cDNA as previous described using an specific primer for CtIP mRNA (S4 Table). The 50-UTR regions corresponding to the G4less and G4 isoforms were amplified by PCR by adding specific target sites for restriction enzymes (Takara). Amplified regions were cloned into the pEGFP-N1 vector (Clontech). Plasmid DNA was inserted into competent cells of the DH5αstrain of Escherichia coli. Vector DNA was purified using PureYield Plasmid Maxiprep System (Promega) and sequenced to check the proper integration of the insert into the GFP plasmid. Statistical analysis Statistical significance was determined with a Student’s t-test or ANOVA as indicated using PRISM software (Graphpad Software Inc.). Statistically significant differences were labelled with one, two or three asterisks for P<0.05, P<0.01 or P<0.001, respectively. Results ALC1 facilitates homologous recombination by promoting resection ALC1 is rapidly recruited to DSBs, suggesting a very early role for it in the response to such type of damage [20]. This observation raised the question whether ALC1 is affecting the choice between DSB repair pathways, namely between HR and NHEJ. To test this idea, we analyzed previously described, pathway-specific, GFP-based, DSB repair assays [25,26]. Strikingly, ALC1 depletion led to a global decrease in homologous recombination, both in Rad51-depen- dent gene conversion and in Rad51-independent single-strand annealing (Fig 1A and 1B). Reciprocally, downregulation of ALC1 increased NHEJ (Fig 1C). Similar results for homologous recombination were obtained using two additional siRNAs against ALC1, including one against the 30-UTR (S1A Fig). However, NHEJ was more variable using these additional siR- NAs (S1B Fig) (the levels of ALC1 depletion in U2OS cells with different siRNAs are shown in S1C and S1D Fig). Thus, our data agreed with ALC1 playing a role in the decision between HR and NHEJ at an initial level, and mostly at the level of recombination. Such unbalancing of DSB repair pathways has been observed mainly when DNA resection regulation is affected. In agreement with a role of ALC1 in DNA end processing, ALC1 depletion qualitatively resembled the downregulation of the key resection factor CtIP (Fig 1A–1C and S1A and S1B Fig). Thus, one possibility was that ALC1 was affecting DNA end resection. To test this, we analyzed DNA end resection by checking the formation of RPA foci in cells exposed to DNA damage. We used phosphorylation of the histone variant H2AX (γH2AX) as a positive marker of DNA damage. Indeed, ALC1 depletion impaired resection in U2OS cells treated with ionizing radiation (Fig 2A) or the topoisomerase II inhibitor etoposide (Fig 2B). DNA end resection is regulated by the cell cycle, as G1 cells do not resect extensively [4]. We did not observe a significant change in the amount of G1 cells when ALC1 was downregulated (S1E Fig); however, to PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 6 / 22 be absolutely sure that the observed effects were not due to cell cycle perturbations, we repeated the RPA foci assay after irradiation but considering only the cells positive for the cell cycle marker CENPF, a kinetochore protein that accumulates in S and G2 cells [31]. Even analyzing exclusively S/G2 cells, the DNA resection defect caused by ALC1 depletion was maintained (Fig 2C). Moreover, in HeLa cells, RPA foci formation after ionizing radiation was also compromised in cells downregulated for ALC1 (Fig 2D; see also S1C Fig for depletion, and S1F Fig for cell cycle). From this point onwards, all the experiments shown were performed in U2OS cells. To demonstrate that the effect in DNA end resection was indeed mediated by ALC1, we complemented the depletion of the mRNA using a siRNA against the 30-UTR with a siRNA- resistant YFP-ALC1 construct (Fig 2E). We observed a reduction not only in the number of breaks that were processed in ALC1-depleted cells but also in the length of the resected DNA, measured by SMART [29,32] (Fig 2F). Moreover, we observed a decrease after ALC1 depletion not only in the number of RPA foci–positive cells, but also in the average number of RPA foci per cell (S1G Fig). ALC1 depletion increases the loading of anti-resection factors For resection to proceed, it needs to overcome the barriers caused by several anti-resection pro-NHEJ proteins that are recruited to damaged chromatin, such as 53BP1, RIF1 and CCAR2 [33,34]. Thus, we wondered if the impaired resection observed in ALC1-depleted cells Fig 1. ALC1 facilitates homologous recombination and impairs NHEJ. A Classical homologous recombination was measured as described in the methods section using the DR-GFP reporter (top) [25]. An active GFP gene is formed upon gene conversion of an I-SceI–induced DSB. Briefly, cells constitutively transfected with a single copy of the reporter were depleted for the indicated proteins, and after two days were infected with viral particles containing an I-SceI–BFP expression construct. Two day later, fluorescence accumulation was scored by FACs. The percentage of blue fluorescent cells (bearing the I-SceI) that became green was scored and normalized as described in the Methods section. The experiment was repeated three times, and the average and standard deviation is plotted (bottom). Statistical significance was calculated using a one-way ANOVA test. Two or three asterisks represent P<0.01 and P<0.001, respectively. BSame as (A) but using the SSA reporter SA-GFP [26]. CSame as (A) but using the NHEJ reporter EJ5-GFP [26]. https://doi.org/10.1371/journal.pgen.1008787.g001 PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 7 / 22 Fig 2. ALC1 is required for DNA end resection. A DNA end resection was scored by the accumulation of RPA foci 1 h after 10 Gy of ionizing radiation in U2OS cells depleted for two days for the indicted factors. γH2AX was used as a mark of DNA damage. The average and standard deviation of three independent experiments is plotted (top), and representative images are shown (bottom). Scale Bar represent 25 μm. Statistical significance was calculated using a one-way ANOVA test. One, two or three asterisks represent P<0.05, P<0.01 and P<0.001 respectively. BSame as (A) but after 1 h treatment of 10 μM etoposide. CSame as (A) but using CENPF as a marker of the S and G2 phases of the cell cycle. The percentage of RPA-positive cells from those positive for CENPF is represented in the graph. DSame as (A) but in HeLa cells. ESame as (A) but in cells transfected or not with a YFP-tagged version of wild-type ALC1. In this case, an siRNA against the 30-UTR of ALC1, absent in the YFP-fusion, was used to deplete endogenous ALC1. F Resection length on individual DNA fibers was calculated as previously published using SMART. Briefly, cells depleted for the indicated proteins were incubated for 24 h with BrdU. Cells were then irradiated with 10 Gy, and after 1 h, DNA was extracted as described in the Method section. DNA was then stretched on coverslips, and ssDNA was detected using a BrdU antibody. At least 250 DNA fibers were scored per condition. Resection length was normalized to the control sample. The average and standard deviation of three independent experiments were plotted (top). and representative images are shown (bottom). Statistical significance was calculated using a one-way ANOVA test. Three asterisks represent P<0.001. https://doi.org/10.1371/journal.pgen.1008787.g002 PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 8 / 22 correlated with an increased loading of such factors, as suggested by the increase in the NHEJ pathway (Fig 1C). Indeed, ALC1 downregulation, similar to CtIP depletion, increased the number of foci of both 53BP1 and RIF1 in irradiated cells (Fig 3A and 3B). This was not due to an accelerated kinetic of recruitment, as the levels of 53BP1 remained equally elevated at different times after irradiation (S2A Fig). Moreover, for 53BP1 (but not for RIF1), these foci were brighter and bigger (S2B–S2E Fig). Along the same lines, ALC1 depletion increased the number of foci of the resection antagonist CCAR2 (Fig 3C). Neither the size of CCAR2 foci nor their intensity were significantly different in siRNA-depleted ALC1 cells as compared to control cells (S2F–S2G Fig). There are two possible explanations for the resection phenotype observed in ALC1 downregulated cells: 1) the chromatin environment favored either the recruitment or retention of anti-resection proteins, which in turn blocked processing of the DNA ends; and 2) the resection impairment caused an accumulation of 53BP1, RIF1 and CCAR2. In the first scenario, depletion of such resection barriers should suppress, or at least alleviate, the decrease in RPA foci caused by ALC1 reduction. As this was not observed (S3A–S3C Fig), we concluded that ALC1 facilitates resection progression directly, and that this resection is responsible for displacing 53BP1, RIF1 and CCAR2 from damaged DNA. The role of ALC1 in resection is independent of its catalytic activity and DNA binding Several domains have been previously described in ALC1 as important for its role in the DNA damage response. This include two tandem catalytic domains with helicase activity, and a Fig 3. The recruitment of anti-resection factors is increased upon ALC1 depletion. A–C The formation of 53BP1 foci (A) or RIF1 foci (B), or the recruitment of CCAR2d (C) was tested in U2OS cells transfected with the indicated siRNAs at 1 h after irradiation (10 Gy). Cells were treated as described in Fig 2A. The number of foci per cell was scored automatically using the software Metamorph. The average and standard deviation of three independent experiments were plotted (left), and representative images of one cell are shown (right). Scale Bar represent 10 μm. Statistical significance was calculated using a one-way ANOVA test. Two or three asterisks represent P<0.01 and P<0.001, respectively. https://doi.org/10.1371/journal.pgen.1008787.g003 PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 9 / 22 ALC1 and eIF4A1, whereas G4-GFP mRNA required both proteins for proper accumulation (Fig 7D). Thus, the presence of the G4 from CtIP in the unrelated 50-UTR of the GFP gene is sufficient to make the expression of that gene dependent on ALC1 and eIF4A1. Discussion The balance between HR and NHEJ is critical for cell fitness and an appropriate response to chromosome breaks. Thus, it is not surprising that a tight regulatory network controls the choices between the two DSB repair pathways. In the last decade, it has become obvious that the preferred regulatory point is DNA end resection, and more specifically, the control of the activity and levels of the key resection factor CtIP. CtIP is heavily post-transcriptionally modified, allowing it to specifically respond to cell cycle positioning and the DNA damage response [6]. Constitutive post-transcriptional modifications also impact in its role in resection [14]. However, it is also heavily controlled both transcriptionally and at the level of protein stability [7–12]. Here, we show an additional layer of regulation of CtIP homeostasis, which acts post-transcriptionally at the level of mRNA stability. Of the two known splicing variants that code for a full-length protein, we show that the most abundant form (the mRNA of which contains the G4 structure; termed here G4) is intrinsically less stable. Moreover, both variants (but mostly the most abundant one) require additional factors that control their stability, namely the helicases ALC1 and the transcription factor eIF4A1. ALC1 depletion has little effect on the bulk of CtIP mRNA, as detected using a pair of oligonucleotides that recognize all CtIP isoforms and not only the two that code for full-length CtIP; this argues that it does not affect general CtIP transcription. But a different picture emerges with a careful analysis specifically of the two splicing variants that code for a fully functional CtIP. In this case, changes in mRNA levels can be readily observed in the G4 variant. Thus, although ALC1 has little effect on the bulk of CtIP splicing forms, it is extremely important for maintaining the levels of the full-length CtIP protein, which it does by affecting the stability of the mRNA variants that encode the full-length protein and, particularly, for the G4 isoform. Most of this depends on the presence of specific 50-UTR sequences. Indeed, of the two isoforms analyzed here, termed G4 and G4less, the one containing RNA sequences that are prone to forming G-quadruplexes (G4) is especially relevant. The presence of the G4 sequence, even in a chimeric fusion with GFP, increases the levels of mRNA and protein as compared with the sequence of G4less, but, on the negative side, it makes such splicing variant more dependent on ALC1. Interestingly, this is more evident in the context of the GFP constructs than in the endogenous CtIP mRNA, particularly for the requirement of eIF4A1. One likely explanation is that the CtIP mRNA contains additional signals that control its stability, which could be dependent as well as independent of ALC1 and eIF4A1. The analysis of the GFP construct, at the level of mRNA and protein accumulation, also indicates that, superseded to the regulation of mRNA stability, an additional layer of control seems to exist at the level of protein translation that relies on eIF4A1 but not on ALC1. Indeed, siRNA- mediated depletion of ALC1 and eiF4A1 rendered similar effects on the mRNA amount from the GFP constructs but showed dramatic differences at the protein level. Specifically, whereas downregulation of eIF4A1 increased GFP levels from a G4less-harboring mRNA, it severely reduced protein production from the G4 construct as compared with cells depleted for ALC1 and despite having similar mRNA levels. Moreover, even though both GFP constructs are severely decreased at the level of mRNA after eIF4A1 depletion, a reduction in protein accumulation is only evident in the G4 GFP-fusion. As mentioned above, both isoforms contain the (CGG) motif that can be recognized by eiF4A1, but only the G4 variant can form a secondary structure of the RNA G-quadruplex. Thus, it is not surprising that the PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 16 / 22 stability of both mRNA species can depend on eIF4A1, but the translation of only the G4-containing one is affected. This agrees with the known role of eIF4A1 in translation of mRNAs that contain G-quadruplexes in their 50-UTR [35]. However, and as mentioned above, these results are less clear in the context of the endogenous CtIP mRNA splice variants, suggesting additional effects of ALC1, eIF4A1 and possibly other proteins in controlling CtIP mRNA stability and maybe translation efficiency. In fact, the splicing factors SF3B and Aquarius were recently shown to maintain CtIP levels and proper HR rates [10,39]. Interestingly, SF3B affect resection mainly through CtIP, but as we observe here with ALC1, SF3B effect on HR cannot be rescued by CtIP overexpression [39]. Moreover, the mRNA binding protein SERBP1, associated to the 40S subunit of active ribosomes in normal and tumor cells [40], binds to CtIP mRNA and promotes its translation specifically in S phase by binding to its 30-UTR [41]. These additional factors might explain some discrepancies observed between ALC1 and CtIP depletion for DNA end resection. Chiefly among them, ALC1-depleted cells are not sensitive to camptothecin treatment [20], but CtIP has been shown to be particularly important for CPT-induced DSB resection and survival [42]. CPT adducts only become DNA DSBs in S phase associated to replication. In contrast, IR or etoposide promote DSB formation in all of the cell cycle stages. Therefore, ALC1 may have a more important role in maintaining CtIP levels in G2, whereas it could be counteracted in S phase by SERBP1 [41]. One likely explanation is that in S phase, SERB- P1-induced increases in translation might outweigh the reduction of CtIP mRNA levels caused by ALC1 depletion, allowing a normal expression of the protein and sufficient resection of DSB in S phase. This additional layer of regulation of CtIP expression at the level of mRNA stability/translation probably reflects its importance for replication. Indeed, CtIP is known to be enriched in ongoing replication forks [43], and to be important for rescuing stalled replication forks [44]. Further, CtIP protects the forks from degradation when a replication stress is present [45]. Thus, another possibility is that, despite the reduced levels of CtIP after ALC1 depletion, all the remaining protein remains associated with the replication machinery, where it will be readily available to repair CPT-induced damage, but it is not free to support resection outside of the replication context. Our data also indicate that ALC1 has an additional role in controlling the response to DNA breaks that is independent from its DDR function of relaxing chromatin in the vicinity of DSBs [21]. This new role relies in controlling CtIP to regulate resection. Whether this novel activity is connected with the DNA damage response remains to be explored. Additionally, our data also suggest that ALC1 affects HR independently of CtIP levels, and this effect could rely in the regulation of the levels of other HR proteins or in the role in the DDR previously described [20,21]. One interesting hypothesis is that the G4less variant serves as a reservoir of CtIP mRNA, in order to grant the production of a basal level of the protein that is less affected by external signals. In contrast, the G4 50-UTR specie is responsible for the bulk of protein expression but might be more sensitive to the presence of specific factors. One tantalizing idea is that this provides for an alternative regulatory step in DNA end resection and that, in fact, the G4 50-UTR isoform abundance might react to yet unknown signals. ALC1 and eIF4A1 might participate in this unknown regulation. In fact, it is plausible that ALC1 retention at sites of DSB might modulate its ability to stabilize CtIP mRNA. Usually, ALC1 is quickly recruited to DNA damage, but it is also quickly released [20]. Thus, normally CtIP expression would not be affected. However, in this scenario, trapping ALC1 at damaged chromatin (for example, because the breaks cannot be repaired) could destabilize the mRNA, causing a drop in the CtIP protein level in the long term, and thus preventing hyper-resection. PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 17 / 22 Supporting information S1 Table. siRNAs used in this study. (DOCX) S2 Table. Primary antibodies used in this study. WB: Western blot; IF; Immunofluorescence; SMART: Single Molecule Analysis of Resection Tracks. (DOCX) S3 Table. Secondary antibodies used in this study. WB: Western blot; IF; Immunofluorescence; SMART: Single Molecule Analysis of Resection Tracks. (DOCX) S4 Table. Primers used in this study. (DOCX) S1 Fig. ALC1 depletion. A, Gene conversion, measured as described in Fig 1A in cells depleted of ALC1 using two additional siRNAs. siALC1-2 targets the coding region, whereas siALC1- 3’ UTR targets the 3’ UTR of the RNA. B, Same as A, but using the EJ5 reproter to check NHEJ efficiency. C, Depletion efficiency of ALC1 in cells transfected with the indictaed siRNAs. D, Same as A, but using additional siRNA against ALC1, either ALC1-2 (left) or an siRNA targeting the 3’ UTR (right) in U2OS cells. E, Cell cycle profile of U2OS cells transfected either with siNT control sequence or with siRNAs against ALC1 and CtIP as indicated. The average and standard deviation of three independent experiment are plotted. F, Same as C but in HeLa cells. G, The average number of RPA foci per cell formed 1h upon exposure to 10Gy of ionizing radiation was scored automatically using the software Metamorph. The average and standard deviation of three independent experiments are plotted (left) and representative images of one cell are shown on the right. Statistical analysis as described in Fig 2A. (PDF) S2 Fig. Accumulation of anti-resection factors in ALC1 depleted cells. A, The average number of 53BP1 foci per cell was calculated as described in Fig 3A in cells at different times after exposure to 2 Gy of ionizing radiation in control cells and cells depleted for ALC1 (red). B, Computer-based measurement of the size of 53BP1 foci in cells depleted for the indicated factors using the Metamorph software. Size was normalized with the control sample. The average and standard deviation of three independent experiments is plotted. Other details as Fig 1A.C, Computer-based analysis of the intensity of 53BP1 foci. Other details as panel B. D, Same as B but for RIF1 foci. E, Same as C but for RIF1. F, Same as B but for CCAR2 foci. G, Same as C but for CCAR2. (PDF) S3 Fig. Resection impairment in ALC1 depleted cells does not depend on the loading of anti-resection factors. A, Resection was measured 1h after irradiation in cells infected with shRNA against 53BP1, CCAR2 or a control sequence and transfected with an siRNA against either ALC1 (black bars) or a control sequence (white bars). Representative images are shown on the right. Scale Bar represent 25 μm. Other details as in Fig 5B.B, Western blot showing the downregulation of ALC1, CCAR2 and 53BP1 of the cells described in A. HSP70 was used as loading control. C, Cell cycle analysis of the cells described in panel A. (PDF) S4 Fig. Resection impairment in ALC1 depleted cells is suppressed by different ALC1 constructs. A, Resection was measured 1h after irradiation in cells transfected with siRNA against ALC1 (black bars) or a control sequence (white bars) and then transfected with the different PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 18 / 22 YFP-ALC1 constructs as mentioned in Fig 2. Representative images are shown on the right. Scale Bar represent 25 μm. Other details as in Fig 2A.B, The average number of RPA foci per cell in samples treated as in panel A and formed 1h upon exposure to 10Gy of ionizing radiation was scored automatically using the software Metamorph. The average and standard deviation of three independent experiments are plotted. C, Cell cycle analysis of the cells described in panel A. D, Western blot showing the expression of YFP-tagged version of ALC1. Protein samples from cells transfected with the indicated ALC1 variants, downregulated or not for the endogenous version using siRNA as depicted in the figures, were resolved in SDS-PAGE and blotted with an ALC1 antibody. The YFP-tagged and endogenous proteins are marked in the blot with triangles. α-tubulin was used as loading control. E, The levels of CtIP protein in cells transfected with the mentioned version of YFP-ALC1 and depleted (black bars) or not (white bars) of endogenous ALC1 with an siRNA targeting the 3’ end of the mRNA was determined by Western blot quantification using the Odissey Li-Cor Infrared system and normalized to control cells transfected with an empty plasmid. The average and standard deviation of three independent experiment are plotted. (PDF) S5 Fig. Cell cycle analysis of ALC1 and eIF4A1 depleted cells bearing GFP and GFP-CtIP. A, The levels of CtIP protein in cells depleted for the indicated proteins was determined by western blot quantification using the Odissey Li-Cor Infrared system and normalized to control cells. A representative image is shown on the right side and the average and standard deviation of three independent experiment are plotted on the left. Statistical significance was calculated using a one-way ANOVA test. One, two or three asterisks represent p<0.05, p<0.01 and p<0.001, respectively. B, Cell cycle analysis of the cells described in Fig 5B.C, Gene conversion was measured as described in Fig 1A in cells bearing with a FLAG-CtIP or an empty FLAG plasmid and transfected with the indicated siRNAs, D, RPE1 cells synchronized by serum starvation and release as described in the methods section were collected 0h or 22h after serum addition and cell cycle distribution was assayed by FACs analysis. At 0h an enrichment in G1 cells was observed, whereas 22 h later an enrichment on S and G2 was detected. (PDF) Acknowledgments We wish to thank Simon Boulton for kindly providing YFP-ALC1 variants, and Nestor Garcı ´a-Rodrı ´guez and Sabrina Rivero for critical reading of the manuscript. Author Contributions Funding acquisition: Pablo Huertas. Investigation: Fernando Mejı ´as-Navarro, Guillermo Rodrı ´guez-Real, Javier Ramo ´n, Rosa Camarillo. Supervision: Pablo Huertas. Writing – original draft: Pablo Huertas. Writing – review & editing: Pablo Huertas. References 1. Ciccia A, Elledge SJ. The DNA Damage Response: Making It Safe to Play with Knives. Mol Cell. 2010; 40: 179–204. https://doi.org/10.1016/j.molcel.2010.09.019 PMID: 20965415 PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 19 / 22 2. Davis AJA, Chen DDJ. DNA double strand break repair via non-homologous end-joining. Transl Cancer Res. 2013; 2: 130–143. https://doi.org/10.3978/j.issn.2218-676X.2013.04.02 PMID: 24000320 3. Jasin M, Rothstein R. Repair of strand breaks by homologous recombination. Cold Spring Harb Perspect Biol. 2013; 5. https://doi.org/10.1101/cshperspect.a012740 PMID: 24097900 4. Huertas P. DNA resection in eukaryotes: Deciding how to fix the break. Nat Struct Mol Biol. 2010; 17: 11–16. https://doi.org/10.1038/nsmb.1710 PMID: 20051983 5. Symington LS. Mechanism and regulation of DNA end resection in eukaryotes. Crit Rev Biochem Mol Biol. 2016; 51: 195–212. https://doi.org/10.3109/10409238.2016.1172552 PMID: 27098756 6. Makharashvili N, Paull TT. CtIP: A DNA damage response protein at the intersection of DNA metabolism. DNA Repair (Amst). 2015; 32: 75–81. https://doi.org/10.1016/j.dnarep.2015.04.016 PMID: 25957490 7. Liu F, Lee W. CtIP Activates Its Own and Cyclin D1 Promoters via the E2F / RB Pathway during G 1 / S Progression. Mol Cell Biol. 2006; 26: 3124–3134. https://doi.org/10.1128/MCB.26.8.3124-3134.2006 PMID: 16581787 8. Zhang F, Tang H, Jiang Y, Mao Z. The transcription factor GATA3 is required for homologous recombination repair by regulating CtIP expression. Oncogene. 2017; 36: 5168–5176. https://doi.org/10.1038/ onc.2017.127 PMID: 28481869 9. Hu¨hn D, Kousholt AN, Sørensen CS, Sartori AA. MiR-19, a component of the oncogenic miR-17*92 cluster, targets the DNA-end resection factor CtIP. Oncogene. 2015; 34: 3977–3984. https://doi.org/10. 1038/onc.2014.329 PMID: 25308476 10. Hashimoto M, Iwabuchi K, Isono M, Matsui T, Matsunaga T, Wakasugi M, et al. Aquarius is required for proper CtIP expression and homologous recombination repair. Sci Rep. 2017; 7: 1–11. https://doi.org/ 10.1038/s41598-016-0028-x PMID: 28127051 11. Steger M, Murina O, Hu¨hn D, Ferretti LP, Walser R, Ha ¨nggi K, et al. Prolyl isomerase PIN1 regulates DNA double-strand break repair by counteracting DNA end resection. Mol Cell. 2013; 50: 333–343. https://doi.org/10.1016/j.molcel.2013.03.023 PMID: 23623683 12. Lafranchi L, de Boer HR, de Vries EG, Ong S, Sartori AA, van Vugt MA. APC/C C dh1 controls CtIP stability during the cell cycle and in response to DNA damage. EMBO J. 2014; 33: 2860–2879. https://doi. org/10.15252/embj.201489017 PMID: 25349192 13. Huertas P, Jackson SP. Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair. J Biol Chem. 2009; 284: 9558–9565. https://doi.org/10.1074/jbc.M808906200 PMID: 19202191 14. Soria-Bretones I, Cepeda-Garcı ´a C, Checa-Rodriguez C, Heyer V, Reina-San-Martin B, Soutoglou E, et al. DNA end resection requires constitutive sumoylation of CtIP by CBX4. Nat Commun. 2017; 8. https://doi.org/10.1038/s41467-017-00183-6 PMID: 28740167 15. Peterson SE, Li Y, Wu-Baer F, Chait BT, Baer R, Yan H, et al. Activation of DSB Processing Requires Phosphorylation of CtIP by ATR. Mol Cell. 2013; 49: 657–667. https://doi.org/10.1016/j.molcel.2012.11. 020 PMID: 23273981 16. Yu X, Fu S, Lai M, Baer R, Chen J. BRCA1 ubiquitinates its phosphorylation-dependent binding partner CtIP. Genes Dev. 2006; 20: 1721–1726. https://doi.org/10.1101/gad.1431006 PMID: 16818604 17. Yu X, Chen J. DNA Damage-Induced Cell Cycle Checkpoint Control Requires CtIP, a Phosphorylation- Dependent Binding Partner of BRCA1 C-Terminal Domains DNA Damage-Induced Cell Cycle Checkpoint Control Requires CtIP, a Phosphorylation-Dependent Binding Partner of BRCA1. Mol Cell Biol. 2004; 24: 9478–9486. https://doi.org/10.1128/MCB.24.21.9478-9486.2004 PMID: 15485915 18. Cejka P. DNA end resection: Nucleases team up with the right partners to initiate homologous recombination. J Biol Chem. 2015; 290: 22931–22938. https://doi.org/10.1074/jbc.R115.675942 PMID: 26231213 19. Dantuma NP, van Attikum H. Spatiotemporal regulation of posttranslational modifications in the DNA damage response. EMBO J. 2015; 35: 6–23. https://doi.org/10.15252/embj.201592595 PMID: 26628622 20. Ahel D, Hořejs ˇı ´Z, Wiechens N, Polo SEE, Garcia-Wilson E, Ahel I, et al. Poly(ADP-ribose)-dependent regulation of DNA repair by the chromatin remodeling enzyme ALC1. Science (80-). 2009; 325: 1240– 1243. https://doi.org/10.1126/science.1177321 PMID: 19661379 21. Sellou H, Lebeaupin T, Chapuis C, Smith R, Hegele A, Singh HR, et al. The poly(ADP-ribose)-depen- dent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage. Mol Biol Cell. 2016; 27: 3791–3799. https://doi.org/10.1091/mbc.E16-05-0269 PMID: 27733626 22. Li Y, Chen L, Chan THM, Liu M, Kong K, Qiu J, et al. SPOCK1 Is Regulated by CHD1L and Blocks Apoptosis and Promotes HCC Cell Invasiveness and Metastasis in Mice. Gastroenterology. 2013; 144: 179–191.e4. https://doi.org/10.1053/j.gastro.2012.09.042 PMID: 23022495 PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 20 / 22 23. Chan THM, Chen L, Liu M, Hu L, Zheng BJ, Poon VKM, et al. Translationally controlled tumor protein induces mitotic defects and chromosome missegregation in hepatocellular carcinoma development. Hepatology. 2012; 55: 491–505. https://doi.org/10.1002/hep.24709 PMID: 21953552 24. Ma N-F, Hu L, Fung JM, Xie D, Zheng B-J, Chen L, et al. Isolation and characterization of a novel oncogene, amplified in liver cancer 1, within a commonly amplified region at 1q21 in hepatocellular carcinoma. Hepatology. 2008; 47: 503–510. https://doi.org/10.1002/hep.22072 PMID: 18023026 25. Pierce AJ, Johnson RD, Thompson LH, Jasin M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells service XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. 1999; 2633–2638. 26. Bennardo N, Cheng A, Huang N, Stark JM. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 2008; 4. https://doi.org/10.1371/journal.pgen. 1000110 PMID: 18584027 27. Stark JM, Pierce AJ, Oh J, Pastink A, Jasin M. Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol Cell Biol. 2004; 24: 9305–16. https://doi.org/10.1128/MCB.24.21. 9305-9316.2004 PMID: 15485900 28. Pierce AJ, Johnson RD, Thompson LH, Jasin M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev. 1999; 13: 2633–8. https://doi.org/10.1101/gad.13.20.2633 PMID: 10541549 29. Huertas P, Cruz-Garcia A. Single Molecule Analysis of Resection Tracks. 2018. pp. 147–154. 30. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001; 25: 402–408. https://doi.org/10.1006/meth.2001.1262 PMID: 11846609 31. Varis A, Salmela AL, Kallio MJ. Cenp-F (mitosin) is more than a mitotic marker. Chromosoma. 2006; 115: 288–295. https://doi.org/10.1007/s00412-005-0046-0 PMID: 16565862 32. Cruz-Garcı ´a A, Lo ´pez-Saavedra A, Huertas P. BRCA1 accelerates CtIP-ediated DNA-end resection. Cell Rep. 2014; 9: 451–459. https://doi.org/10.1016/j.celrep.2014.08.076 PMID: 25310973 33. Zimmermann M, De Lange T. 53BP1: Pro choice in DNA repair. Trends Cell Biol. 2014; 24: 108–117. https://doi.org/10.1016/j.tcb.2013.09.003 PMID: 24094932 34. Lo ´pez-Saavedra A, Go ´mez-Cabello D, Domı ´nguez-Sa ´nchez MS, Mejı ´as-Navarro F, Ferna ´ndez-A ´vila MJ, Dinant C, et al. A genome-wide screening uncovers the role of CCAR2 as an antagonist of DNA end resection. Nat Commun. 2016; 7. https://doi.org/10.1038/ncomms12364 PMID: 27503537 35. Wolfe AL, Singh K, Zhong Y, Drewe P, Rajasekhar VK, Sanghvi VR, et al. RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer. Nature. 2014; 513: 65–70. https://doi.org/10.1038/ nature13485 PMID: 25079319 36. Modelska A, Turro E, Russell R, Beaton J, Sbarrato T, Spriggs K, et al. The malignant phenotype in breast cancer is driven by eIf4A1-mediated changes in the translational landscape. Cell Death Dis. 2015; 6: 1–12. https://doi.org/10.1038/cddis.2014.542 PMID: 25611378 37. Wong AK, Ormonde PA, Pero R, Chen Y, Lian L, Salada G, et al. Characterization of a carboxy-terminal BRCA1 interacting protein. Oncogene. 1998; 17: 2279–2285. https://doi.org/10.1038/sj.onc.1202150 PMID: 9811458 38. Chen L, Nievera CJ, Lee AYL, Wu X. Cell cycle-dependent complex formation of BRCA1�CtIP�MRN is important for DNA double-strand break repair. J Biol Chem. 2008; 283: 7713–7720. https://doi.org/10. 1074/jbc.M710245200 PMID: 18171670 39. Prados-Carvajal R, Lo ´pez-Saavedra A, Cepeda-Garcı ´a C, Jimeno S, Huertas P. Multiple roles of the splicing complex SF3B in DNA end resection and homologous recombination. DNA Repair (Amst). 2018; 66–67: 11–23. https://doi.org/10.1016/j.dnarep.2018.04.003 PMID: 29705135 40. Muto A, Sugihara Y, Shibakawa M, Oshima K, Matsuda T, Nadano D. The mRNA-binding protein Serbp1 as an auxiliary protein associated with mammalian cytoplasmic ribosomes. Cell Biochem Funct. 2018; 36: 312–322. https://doi.org/10.1002/cbf.3350 PMID: 30039520 41. Ahn J-W, Kim S, Na W, Baek S-J, Kim J-H, Min K, et al. SERBP1 affects homologous recombinationmediated DNA repair by regulation of CtIP translation during S phase. Nucleic Acids Res. 2015; 43: 6321–6333. https://doi.org/10.1093/nar/gkv592 PMID: 26068472 42. Sartori AA, Lukas C, Coates J, Mistrik M, Fu S, Bartek J, et al. Human CtIP promotes DNA end resection. Nature. 2007; 450: 509–514. https://doi.org/10.1038/nature06337 PMID: 17965729 43. Dungrawala H, Rose KL, Bhat KP, Mohni KN, Glick GG, Couch FB, et al. The Replication Checkpoint Prevents Two Types of Fork Collapse without Regulating Replisome Stability. Mol Cell. 2015; 59: 998– 1010. https://doi.org/10.1016/j.molcel.2015.07.030 PMID: 26365379 PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 21 / 22 44. Yeo JE, Lee EH, Hendrickson EA, Sobeck A. CtIP mediates replication fork recovery in a FANCD2-reg- ulated manner. Hum Mol Genet. 2014; 23: 3695–3705. https://doi.org/10.1093/hmg/ddu078 PMID: 24556218 45. Przetocka S, Porro A, Bolck HA, Walker C, Lezaja A, Trenner A, et al. CtIP-Mediated Fork Protection Synergizes with BRCA1 to Suppress Genomic Instability upon DNA Replication Stress. Mol Cell. 2018; 72: 568–582.e6. https://doi.org/10.1016/j.molcel.2018.09.014 PMID: 30344097 PLOS GENETICS ALC1 regulates CtIP mRNA stability PLOS Genetics | https://doi.org/10.1371/journal.pgen.1008787 May 11, 2020 22 / 22