Review

DNA damage: RNA-binding proteins protect from near and far Martin Dutertre1,2, Sarah Lambert1,2, Aura Carreira1,2, Mounira Amor-Gue´ret1,2, and Ste´phan Vagner1,2 1 2

Institut Curie, Centre de Recherche, Orsay, France CNRS UMR3348, Genotoxic Stress and Cancer, Centre Universitaire, Orsay, France

Recent work, including large-scale genetic and molecular analyses, identified RNA-binding proteins (RBPs) as major players in the prevention of genome instability. These studies show that RBPs prevent harmful RNA/ DNA hybrids and are involved in the DNA damage response (DDR), from DNA repair to cell survival decisions. Indeed, specific RBPs allow the selective regulation of DDR genes at multiple post-transcriptional levels (from pre-mRNA splicing/polyadenylation to mRNA stability/translation) and are directly involved in DNA repair. These multiple activities are mediated by RBP binding to mRNAs, nascent transcripts, noncoding RNAs, and damaged DNA. Finally, because DNA damage modifies RBP localization and binding to different RNA/ DNA molecules, we propose that upon DNA damage, RBPs coordinately regulate various aspects of both RNA and DNA metabolism. The rise of RNA-binding proteins in the DNA damage response DNA lesions are continuously generated in living cells as a result of replication errors and oxidative metabolism [1]. They also arise as a consequence of exposure to environmental agents (e.g., ultraviolet, ionizing radiation), radiation therapy, and chemotherapeutic drugs. Accumulation of DNA insults is associated with multiple diseases from neurodegenerative disorders to cancers, immune deficiencies, and infertility. It is therefore crucial for the cell to detect DNA damage, signal its presence, and effect DNA repair, cell cycle arrest, and ultimately cell fate decisions, which are together called the DNA damage response (DDR; Box 1). For simplicity, we will use the term ‘DNA damage’ to refer to the action of different DNA-damaging agents (Box 2). In addition to the rapid DNA damage-dependent posttranslational regulation of the activity of DDR proteins, the expression of DDR genes must be precisely regulated. This regulation involves a transcriptional program orchestrated Corresponding author: Vagner, S. ([email protected]). Keywords: DNA damage response; post-transcriptional control of gene expression; RNA processing; mRNA translation. 0968-0004/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibs.2014.01.003

in part by the p53 transcription factor. Abundant literature also documents that DNA damage affects post-transcriptional gene expression, including the processing of the primary transcript in the nucleus (pre-mRNA splicing and 30 end processing also called cleavage/polyadenylation) and the control of mRNA stability, and translation in the cytoplasm. Post-transcriptional gene expression is regulated, in large, by RNA-binding proteins (RBPs), which directly bind specific RNA sequences and secondary structures in premRNAs, mRNAs, or regulatory noncoding RNAs (ncRNAs; Box 3) [2,3]. Strikingly, various unbiased proteomic approaches and functional screens have identified RNA processing and/or translation factors, including RBPs, as major functional categories of gene products that are posttranslationally modified by DNA damage-signaling proteins [4,5] and are required for the DDR [6,7]. In parallel with these global approaches, studies focusing on individual RBPs have begun to reveal that RBPs protect cells from DNA damage by acting from near (i.e., at the site of DNA damage) and far [i.e., bound to (pre-)mRNAs encoding DDR proteins]. RBPs allow the selective expression of DDR genes upon DNA damage Repression of gene expression in response to DNA damage DNA damage tends to globally repress gene expression. This is mainly achieved through a decrease in the levels of mRNAs, which occurs through several mechanisms. DNA damage leads to inhibition of transcription and also triggers a repression of pre-mRNA 30 -end processing [8,9]. The latter effect is mediated by the direct inhibitory interaction between a basic component of the 30 -end processing machinery (cleavage stimulatory factor CSTF1) and phosphorylated BARD1 (BRCA1-associated RING domain 1) within complexes that also contain the BRCA1 (breast cancer 1) DNA repair protein and the p53 tumor protein [10]. Whereas premRNA splicing has not been reported to be globally inhibited by DNA damage, alternative splicing of specific pre-mRNAs is altered (discussed later). In particular, for many genes, DNA damage results in a decrease in the expression of functional gene products by switching alternative splicing towards variants harboring premature stop codons, which are subject to nonsense-mediated mRNA decay (NMD) [11,12]. Beyond inhibitory actions at the pre-mRNA level, DNA damage leads to a decrease in the stability of many Trends in Biochemical Sciences, March 2014, Vol. 39, No. 3

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Box 1. The DNA damage response (DDR) The DDR relies on a highly coordinated network of factors that detect and signal the presence of different types of DNA lesions to promote their repair or tolerance, control chromatin structure, and regulate cell cycle progression, gene expression, and cell fate. Within minutes after DNA damage, a cascade of protein phosphorylation and other posttranslational modifications (e.g., parylation, acetylation, ubiquitylation, sumoylation) occur to recruit and precisely regulate the activity of DDR factors in a given chromatin context. In the longer term, global gene expression is regulated to sustain the DDR and control cell fate. DNA lesions affect either one or two DNA strands, which are repaired by different mechanisms (Figure I). Single-strand DNA damage is repaired by excision of either the base (base

excision repair, BER) or the nucleotide (nucleotide excision repair, NER, and mismatch repair, MMR). During transcription, DNA lesions located on a transcribed strand are repaired faster than lesions located on the nontranscribed strand owing to transcription-coupled repair (TCR). TCR involves some components of the NER machinery and TCR factors such as CSA and CSB (Cockayne syndrome). The repair of damage affecting both DNA strands, such as double-strand DNA breaks, is supported by homologous recombination (HR) and by non-homologous end joining (NHEJ). Inter-strand crosslinks (ICLs) are repaired by the concomitant action of HR and the Fanconi anemia (FA) pathway.

Single-stranded breaks / stalled replicaon forks

PCNA-like

Double-stranded breaks (DSBs)

MRN

MRN

RPA RPA

RFC-like

ATRIP ATR

PALB2 BRCA2

ATM HR

Key: Sensors TopBP1

Mediators Mediato ors

53BP1

RAD51

MDC1

BRCA1

Transduc cers Transducers Claspin A repair DNA proteins

NHEJ Ku70–Ku80 DNAPKcs

Sensing protein kinases

CHK2

CHK1

Cell cycle/ senescence

Apoptosis

Chroman remodeling

Genome maintenance

Gene expression Ti BS

Figure I. Scheme of the DNA damage response (DDR) to double-strand DNA breaks, single-strand DNA breaks, and stalled replication forks. DNA damage is sensed and signaled by sensors (blue text), mediators (green text), and transducers (purple text). Sensors recognize the DNA damage and are thought to detect either double-strand break or single-stranded break DNA exposed during the repair of DNA damage. Sensors include the MRN (MRE11/RAD50/NBS1) complex, the Ku70–Ku80 heterodimer, the RFC-like (replication factor C) complex, and the PCNA-like (proliferating cell nuclear antigen) complex. These contribute to the activation of three main sensing protein kinases (red icon): ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3 related), and DNAPKcs (DNA-dependent protein kinase catalytic subunit). Sensor kinases locally phosphorylate several targets to amplify the signal and together with mediators {such as TopBP1 [topoisomerase (DNA) II binding protein 1], MDC1 (mediator of DNA-damage checkpoint 1), Claspin, 53BP1 (tumor protein p53 binding protein 1), and BRCA1 (breast cancer 1}} activate transducers, including notably the checkpoint kinases CHK1 and CHK2. This DDR protein kinase cascade regulates the activity of a plethora of effectors involved in cell cycle arrest, DNA repair, chromatin remodeling, gene expression, and cell death. Blue icons represent proteins with DNA repair function: MRN complex and RPA (replication protein A) have an early function in signaling DNA damage and repair; RAD51, BRCA2 (breast cancer 2), and PALB2 (partner and localize of BRCA2) are involved in HR (homologous recombination); Ku70–Ku80 is involved in NHEJ (non-homologous end joining); and BRCA1 is involved in both repair pathways.

mRNAs [13] and to an inhibition of the activity of general translation factors involved in initiation [14] and elongation [15]. The DNA damage-induced decrease in mRNA stability and translation is more widespread than transcriptional repression [13,16] and can be strengthened by RBPs in a gene selective manner through two mechanisms. First, human antigen R (HuR), an RBP that promotes mRNA 142

stability and translation, is dissociated from many mRNAs in response to DNA damage (see Table S1 in the supplementary material online) [17]. Second, RBPs such as the KH-type splicing regulatory protein (KSRP) enhance the maturation of a large subset of miRNAs that would in turn inhibit the stability and/or translation of their target mRNAs in response to DNA damage [18].

Review

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Box 2. DNA-damaging agents UV-C radiation: ultraviolet, usually 254 nm; leads to CPD (cyclobutane pyrimidine dimers) and 6–4 PP (photo-products) that are mainly excised by NER. UV-induced DNA damage can also be bypassed (and thus tolerated but not repaired) during DNA replication by translesion synthesis (TLS), DNA polymerases, or HR. 4-Nitroquinoline 1-oxide (4NQO) is considered a UV mimetic. UV-A radiation: ultraviolet, 320–400 nm; induces oxidative stress and the formation of reactive oxygen species (ROS) in lipids, proteins, and DNA. This results in DNA lesions such as 8-oxo-7,8-dihydro-20 deoxyguanosines (8-oxoG), leading to G-to-T transversion mutations. This type of DNA damage is mainly removed by the NER pathway. Ionizing radiation (IR): induces several types of DNA damage. For example, 1 Gy of IR induces 1000 single-strand breaks, 2000 oxidized bases, 250 abasic sites, and 40 double strand breaks (DSBs) per cell. DSBs are the most deleterious DNA lesions caused by IR. Neocarzinostatin is a radiomimetic drug that causes DSBs. Crosslinking agents: mitomycin C (MMC), cisplatin, and nitrogen mustard lead to monoadducts and intra- and inter-strand crosslinks, the latter being the most deleterious. Alkylating agents: methyl methanesulfonate (MMS), methylnitrosourea (MNU), and ethyl methanesulfonate (EMS) induce base alkylation, most commonly N7-methylguanine and N3 methylade-

RBPs upregulate DDR genes at multiple posttranscriptional levels Cumulatively, these different breaks should significantly decrease overall gene expression. It is therefore crucial that specific compensatory mechanisms exist to allow DDR genes to be properly expressed during DNA damage. Large-scale approaches based on microarray analysis of polysome-bound mRNAs have shown that mRNAs encoding DDR proteins selectively escape translational repression in response to DNA damage [19–21]. RBPs are critical to ensure the expression of DDR genes upon DNA damage through their specific binding to pre-mRNAs and mRNAs. Among various examples (Figure 1), several RBPs are involved at different post-transcriptional steps to control p53 expression during DNA damage. Two related RBPs [heterogeneous nuclear ribonucleoproteins (hnRNP) H and F] interact with a G-quadruplex RNA structure located downstream of the p53 polyadenylation site to keep its 30 end processing efficient [22]. Several other RBPs, including HuR, PTB (polypyrimidine tract-binding protein), the ribosomal protein L26 and MDM2 (the human homolog of the murine double minute-2) promote p53 mRNA translation during DNA damage [23–26]. Strikingly, the MDM2 protein has dual but mutually exclusive roles in p53 regulation: it is the main ubiquitin ligase targeting the p53 protein for degradation, and it enhances the translation of the p53 mRNA. In response to DNA damage, phosphorylation of MDM2 by the ataxia telangiectasia mutated (ATM) kinase inhibits its binding to the p53 protein and enhances its binding to the p53 mRNA [27], and this switch may contribute to the efficient induction of p53 expression. Coordinated regulation of multiple genes by RBPs In precisely the same way as transcription factors regulate a set of genes through a common DNA motif in their regulatory regions, a given RBP can regulate a set of transcripts (or alternative exons) through binding to an RNA motif. This has given rise to a model in which mRNAs that encode functionally related proteins are coordinately regulated as post-transcriptional RNA operons or regulons

nine, the latter being a strong block to replicative DNA polymerases. Cyclophosphamide is an alkylating agent that creates inter-strand DNA crosslinks. Replication inhibiting agents: several obstacles can locally impede the progression of replication forks, including protein/DNA complexes, structure-forming sequences, DNA lesions, chromatin structures, and RNA/DNA hybrids as byproducts of transcription. In addition, drugs are frequently used to impose a global replication stress, such as HU (hydroxyurea), an inhibitor of the ribonucleotide reductase, leading to an inhibition of dNTP synthesis during S phase. CPT (camptothecin), a TOP-I inhibitor that covalently links TOP-I to DNA, induces replicative DSBs and torsional stress during DNA replication. CPT also inhibits transcription elongation, especially at long and highly transcribed genes, and this effect can induce DSBs even in nonproliferating cells. Topoisomerase II (TOP-II) inhibitors: doxorubicin, etoposide (VP16), and mitoxantrone trap TOP-II in a form that is covalently linked to DNA, resulting in DSBs that cannot be resealed. Doxorubicin and mitoxantrone also intercalate in duplex DNA. Many of these DNA-damaging agents are currently used in chemotherapy and radiotherapy in cancer. For experimental work, the I-SceI endonuclease can also be used to generate site-specific DSBs.

[28]. In this context, there is increasing evidence that a specific RBP can control the expression of a subset of DDR genes. For example, following DNA damage HuR directly regulates the stability and/or translation of mRNAs encoding p53, p21 (cyclin-dependent kinase inhibitor 1A), and RhoB (ras homology family member B) and promotes cell survival [25,29,30] (Figure 1). Likewise, DNA damagedependent alternative splicing of the DDR genes MDM2 and CHK2 (checkpoint kinase 2) is mediated by the EWS (Ewing’s sarcoma proto-oncoprotein) RBP whose depletion leads to enhanced sensitivity to DNA damage [31,32]. Dependence on specific RBPs provides posttranscriptional rate-limiting steps for DDR gene expression The expression of some DDR genes appears to be particularly dependent on specific RBPs, and this may have important consequences for the DDR. Genes that are upregulated by DNA damage at the transcriptional level may differentially depend on RBPs for post-transcriptional expression, creating a post-transcriptional rate-limiting step for some but not other targets. For instance, in response to DNA damage p53 activates the transcription of various genes involved in DNA repair, cell cycle arrest (e.g., p21), cell death (e.g., the BCL2-associated X protein BAX and the p53-upregulated modulator of apoptosis PUMA), and feedback regulation of p53 (e.g., MDM2), whereas RBPs ultimately control the splicing efficiency of some of these targets. First, the RBP and splicing factor SKIP (Ski-interacting protein) was shown to be required for the splicing of p21, but not PUMA, transcripts thereby promoting cell survival following DNA damage [33]. Second, two RBPs (EWS and YB-1) that couple transcription and splicing are required for the correct splicing of MDM2 transcripts. In response to DNA damage, the interaction between EWS and YB-1 (Y box-binding protein) is inhibited, and eight of 12 exons are excluded from the MDM2 transcript. This striking, gene selective alteration of splicing counteracts the transcriptional stimulation of the MDM2 gene and limits the increase in full-length 143

Review Box 3. RNA-binding proteins and post-transcriptional control of gene expression RNA binding domains (RBDs) bind RNA through a limited number of RBDs such as the RNA recognition motif (RRM), the K-homology (KH) domain, the RGG (Arg-Gly-Gly) box, the Sm domain, the DEAD/DEAH box, the double-stranded RNA-binding domain (dsRBD), and a few others. RBDs often bind short sequences of RNA, either single- or double-stranded. RBPs generally contain more than one RBD, thereby increasing their binding specificity. The direct binding of a given RBP to transcripts can be mapped on a genome-wide scale at nucleotide resolution by so-called CLIP (crosslinking immunoprecipitation) procedures [89,90] and such analyses reveal RBP-specific binding maps. Specific RBP depletion followed by RNA-seq (or microarray) analysis reveals even greater specificity of gene (or exon) regulation by a given RBP. The most abundant and promiscuous RBPs are hnRNPs, but even hnRNPs have specific effects on gene expression. By recruiting core machineries involved in specific steps of gene expression, or sometimes through their own enzymatic domains (e.g., RNA helicase), RBPs regulate the post-transcriptional steps of gene expression, including: the cotranscriptional addition of the ‘cap’ structure at the 50 -end of the nascent transcript; the splicing of pre-mRNAs which often occurs cotranscriptionally; the cotranscriptional processing of the 30 -end of premRNAs (endonucleolytic cleavage and polyadenylation); the export of the mature transcript (mRNA) to the cytoplasm; and the translation and turnover of the mRNA in the cytoplasm. All of these post-transcriptional steps are subject to gene-specific regulation that is mediated, in large, by RBPs acting in a combinatorial manner and in concert with core machineries. RBPs also control the processing and activity of an increasing variety of ncRNAs (e.g., miRNAs and lncRNAs), which in turn regulate pre-mRNAs. Any given RBP is often involved in multiple steps of gene expression and in the processing of both coding and ncRNAs. Although >700 RBPs have been identified, most of them are poorly characterized.

MDM2 mRNA levels upon DNA damage [34]. Importantly, this splicing regulation is independent of p53, and when the damaging agent is withdrawn normal splicing of MDM2 is restored, full-length MDM2 mRNA and protein levels increase, and the p53 protein is targeted for degradation. Thus, specific RBP requirements allow differential kinetics of p53 target gene regulation in response to DNA damage. Regulation through binding to long ncRNAs In addition to their key role in post-transcriptional gene regulation, RBPs also regulate transcription and chromatin remodeling in response to DNA damage through their binding to a growing list of long ncRNAs (lncRNAs). In response to DNA damage, TLS/FUS (translocated in liposarcoma/fused in sarcoma), an RBP and transcriptional coregulator, interacts with an lncRNA called ncRNA– CCND1, transcribed upstream of the CCND1 gene encoding cyclin D1, to repress in cis its transcription [35]. The hnRNP K RBP binds to lncRNA–p21 and mediates in trans the transcriptional repression of a large set of genes [36], although it can also serve as a transcriptional coactivator of the p53 transcriptional factor [37]. Another lncRNA called PANDA is induced by DNA damage in a p53-dependent manner and sequesters the transcription factor NFYA (nuclear factor YA) thereby inhibiting the expression of apoptosis genes [38]. Other lncRNAs are induced by DNA damage [36,38]; therefore, a significant role of RBPs in the DDR may be mediated through interactions with lncRNAs. 144

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Altogether, in response to DNA damage RBPs play major roles in the regulation of expression of DDR and cell fate genes at multiple transcriptional and post-transcriptional levels. Furthermore, RBPs play roles in various aspects of DNA-based processes beyond transcription that are closely linked to the DDR, as discussed in the following section. RBPs in the prevention and repair of DNA damage In addition to their key role in the regulation of DDR gene expression at both post-transcriptional and transcriptional levels, RBPs can prevent the accumulation of DNA damage and genome instability by several mechanisms. RBPs prevent DNA breaks by controlling R loop formation, DNA topology, and DNA metabolism A prominent recognized source of double-strand DNA breaks and genome instability is the collision between replication and transcription at the level of genes that are transcribed during S phase, such as highly expressed or very long genes. Indeed, during transcription, nascent RNA can hybridize with the transcribed DNA strand thus leaving the other strand unpaired. Such DNA:RNA hybrids, called R loops, are favored by G-rich DNA repeats and topological constraints and can lead to DNA breaks when hit by replication forks [39,40]. Several RBPs, including the serine/arginine-rich splicing factor 1 (SRSF1), components of the cleavage/polyadenylation machinery, and components of the TREX (transcription export) complex involved in the coupling of transcription, processing, and nuclear export of transcripts, prevent R loop formation and DNA breaks (reviewed in [39]). To avoid the formation of R loops, it has been proposed that RBPs may package nascent pre-mRNA and/or promote their cotranscriptional processing thereby avoiding the presence of unprocessed transcripts that could hybridize with the DNA template. SRSF1 cooperates with DNA topoisomerase I (TOP-I) to prevent the formation of R loops [41]. TOP-I relaxes DNA and transiently cleaves DNA to relieve the topological constraints induced by transcribing and replicating polymerases. A proteomic study has found that many proteins interacting with TOP-I are RBPs; TOP-I phosphorylates SRSF1 and, conversely, SRSF1 and other RBPs such as PSF (PTB-associated splicing factor) and NonO/p54nrb (non-POU domain-containing octamer-binding protein) modulate DNA relaxation or cleavage by TOP-I ([42] and references therein). Thus, interactions between RBPs and TOP-I may prevent DNA damage at highly transcribed loci by removing topological constraints. RBPs could also prevent DNA damage through their role in DNA metabolism processes such as DNA replication and telomere biogenesis, the alteration of which triggers the DDR. During replication, hnRNP A1 is involved in Okazaki fragment maturation through interaction with FEN-1 (flap endonuclease-1) and stimulating its enzymatic activity [43]. The RBP and RNA helicase UPF1 (for upframeshift) interacts with DNA polymerase delta and associates with chromatin upon DNA damage in an ATR (ataxia telangiectasia and Rad3 related)-dependent manner [44]. Several RBPs play a role in the maintenance of telomere integrity not only through interactions with the

Review

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known as hnRNP G) binds to double-strand DNA breaks, protects them from further degradation, and increases the fidelity of DNA end joining [63]; another study indicates that it is recruited to sites of DNA damage, but this is not a prerequisite for its promotion of HR [6]. hnRNP C and hnRNP U-like, a homolog of the hnRNP U RBP, promote HR-mediated repair and are components of the BRCA1/ BRCA2/PALB2 (partner and localizer of BRCA2) and MRE11/RAD50/NBS1 (MRN) complexes, respectively, that promote HR [47,52]. Altogether, several RBPs are directly involved in the maintenance of genome integrity and in DNA repair through interactions with nascent transcripts, ncRNA, damaged DNA, and DNA repair proteins. Conversely, RBPs are also regulated by the DDR, as presented next.

pre-mRNA (nucleus) or mRNA (cytoplasm)

pre-mRNA splicing

pre-mRNA 3′ end processing

SKIP

hnRNP H/F

p21 CHK2

EWS

MDM2

YB-1

p53

Nucleus Cytoplasm

mRNA stability

hnRNP A0

Gadd45α

TIAR p21

PCBP4

rhoB

HuR PTB

mRNA translaon

p53

RNPC1

RPL26 MDM2

XIAP Apaf-1

hnRNP A1 Ti BS

Figure 1. Role of RNA-binding proteins (RBPs) in post-transcriptional regulation of DNA damage response (DDR) genes following DNA damage. Only direct effects of RBPs on the indicated target gene transcripts are shown. Upregulation and downregulation are shown by green and red arrows, respectively; RBPs are shown in blue. The DNA-damaging agents that were used and references are given in Table S1 in the supplementary material online.

ncRNA component of telomerase (hTR) but also with single-stranded telomere DNA through their RNA-binding domains [45,46]. A direct role of RBPs in DNA repair Several RBPs were reported to be located at sites of DNA lesions [6,47–53]. Surprisingly, RBP recruitment to sites of DNA damage has not yet been linked to RNA binding. However, DNA damage-induced foci are sensitive to RNAse and require small ncRNAs produced locally [54,55]. The production and function of such ncRNAs at sites of DNA damage probably involves RBPs. In addition, several RBPs interact with DNA and repair proteins, suggesting that RBPs might be recruited to sites of DNA damage independently of RNA (Figure 2). For instance, the RBP YB-1 interacts with mismatched DNA and can modulate its repair by mismatch repair (MMR); YB-1 may also be involved in other repair pathways, because it interacts with other types of damaged DNA and several DNA repair proteins, and it exhibits strand separation and endonuclease activity in vitro [56–58]. Another RBP, PSF, interacts with DNA and the RAD51D and TopBP1 [topoisomerase (DNA) II binding protein 1] proteins, and is involved in double-strand break (DSB) repair pathways such as non-homologous end joining (NHEJ) and homologous recombination (HR) [59,60]. NonO/p54nrb, a PSF partner protein, stimulates NHEJ and represses HR [61,62]. RBMX (RNA-binding motif protein, X-linked, also

Regulation of RBPs by DNA damage Rapid post-translational modifications of RBPs in the DDR Post-translational modifications are a key part of DDR signaling (Box 1). Several phosphoproteomic screens identified RBPs as a major category of proteins whose phosphorylation is regulated by DNA damage [5,64–67]. Upon DNA damage, some RBPs are directly phosphorylated by major DDR signaling kinases such as sensors (ATM, ATR, DNA-PK) and transducers (CHK1, CHK2), whereas others are phosphorylated by downstream kinases. The SRPK protein kinases that phosphorylate the SR (splicing regulator) family of RBPs are also targeted by DDR signaling [68,69]. DNA damage-dependent RBP phosphorylation can regulate their activities. For example, phosphorylation of KSRP by ATM promotes its function in the maturation of miRNA precursors [18]. Phosphorylation of hnRNP A0 and TIAL1 (TIA1 cytotoxic granule-associated RNA-binding protein-like 1, also called TIAR) by MK2 [mitogen-activated protein kinase (MAPK)-activated protein kinase-2] and p38 MAPK, respectively, promotes the stabilization of Gadd45a (growth arrest and DNA damage-inducible, alpha) mRNA [70]. RBP post-translational modifications can also alter RNA binding. For example, DNA damage widely remodels HuR association with target mRNAs in an ATM- and CHK2-dependent manner [17,71]. Similarly, ATM-dependent phosphorylation of MDM2 at Ser395 switches its interaction from the p53 protein to the p53 mRNA [27]. In addition to phosphorylation, RBPs can be targeted by other post-translational modifications in response to DNA damage. Poly(ADP-ribosylation) (also called parylation) of proteins by poly(ADP-ribose) polymerases (PARPs) plays an important role in the DDR, and RBPs are a major category of proteins whose parylation is regulated by DNA damage [4]. The recruitment of several RBPs to DNA damage sites depends on PARP (including at least PARP1) activity, and SRSF1 interacts with poly(ADPribose) [6,51,53,61,72]. Finally, in response to DNA damage, various RBPs were found to be more acetylated in a large-scale analysis [64], although acetylation of SRSF2 by KAT5 [K(lysine) acetyltransferase 5] decreases [68]. Moreover, some RBPs are targeted by several DDR protein kinases, for example, HuR by CHK2, p38 MAPK, PKC (protein kinase C), and CDK1 (cyclin-dependent 145

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DNA lesions

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Fork arrest

Alkylated base/mismatch

Crosslink

Single-strand break

Photoproduct

T

Repair pathways

HR TLS

MMR BER

FA HR

YB-1

BER

T

NER

hnRNP U -like

Double-strand break

HR NHEJ

hnRNP C

MRN

NonO /p54nrb

RAD51D

complex

PSF

RBMX

TOPBP1

PALB2 BRCA2 BRCA1 Ti BS

Figure 2. Implication of RNA-binding proteins (RBPs) in DNA repair pathways. Interactions (interrupted blue lines) between RBPs (blue icon) and repair factors (gray icon) are shown. DNA lesions are repaired by homologous recombination (HR), translesion DNA synthesis (TLS), mismatch repair (MMR), base excision repair (BER), Fanconi anemia (FA) pathway, nucleotide excision repair (NER), and non-homologous end joining (NHEJ), as indicated below each type of DNA lesion and as detailed in Box 1. The DNA-damaging agents that were used and references are given in Table S1 in the supplementary material online.

kinase 1) [73] or by several types of modifications [64,68], suggesting that RBPs can be nodes in DDR signaling networks. Subcellular relocalization of RBPs may coordinately regulate several aspects of RNA and DNA metabolism DNA damage leads to changes in the subcellular localization of RBPs that may affect their activities (Figure 3). Many of these changes involve intranuclear relocalization. For instance, a growing list of RBPs are recruited to DNA damage sites (Figure 3), where six of them have been involved in DNA repair (see above). Three of these six RBPs were also shown to promote the expression of DDR genes. For example, as mentioned above, MDM2 pre-mRNA splicing is dependent on YB-1 and becomes aberrant upon DNA damage as YB-1 recruitment to the MDM2 gene is decreased [34]. Likewise, hnRNP C is required for correct pre-mRNA splicing and elevated expression of several HR factors (e.g., BRCA2, BRCA1, and RAD51) [47], and BRCA2 expression is also highly sensitive to RBMX depletion [6]. These data suggest a model where the relocalization of a specific RBP to DNA damage sites may not only participate to DNA repair but also impact the processing of DDR gene pre-mRNAs that 146

are highly sensitive to a decrease in the amount of this RBP available for pre-mRNA processing. DNA damage also induces intranuclear relocalization of several RBPs to the nucleolus, such as several members of the SR family of RBPs [74] and EWS, the latter leading to altered splicing of MDM2 and CHK2 pre-mRNAs [32,34]. Likewise, nucleolar relocalization of the MDM2 protein is linked to its binding switch from p53 protein to p53 mRNA, leading to increased p53 mRNA translation [27]. Conversely, DNA damage causes several nucleolar RBPs involved in ribosome biogenesis to exit nucleoli, which may link the repression of ribosome biogenesis to specific mRNA regulation, for example, nucleolin specifically binds p53 mRNA and regulates its translation [26,75]. Additionally, in response to DNA damage, the splicing factor Sam68 (Src-associated in mitosis) is relocalized to nuclear stress granules thereby impacting on alternative splicing of specific pre-mRNAs [76]. DNA damage-induced relocalization also involves nuclear–cytoplasmic shuttling. Indeed, DNA damage induces nucleus-to-cytoplasm redistribution of several RBPs, which is a common theme in stress responses [77] and might allow the coordinated regulation of premRNA maturation and mRNA stability/translation. For

Review

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Nucleolus RBP

Nucleolin Sites of DNA damage

RBP

Nucleus Cytoplasm

SRSF1 MDM2 EWS DDX5

RBMX DDX17 PSF TLS/FUS hnRNP U-like hnRNP C YB-1 nucleolin

Sam68 RBP

Nuclear bodies RBP

YB-1

HuR hnRNP A1 PTB RBP Ti BS

Figure 3. Changes in RNA-binding protein (RBP) subcellular localization following DNA damage. RBPs are indicated in blue; arrows indicate DNA damage-induced movements of RBPs. The DNA-damaging agents that were used and references are given in Table S1 in the supplementary material online.

example, DNA damage-induced export of PTB and hnRNP A1 from the nucleus promotes p53 mRNA translation and inhibits translation of proapoptotic Apaf1 (apoptotic peptidase activating factor 1) mRNA, respectively [24,78], and is also expected to impact nuclear pre-mRNA processing such as alternative splicing choices. Likewise, DNA damage-induced nuclear export of HuR [73] might coordinately regulate pre-mRNA maturation and mRNA stability/translation. Finally, the relocalization of the YB-1 protein from cytoplasm to nucleus, where it plays a role in DNA repair (see above), is expected to impact YB-1regulated translation of specific mRNAs. Altogether, we propose that in response to DNA damage, the relocalization of multifunctional RBPs may play a role in coordinating the regulation of various aspects of RNA and DNA metabolism. Regulation of RBP abundance may play a role in the long-term effects of DNA damage The regulation of RBP abundance may play a role in the long-term effects of DNA damage. First, in response to DNA damage, the expression of many splicing factors is decreased by alternative splicing-induced NMD, and such regulation might constitute a feedback to adapt RBP levels to RNA levels [12]. Second, some DNA damageinduced RBPs may be involved in the termination (shut off) of the DDR. For instance, p53 induces the expression of RNPC1 (also called RBM38) and PCBP4 [poly(rC)-binding protein 4], which in turn repress the translation of the mRNA encoding p53 and the stability of the mRNA encoding p21, respectively [79,80]. Third, RBPs can contribute to cell fate decisions such as apoptosis or permanent cell cycle arrest (senescence). For example, SRSF2 regulation by DNA damage determines whether tumor cells undergo apoptosis or cell cycle arrest [68]. Fourth, some RBPs, such as YB-1 and DDX5 [DEAD (Asp-Glu-Ala-Asp) box helicase], may play a role in the adaptation and resistance of cancer cells to radiotherapeutic and chemotherapeutic agents [81,82].

Concluding remarks and future perspectives The importance of RBPs in the maintenance of genome integrity is increasingly recognized, with three major roles: (i) RBPs refine the proteomic complexity required for the DDR through transcriptional and post-transcriptional control of gene expression; (ii) they prevent harmful DNA/ RNA hybrids; and (iii) they are directly involved in DNA repair. Consistently, many RBPs are targeted by DNA damage signaling at the levels of post-translational modifications, subcellular localization, and expression. In particular, we propose that DNA damage-induced relocalization of multifunctional RBPs allows the coordinated regulation of various aspects of RNA and DNA metabolism (e.g., DNA repair and DDR gene expression). There are several important areas of future research (Box 4). One is the role of RBPs in controlling DNA damage-dependent RNA metabolism and fate. There is a need for large-scale studies of which pre-mRNAs, mRNAs, and ncRNAs interact with and are regulated by specific RBPs in the presence and absence of DNA damage. Major outcomes of such studies would include the identification of large-scale post-transcriptional gene regulatory networks and key regulatory events that may drive cell responses to DNA damage. Relatedly, whether and how RBPs involved in other aspects of RNA metabolism (e.g., alternative polyadenylation, export) recently linked to the DDR [83,84] also warrant further investigation. Another important area of research is how RBPs indirectly sense DNA damage. In many cases the signaling pathways that connect sites of DNA damage to the RBPs and how different types of DNA-damaging agents and DNA lesions activate these pathways remain to be determined. In addition, it is crucial to understand to what extent RBPs located at the sites of DNA damage contribute to DNA repair efficiency. For instance, an interesting question is whether RBPs associated with sites of transcription may play a role in transcription-coupled repair (TCR), as was suggested for the RNA-processing protein CSTF1 [9]. Finally, several proteins involved in the DDR were recently identified as RBPs [e.g., BRCA1, XRCC5 and 6 (X-ray repair complementing defective repair in Chinese hamster cells 5 and 6) also called Ku70 and Ku80] [85–87], but whether the RNAbinding activity of these proteins is important for the DDR remains to be evaluated. For instance, 53BP1 (tumor protein p53-binding protein 1), a protein involved in checkpoint signaling at sites of DNA damage, binds RNA and is recruited to chromatin in an RNA-dependent manner [88]. Clearly, further investigating the role of RBPs, including the integration of their different activities, will deepen our understanding of the fundamental processes controlling Box 4. Outstanding questions  How do RBPs sense DNA damage?  Which RNAs (including ncRNAs) interact with and are regulated by specific RBPs in the presence of DNA damage?  To what extent do RBPs present at the sites of DNA damage contribute to DNA repair efficiency?  How are the multiple activities of a given RBP integrated during the DDR?  Does the potential RNA-binding activity of some DDR proteins contribute to the DDR? 147

Review cell responses to DNA damage and of the numerous diseases and cancer treatments that are linked to DNA damage. Acknowledgements We apologize to all colleagues who have made contributions in the field and could not be cited owing to space constraints.

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