RESUMEN
Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.
Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Núcleo Celular/enzimología , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias/enzimología , Proteínas Nucleares/metabolismo , Procesamiento Postranscripcional del ARN , ARN Neoplásico/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Núcleo Celular/genética , ADN Helicasas/genética , Metilación de ADN , Proteínas de Unión al ADN/genética , Células HEK293 , Células HeLa , Humanos , Metilación , Neoplasias/genética , Proteínas Nucleares/genética , Estructuras R-Loop , ARN Neoplásico/genética , Empalmosomas/genética , Empalmosomas/metabolismo , Transcripción Genética , UbiquitinaciónRESUMEN
Ubiquitination is a major mechanism that regulates numerous cellular processes, including autophagy, DNA damage signaling, and inflammation. While hundreds of ubiquitin ligases exist to conjugate ubiquitin onto substrates, approximately 100 deubiquitinases are encoded by the human genome. Thus, deubiquitinases are likely regulated by unidentified mechanisms to target distinct substrates and cellular functions. Here, we demonstrate that the deubiquitinase OTUD4, which nominally encodes a K48-specific deubiquitinase, is phosphorylated near its catalytic domain, activating a latent K63-specific deubiquitinase. Besides phosphorylation, this latter activity requires an adjacent ubiquitin-interacting motif, which increases the affinity of OTUD4 for K63-linked chains. We reveal the Toll-like receptor (TLR)-associated factor MyD88 as a target of this K63 deubiquitinase activity. Consequently, TLR-mediated activation of NF-κB is negatively regulated by OTUD4, and macrophages from Otud4-/- mice exhibit increased inflammatory signaling upon TLR stimulation. Our results reveal insights into how a deubiquitinase may modulate diverse processes through post-translational modification.
Asunto(s)
Factor 88 de Diferenciación Mieloide/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Células HEK293 , Humanos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteolisis , Transducción de Señal , Receptores Toll-Like , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE; associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high-resolution details and features conserved over 500 million years of evolution. Extending our understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence-specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small-angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1's roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.
Asunto(s)
Hidrolasas Diéster Fosfóricas , Humanos , Biología Computacional/métodos , Cristalografía por Rayos X , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Motivos de Unión al ARN/genéticaRESUMEN
DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.
Asunto(s)
Enzimas AlkB/metabolismo , Aductos de ADN/metabolismo , Reparación del ADN , Complejos Multiproteicos/metabolismo , Transducción de Señal , Síndromes de Tricotiodistrofia/genética , Ubiquitina/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Alquilantes/farmacología , Alquilación , Secuencia de Aminoácidos , Aductos de ADN/química , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Genes Ligados a X , Humanos , Cinética , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poliubiquitina/metabolismo , ARN Polimerasa II/metabolismo , Empalme del ARN , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patología , UbiquitinaciónRESUMEN
Alkylation chemotherapy is one of the most widely used systemic therapies for cancer. While somewhat effective, clinical responses and toxicities of these agents are highly variable. A major contributing factor for this variability is the numerous distinct lesions that are created upon alkylation damage. These adducts activate multiple repair pathways. There is mounting evidence that the individual pathways function cooperatively, suggesting that coordinated regulation of alkylation repair is critical to prevent toxicity. Furthermore, some alkylating agents produce adducts that overlap with newly discovered methylation marks, making it difficult to distinguish between bona fide damaged bases and so-called 'epigenetic' adducts. Here, we discuss new efforts aimed at deciphering the mechanisms that regulate these repair pathways, emphasizing their implications for cancer chemotherapy.
Asunto(s)
Daño del ADN , Reparación del ADN , ADN de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Alquilación/efectos de los fármacos , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Humanos , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
Multiple DNA damage response (DDR) pathways have evolved to sense the presence of damage and recruit the proper repair factors. We recently reported a signaling pathway induced upon alkylation damage to recruit the AlkB homolog 3, α-ketoglutarate-dependent dioxygenase (ALKBH3)-activating signal cointegrator 1 complex subunit 3 (ASCC3) dealkylase-helicase repair complex. As in other DDR pathways, the recruitment of these repair factors is mediated through a ubiquitin-dependent mechanism. However, the machinery that coordinates the proper assembly of this repair complex and controls its recruitment is still poorly defined. Here, we demonstrate that the ASCC1 accessory subunit is important for the regulation of ASCC complex function. ASCC1 interacts with the ASCC complex through the ASCC3 helicase subunit. We find that ASCC1 is present at nuclear speckle foci prior to damage, but leaves the foci in response to alkylation. Strikingly, ASCC1 loss significantly increases ASCC3 foci formation during alkylation damage, yet most of these foci lack ASCC2. These results suggest that ASCC1 coordinates the proper recruitment of the ASCC complex during alkylation, a function that appears to depend on a putative RNA-binding motif near the ASCC1 C terminus. Consistent with its role in alkylation damage signaling and repair, ASCC1 knockout through a CRISPR/Cas9 approach results in alkylation damage sensitivity in a manner epistatic with ASCC3. Together, our results identify a critical regulator of the ALKBH3-ASCC alkylation damage signaling pathway and suggest a potential role for RNA-interacting domains in the alkylation damage response.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas Nucleares/metabolismo , Mapas de Interacción de Proteínas , Factores de Transcripción/metabolismo , Alquilación , Secuencia de Aminoácidos , Línea Celular , Daño del ADN , Desmetilación del ADN , Reparación del ADN , Humanos , Modelos Moleculares , Dominios Proteicos , ARN/metabolismo , ARN Ligasa (ATP)/química , ARN Ligasa (ATP)/metabolismo , Factores de Transcripción/químicaRESUMEN
Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors.
Asunto(s)
Alquilación/fisiología , Daño del ADN/fisiología , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN/fisiología , Dioxigenasas/metabolismo , Regulación de la Expresión Génica/fisiología , Proteasas Ubiquitina-Específicas/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB , Alquilación/genética , Western Blotting , Daño del ADN/genética , Reparación del ADN/genética , Células HEK293 , Humanos , Inmunoprecipitación , Microscopía Fluorescente , Modelos Biológicos , Espectrometría de Masas en TándemRESUMEN
Cellular RNAs are modified by both physiological factors and exogenous agents, such as methyl methanesulfonate (MMS). However, techniques for analyzing how proteins may interact with these modified RNAs are limited. Here, we provide a protocol combining RNA immunoprecipitation (RIP) with mass spectrometry (MS) to analyze the methylation state of the RNAs bound by Flag-tagged proteins in mammalian cells. The approach is highly quantitative and can simultaneously detect several methylated nucleosides in a single experiment. For complete details on the use and execution of this protocol, please refer to Tsao et al. (2021).
Asunto(s)
Nucleósidos , ARN , Animales , Inmunoprecipitación , Mamíferos/genética , Espectrometría de Masas/métodos , Metilación , Nucleósidos/análisis , Proteínas , ARN/químicaRESUMEN
Immunoaffinity purification allows for the purification of epitope-tagged proteins and their associated multisubunit complexes from mammalian cells. Subsequent identification of the proteins by proteomic analysis enables unbiased biochemical characterization of their associated partners, potentially revealing the physiological or functional context of any given protein. Here, we use immunoaffinity isolation of the Activating Signal Co-integrator Complex (ASCC) from human cells as an example, demonstrating the utility of the approach in revealing protein complexes involved in genotoxic stress responses.