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1.
Nucleic Acids Res ; 51(19): 10467-10483, 2023 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-37713620

RESUMEN

Proper regulation of replication fork progression is important for genomic maintenance. Subverting the transcription-induced conflicts is crucial in preserving the integrity of replication forks. Various chromatin remodelers, such as histone chaperone and histone deacetylases are known to modulate replication stress, but how these factors are organized or collaborate are not well understood. Here we found a new role of the OTUD5 deubiquitinase in limiting replication stress. We found that OTUD5 is recruited to replication forks, and its depletion causes replication fork stress. Through its C-terminal disordered tail, OTUD5 assembles a complex containing FACT, HDAC1 and HDAC2 at replication forks. A cell line engineered to specifically uncouple FACT interaction with OTUD5 exhibits increases in FACT loading onto chromatin, R-loop formation, and replication fork stress. OTUD5 mediates these processes by recruiting and stabilizing HDAC1 and HDAC2, which decreases H4K16 acetylation and FACT recruitment. Finally, proteomic analysis revealed that the cells with deficient OTUD5-FACT interaction activates the Fanconi Anemia pathway for survival. Altogether, this study identified a new interaction network among OTUD5-FACT-HDAC1/2 that limits transcription-induced replication stress.


Asunto(s)
Cromatina , Replicación del ADN , Humanos , Línea Celular , Cromatina/genética , Inestabilidad Genómica , Proteómica
2.
Nucleic Acids Res ; 50(3): 1501-1516, 2022 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-35061896

RESUMEN

Homologous recombination (HR) is critical for error-free repair of DNA double-strand breaks. Chromatin loading of RAD51, a key protein that mediates the recombination, is a crucial step in the execution of the HR repair. Here, we present evidence that SUMOylation of RAD51 is crucial for the RAD51 recruitment to chromatin and HR repair. We found that topoisomerase 1-binding arginine/serine-rich protein (TOPORS) induces the SUMOylation of RAD51 at lysine residues 57 and 70 in response to DNA damaging agents. The SUMOylation was facilitated by an ATM-induced phosphorylation of TOPORS at threonine 515 upon DNA damage. Knockdown of TOPORS or expression of SUMOylation-deficient RAD51 mutants caused reduction in supporting normal RAD51 functions during the HR repair, suggesting the physiological importance of the modification. We found that the SUMOylation-deficient RAD51 reduces the association with its crucial binding partner BRCA2, explaining its deficiency in supporting the HR repair. These findings altogether demonstrate a crucial role for TOPORS-mediated RAD51 SUMOylation in promoting HR repair and genomic maintenance.


Asunto(s)
Recombinasa Rad51 , Reparación del ADN por Recombinación , Cromatina , ADN/metabolismo , Daño del ADN , Reparación del ADN/genética , Recombinación Homóloga , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Sumoilación
3.
Nucleic Acids Res ; 50(18): 10469-10486, 2022 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-36155803

RESUMEN

Human CtIP maintains genomic integrity primarily by promoting 5' DNA end resection, an initial step of the homologous recombination (HR). A few mechanisms have been suggested as to how CtIP recruitment to damage sites is controlled, but it is likely that we do not yet have full understanding of the process. Here, we provide evidence that CtIP recruitment and functioning are controlled by the SIAH2 E3 ubiquitin ligase. We found that SIAH2 interacts and ubiquitinates CtIP at its N-terminal lysine residues. Mutating the key CtIP lysine residues impaired CtIP recruitment to DSBs and stalled replication forks, DSB end resection, overall HR repair capacity of cells, and recovery of stalled replication forks, suggesting that the SIAH2-induced ubiquitination is important for relocating CtIP to sites of damage. Depleting SIAH2 consistently phenocopied these results. Overall, our work suggests that SIAH2 is a new regulator of CtIP and HR repair, and emphasizes that SIAH2-mediated recruitment of the CtIP is an important step for CtIP's function during HR repair.


Asunto(s)
Reparación del ADN , Replicación del ADN , Endodesoxirribonucleasas/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas/genética , Humanos , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación
4.
PLoS Genet ; 16(3): e1008524, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32142505

RESUMEN

Common fragile sites (CFSs) are breakage-prone genomic loci, and are considered to be hotspots for genomic rearrangements frequently observed in cancers. Understanding the underlying mechanisms for CFS instability will lead to better insight on cancer etiology. Here we show that Polycomb group proteins BMI1 and RNF2 are suppressors of transcription-replication conflicts (TRCs) and CFS instability. Cells depleted of BMI1 or RNF2 showed slower replication forks and elevated fork stalling. These phenotypes are associated with increase occupancy of RNA Pol II (RNAPII) at CFSs, suggesting that the BMI1-RNF2 complex regulate RNAPII elongation at these fragile regions. Using proximity ligase assays, we showed that depleting BMI1 or RNF2 causes increased associations between RNAPII with EdU-labeled nascent forks and replisomes, suggesting increased TRC incidences. Increased occupancy of a fork protective factor FANCD2 and R-loop resolvase RNH1 at CFSs are observed in RNF2 CRISPR-KO cells, which are consistent with increased transcription-associated replication stress in RNF2-deficient cells. Depleting FANCD2 or FANCI proteins further increased genomic instability and cell death of the RNF2-deficient cells, suggesting that in the absence of RNF2, cells depend on these fork-protective factors for survival. These data suggest that the Polycomb proteins have non-canonical roles in suppressing TRC and preserving genomic integrity.


Asunto(s)
Sitios Frágiles del Cromosoma/genética , Replicación del ADN/genética , Complejo Represivo Polycomb 1/genética , Transcripción Genética/genética , Línea Celular , Línea Celular Tumoral , Inestabilidad Genómica/genética , Células HEK293 , Células HeLa , Humanos
5.
Mar Drugs ; 20(1)2022 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-35049906

RESUMEN

Sponges are at the forefront of marine natural product research. In the deep sea, extreme conditions have driven secondary metabolite pathway evolution such that we might expect deep-sea sponges to yield a broad range of unique natural products. Here, we investigate the chemodiversity of a deep-sea tetractinellid sponge, Characella pachastrelloides, collected from ~800 m depth in Irish waters. First, we analyzed the MS/MS data obtained from fractions of this sponge on the GNPS public online platform to guide our exploration of its chemodiversity. Novel glycolipopeptides named characellides were previously isolated from the sponge and herein cyanocobalamin, a manufactured form of vitamin B12, not previously found in nature, was isolated in a large amount. We also identified several poecillastrins from the molecular network, a class of polyketide known to exhibit cytotoxicity. Light sensitivity prevented the isolation and characterization of these polyketides, but their presence was confirmed by characteristic NMR and MS signals. Finally, we isolated the new betaine 6-methylhercynine, which contains a unique methylation at C-2 of the imidazole ring. This compound showed potent cytotoxicity towards against HeLa (cervical cancer) cells.


Asunto(s)
Antineoplásicos/farmacología , Poríferos , Vitamina B 12/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Organismos Acuáticos , Femenino , Células HeLa/efectos de los fármacos , Humanos , Neoplasias del Cuello Uterino/patología , Vitamina B 12/química , Vitamina B 12/uso terapéutico
6.
Nucleic Acids Res ; 47(2): 729-746, 2019 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-30508113

RESUMEN

Timely stalling and resumption of RNA polymerases at damaged chromatin are actively regulated processes. Prior work showed an importance of FACT histone chaperone in such process. Here we provide a new role of OTUD5 deubiquitinase in the FACT-dependent process. Through a DUB RNAi screen, we found OTUD5 as a specific stabilizer of the UBR5 E3 ligase. OTUD5 localizes to DNA double strand breaks (DSBs), interacts with UBR5 and represses the RNA Pol II elongation and RNA synthesis. OTUD5 co-localizes and interacts with the FACT component SPT16 and antagonizes the histone H2A deposition at DSB lesions. OTUD5 interacts with UBR5 and SPT16 independently through two distinct regions, and both interactions are necessary for arresting the Pol II elongation at lesions. These analyses suggested that the catalytic (through UBR5 stabilization) as well as scaffolding (through FACT binding) activities of OTUD5 are involved in the FACT-dependent transcription. We found that a cancer-associated missense mutation within the OTUD5 Ubiquitin Interacting Motif (UIM) abrogates the FACT association and the Pol II arrest, providing a possible link between the transcriptional regulation and tumor suppression. Our work establishes OTUD5 as a new regulator of the DNA damage response, and provides an insight into the FACT-dependent transcription at damaged chromatin.


Asunto(s)
Cromatina/metabolismo , Daño del ADN , Endopeptidasas/metabolismo , Transcripción Genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Roturas del ADN de Doble Cadena , Endopeptidasas/química , Endopeptidasas/genética , Regulación de la Expresión Génica , Inestabilidad Genómica , Histonas/metabolismo , Humanos , Mutación , Neoplasias/genética , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Factores de Transcripción/metabolismo
7.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-34360546

RESUMEN

Promyelocytic leukemia (PML) protein is the core component of subnuclear structures called PML nuclear bodies that are known to play important roles in cell survival, DNA damage responses, and DNA repair. Fanconi anemia (FA) proteins are required for repairing interstrand DNA crosslinks (ICLs). Here we report a novel role of PML proteins, regulating the ICL repair pathway. We found that depletion of the PML protein led to the significant reduction of damage-induced FANCD2 mono-ubiquitination and FANCD2 foci formation. Consistently, the cells treated with siRNA against PML showed enhanced sensitivity to a crosslinking agent, mitomycin C. Further studies showed that depletion of PML reduced the protein expression of FANCA, FANCG, and FANCD2 via reduced transcriptional activity. Interestingly, we observed that damage-induced CHK1 phosphorylation was severely impaired in cells with depleted PML, and we demonstrated that CHK1 regulates FANCA, FANCG, and FANCD2 transcription. Finally, we showed that inhibition of CHK1 phosphorylation further sensitized cancer cells to mitomycin C. Taken together, these findings suggest that the PML is critical for damage-induced CHK1 phosphorylation, which is important for FA gene expression and for repairing ICLs.


Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación G de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Regulación de la Expresión Génica , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Daño del ADN , Reparación del ADN , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Células HeLa , Humanos , Fosforilación , Ubiquitinación
8.
Mar Drugs ; 17(9)2019 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-31480497

RESUMEN

: An Antarctic coral belonging to the order Pennatulacea, collected during the 2013 austral autumn by trawl from 662 to 944 m depth, has yielded three new briarane diterpenes, bathyptilone A-C (1-3) along with a trinorditerpene, enbepeanone A (4), which bears a new carbon skeleton. Structure elucidation was facilitated by one- and two-dimensional NMR spectroscopy, mass spectrometry and confirmed by X-ray crystallography. The three compounds were screened in four cancer cell lines. Bathyptilone A displayed selective nanomolar cytotoxicity against the neurogenic mammalian cell line Ntera-2.


Asunto(s)
Antozoos/química , Terpenos/química , Animales , Regiones Antárticas , Cristalografía por Rayos X/métodos , Diterpenos/química , Células HeLa , Humanos , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas/métodos
9.
Mar Drugs ; 17(4)2019 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-30999651

RESUMEN

The subtidal red alga Plocamium cartilagineum was collected from the Western Antarctic Peninsula during the 2011 and 2017 austral summers. Bulk collections from specific sites corresponded to chemogroups identified by Young et al. in 2013. One of the chemogroups yielded several known acyclic halogenated monoterpenes (2-5) as well as undescribed compounds of the same class, anverenes B-D (6-8). Examination of another chemogroup yielded an undescribed cyclic halogenated monoterpene anverene E (9) as its major secondary metabolite. Elucidation of structures was achieved through one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) spectroscopy and negative chemical ionization mass spectrometry. Compounds 1-9 show moderate cytotoxicity against cervical cancer (HeLa) cells.


Asunto(s)
Monoterpenos/química , Monoterpenos/farmacología , Plocamium/química , Regiones Antárticas , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células HeLa , Humanos , Hidrocarburos Halogenados/química , Hidrocarburos Halogenados/aislamiento & purificación , Hidrocarburos Halogenados/farmacología , Concentración 50 Inhibidora , Conformación Molecular , Monoterpenos/aislamiento & purificación , Resonancia Magnética Nuclear Biomolecular
10.
Proc Natl Acad Sci U S A ; 113(40): 11243-11248, 2016 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-27647897

RESUMEN

BMI1 is a component of the Polycomb Repressive Complex 1 (PRC1), which plays a key role in maintaining epigenetic silencing during development. BMI1 also participates in gene silencing during DNA damage response, but the precise downstream function of BMI1 in gene silencing is unclear. Here we identified the UBR5 E3 ligase as a downstream factor of BMI1. We found that UBR5 forms damage-inducible nuclear foci in a manner dependent on the PRC1 components BMI1, RNF1 (RING1a), and RNF2 (RING1b). Whereas transcription is repressed at UV-induced lesions on chromatin, depletion of the PRC1 members or UBR5 alone derepressed transcription elongation at these sites, suggesting that UBR5 functions in a linear pathway with PRC1 in inducing gene silencing at lesions. Mass spectrometry (MS) analysis revealed that UBR5 associates with BMI1 as well as FACT components SPT16 and SSRP1. We found that UBR5 localizes to the UV-induced lesions along with SPT16. We show that UBR5 ubiquitinates SPT16, and depletion of UBR5 or BMI1 leads to an enlargement of SPT16 foci size at UV lesions, suggesting that UBR5 and BMI1 repress SPT16 enrichment at the damaged sites. Consistently, depletion of the FACT components effectively reversed the transcriptional derepression incurred in the UBR5 and BMI1 KO cells. Finally, UBR5 and BMI1 KO cells are hypersensitive to UV, which supports the notion that faulty RNA synthesis at damaged sites is harmful to the cell fitness. Altogether, these results suggest that BMI1 and UBR5 repress the polymerase II (Pol II)-mediated transcription at damaged sites, by negatively regulating the FACT-dependent Pol II elongation.


Asunto(s)
Cromatina/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Ubiquitina-Proteína Ligasas/metabolismo , Células HCT116 , Células HeLa , Humanos , Unión Proteica , Transducción de Señal , Elongación de la Transcripción Genética , Rayos Ultravioleta
11.
Genes Dev ; 24(16): 1680-94, 2010 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-20713514

RESUMEN

Studying rare human genetic diseases often leads to a better understanding of normal cellular functions. Fanconi anemia (FA), for example, has elucidated a novel DNA repair mechanism required for maintaining genomic stability and preventing cancer. The FA pathway, an essential tumor-suppressive pathway, is required for protecting the human genome from a specific type of DNA damage; namely, DNA interstrand cross-links (ICLs). In this review, we discuss the recent progress in the study of the FA pathway, such as the identification of new FANCM-binding partners and the identification of RAD51C and FAN1 (Fanconi-associated nuclease 1) as new FA pathway-related proteins. We also focus on the role of the FA pathway as a potential regulator of DNA repair choices in response to double-strand breaks, and its novel functions during the mitotic phase of the cell cycle.


Asunto(s)
Anemia de Fanconi/genética , Anemia de Fanconi/fisiopatología , Inestabilidad Genómica/genética , Animales , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas , Exodesoxirribonucleasas/metabolismo , Humanos , Enzimas Multifuncionales , Mutación , Unión Proteica , Proteínas Supresoras de Tumor/metabolismo
12.
Genes Dev ; 23(5): 555-60, 2009 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-19270156

RESUMEN

The 13 Fanconi anemia (FA) proteins cooperate in a common DNA repair pathway. Eight of these proteins are assembled into a multisubunit E3 ligase called the FA core complex. During S phase, the FA core complex is loaded by the FANCM protein into chromatin where it monoubiquitinates its substrates. In mitosis, the FA core complex is released from FANCM by an unknown mechanism. Here we show that FANCM is hyperphosphorylated and degraded during mitosis. beta-TRCP and Plk1 are the key regulators of FANCM degradation. Nondegradable mutant forms of FANCM retain the FA core complex in the chromatin and disrupt the FA pathway. Our data provide a novel mechanism for the cell cycle-dependent regulation of the FA pathway.


Asunto(s)
ADN Helicasas/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Mitosis/fisiología , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Inestabilidad Genómica , Células HeLa , Humanos , Ratones , Complejos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Alineación de Secuencia , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Quinasa Tipo Polo 1
13.
J Biol Chem ; 289(10): 7003-7010, 2014 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-24451376

RESUMEN

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.


Asunto(s)
Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación E de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Anemia de Fanconi/genética , Proteína del Grupo de Complementación E de la Anemia de Fanconi/química , Proteína del Grupo de Complementación E de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación L de la Anemia de Fanconi/metabolismo , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Estructura Terciaria de Proteína , Ubiquitinación
14.
Res Sq ; 2024 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-39070640

RESUMEN

DNA Double-strand breaks (DSBs) are harmful lesions and major sources of genomic instability. Studies have suggested that DSBs induce local transcriptional silencing that consequently promotes genomic stability. Several factors have been proposed to actively participate in this process, including ATM and Polycomb repressive complex 1 (PRC1). Here we found that disrupting PRC1 clustering disrupts DSB-induced gene silencing. Interactome analysis of PHC2, a PRC1 subunit that promotes the formation of the Polycomb body, found several nucleoporins that constitute the Nuclear Pore Complex (NPC). Similar to PHC2, depleting the nucleoporins also disrupted the DSB-induced gene silencing. We found that some of these nucleoporins, such as NUP107 and NUP43, which are members of the Y-complex of NPC, localize to DSB sites. These nucleoporin-enriched DSBs were distant from the nuclear periphery. The presence of nucleoporins and PHC2 at DSB regions were inter-dependent, suggesting that they act cooperatively in the DSB-induced gene silencing. We further found two structural components within NUP107 to be necessary for the transcriptional repression at DSBs: ATM/ATR-mediated phosphorylation at Serine37 residue within the N-terminal disordered tail, and the NUP133-binding surface at the C-terminus. These results provide a new functional interplay among nucleoporins, ATM and the Polycomb proteins in the DSB metabolism, and underscore their emerging roles in genome stability maintenance. *Hongseon Song, Yubin Bae, Sangin Kim, and Dante Deascanis contributed equally to this work.

15.
Mol Cell Oncol ; 9(1): 2054263, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35372672

RESUMEN

RAD51 loading onto chromatin is a key step during the homologous recombination (HR) repair. We recently reported a new mode of RAD51 regulation, which is mediated by TOPORS E3 SUMO ligase and RAD51 SUMOylation. ATM/ATR-induced phosphorylation of TOPORS is necessary for this event, revealing a new role of these master DNA damage response kinases in HR repair.

16.
Cell Death Dis ; 13(11): 933, 2022 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-36344491

RESUMEN

Serine/arginine-rich splicing factor 3 (SRSF3) is an RNA binding protein that most often regulates gene expression at the splicing level. Although the role of SRSF3 in mRNA splicing in the nucleus is well known, its splicing-independent role outside of the nucleus is poorly understood. Here, we found that SRSF3 exerts a translational control of p21 mRNA. Depletion of SRSF3 induces cellular senescence and increases the expression of p21 independent of p53. Consistent with the expression patterns of SRSF3 and p21 mRNA in the TCGA database, SRSF3 knockdown increases the p21 mRNA level and its translation efficiency as well. SRSF3 physically associates with the 3'UTR region of p21 mRNA and the translational initiation factor, eIF4A1. Our study proposes a model in which SRSF3 regulates translation by interacting with eIF4A1 at the 3'UTR region of p21 mRNA. We also found that SRSF3 localizes to the cytoplasmic RNA granule along with eIF4A1, which may assist in translational repression therein. Thus, our results provide a new mode of regulation for p21 expression, a crucial regulator of the cell cycle and senescence, which occurs at the translational level and involves SRSF3.


Asunto(s)
Empalme del ARN , Proteínas de Unión al ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Factores de Empalme de ARN/metabolismo , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , Proteínas de Unión al ARN/metabolismo
17.
J Biol Chem ; 285(15): 11252-7, 2010 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-20147737

RESUMEN

The UAF1 (Usp1-associated factor 1) protein binds and stimulates three deubiquitinating enzymes: USP1, USP12, and USP46. Although the USP1 x UAF1 complex is required for regulation of the Fanconi anemia (FA) DNA repair pathway, less is known about the USP12 x UAF1 and the USP46 x UAF1 complexes. To understand further the nature of the USP12 and USP46 complexes, we attempted to identify proteins that interact with the USP12 and USP46 deubiquitinating enzyme complexes. We identified WDR20, a WD40-repeat containing protein, as a common binding partner of UAF1, USP12, and USP46. Further analysis showed that WDR20 associates exclusively with USP12 and USP46, not with USP1. Furthermore, we demonstrate the purification of a ternary USP12 x UAF1 x WDR20 complex. Interestingly, and consistent with the binding assays, WDR20 stimulated the enzymatic activity of USP12 x UAF1, but not of USP1 x UAF1. Consistent with our previous report that USP12 and USP46 do not regulate the FA pathway, small interference RNA-mediated depletion of WDR20 protein did not affect the FA pathway or DNA damage responses. We provide a model in which WDR20 serves as a stimulatory subunit for preserving and regulating the activity of the subset of the UAF1 x USP complexes.


Asunto(s)
Proteínas Portadoras/fisiología , Regulación Enzimológica de la Expresión Génica , Complejos Multienzimáticos/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina/química , Proteínas Portadoras/metabolismo , Línea Celular , Daño del ADN , Anemia de Fanconi/metabolismo , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo
18.
Blood ; 111(10): 5215-22, 2008 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-18174376

RESUMEN

Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. A total of 13 FA proteins are involved in regulating genome surveillance and chromosomal stability. The FA core complex, consisting of 8 FA proteins (A/B/C/E/F/G/L/M), is essential for the monoubiquitination of FANCD2 and FANCI. FANCM is a human ortholog of the archaeal DNA repair protein Hef, and it contains a DEAH helicase and a nuclease domain. Here, we examined the effect of FANCM expression on the integrity and localization of the FA core complex. FANCM was exclusively localized to chromatin fractions and underwent cell cycle-dependent phosphorylation and dephosphorylation. FANCM-depleted HeLa cells had an intact FA core complex but were defective in chromatin localization of the complex. Moreover, depletion of the FANCM binding partner, FAAP24, disrupted the chromatin association of FANCM and destabilized FANCM, leading to defective recruitment of the FA core complex to chromatin. Our results suggest that FANCM is an anchor required for recruitment of the FA core complex to chromatin, and that the FANCM/FAAP24 interaction is essential for this chromatin-loading activity. Dysregulated loading of the FA core complex accounts, at least in part, for the characteristic cellular and developmental abnormalities in FA.


Asunto(s)
Ciclo Celular , Cromatina/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Anemia de Fanconi/patología , Células HeLa , Humanos , Fosforilación
19.
Mol Biol Cell ; 18(1): 324-35, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17079730

RESUMEN

Ubiquitinated integral membrane proteins are delivered to the interior of the lysosome/vacuole for degradation. This process relies on specific ubiquitination of potential cargo and recognition of that Ub-cargo by sorting receptors at multiple compartments. We show that the endosomal Hse1-Vps27 sorting receptor binds to ubiquitin peptidases and the ubiquitin ligase Rsp5. Hse1 is linked to Rsp5 directly via a PY element within its C-terminus and through a novel protein Hua1, which recruits a complex of Rsp5, Rup1, and Ubp2. The SH3 domain of Hse1 also binds to the deubiquitinating protein Ubp7. Functional analysis shows that when both modes of Rsp5 association with Hse1 are altered, sorting of cargo that requires efficient ubiquitination for entry into the MVB is blocked, whereas sorting of cargo containing an in-frame addition of ubiquitin is normal. Further deletion of Ubp7 restores sorting of cargo when the Rsp5:Hse1 interaction is compromised suggesting that both ubiquitin ligases and peptidases associate with the Hse1-Vps27 sorting complex to control the ubiquitination status and sorting efficiency of cargo proteins. Additionally, we find that disruption of UBP2 and RUP1 inhibits MVB sorting of some cargos suggesting that Rsp5 requires association with Ubp2 to properly ubiquitinate cargo for efficient MVB sorting.


Asunto(s)
Endopeptidasas/metabolismo , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Vesículas Transportadoras/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Complejo Mediador , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Dominios Homologos src
20.
Oncotarget ; 11(21): 2024-2025, 2020 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-32523656

RESUMEN

[This corrects the article DOI: 10.18632/oncotarget.10275.].

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