RESUMEN
LSG1 is a conserved GTPase involved in ribosome assembly. It is required for the eviction of the nuclear export adapter NMD3 from the pre-60S subunit in the cytoplasm. In human cells, LSG1 has also been shown to interact with vesicle-associated membrane protein-associated proteins (VAPs) that are found primarily on the endoplasmic reticulum. VAPs interact with a large host of proteins which contain FFAT motifs (two phenylalanines (FF) in an acidic tract) and are involved in many cellular functions including membrane traffic and regulation of lipid transport. Here, we show that human LSG1 binds to VAPs via a noncanonical FFAT-like motif. Deletion of this motif specifically disrupts the localization of LSG1 to the ER, without perturbing LSG1-dependent recycling of NMD3 in cells or modulation of LSG1 GTPase activity in vitro.
RESUMEN
5'-end modifications play key roles in determining RNA fates. Phospho-methylation is a noncanonical cap occurring on either 5'-PPP or 5'-P ends. We used ChemRAP, in which affinity purification of cellular proteins with chemically synthesized modified RNAs is coupled to quantitative proteomics, to identify 5'-Pme "readers". We show that 5'-Pme is directly recognized by EPRS, the central subunit of the multisynthetase complex (MSC), through its linker domain, which has previously been involved in key noncanonical EPRS and MSC functions. We further determine that the 5'-Pme writer BCDIN3D regulates the binding of EPRS to specific mRNAs, either at coding regions rich in MSC codons, or around start codons. In the case of LRPPRC (leucine-rich pentatricopeptide repeat containing), a nuclear-encoded mitochondrial protein associated with the French Canadian Leigh syndrome, BCDIN3D deficiency abolishes binding of EPRS around its mRNA start codon, increases its translation but ultimately results in LRPPRC mislocalization. Overall, our results suggest that BCDIN3D may regulate the translation of specific mRNA via RNA-5'-Pme.
Asunto(s)
Proteínas de Neoplasias , Biosíntesis de Proteínas , Proteínas de Neoplasias/genética , Canadá , Metilación , ARN Mensajero/genética , ARN/metabolismoRESUMEN
RNAs from various cells and tissues are modified in nearly 200 chemically distinct ways. These modifications can be deposited either on the 5' or 3' ends, or internally on the nucleobases or sugar backbone. 5'-end modifications are crucial for protecting RNAs from untimely degradation/processing, regulating their cellular functions, or discriminating endogenous RNAs from pathogenic RNAs. 5'-end phospho-methylation is a remarkable RNA modification that is enzymatically deposited either on the γ-phosphate of nascent triphosphorylated RNAs by human BCDIN3/MePCE, or on the α-phosphate of processed monophosphorylated RNAs by human BCDIN3D. These 5'-phospho-methyltransferases are part of the BIN3 family of O-methyltransferases conserved from S. pombe to humans and play important cellular and biological roles, many of which await further elucidation. Here, we quickly recapitulate historical methods for the detection of 5'-end phospho-methyl modifications, and focus more specifically on a method that can be used to detect and quantify α-monophosphate methylation from as low as 10-100ng of total RNA from cells or tissues. This method is important for deciphering the roles of BCDIN3D and its homologs across species, as well as serves as starting point for the development of new methods for detection of 5'-end modifications.
Asunto(s)
Metiltransferasas , ARN , Humanos , Metilación , Metiltransferasas/metabolismo , Procesamiento Postranscripcional del ARNRESUMEN
The histone demethylase KDM5A erases histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDRs). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a noncanonical poly(ADP-ribose) (PAR)-binding region unique to KDM5A. Loss of the PAR-binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi's) blocks KDM5A-PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and function at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR binding and macroH2A1.2 in KDM5A recognition of DNA lesion sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity.
Asunto(s)
ADN/genética , Histonas/genética , Reparación del ADN por Recombinación/genética , Proteína 2 de Unión a Retinoblastoma/genética , Cromatina/genética , Roturas del ADN de Doble Cadena , Daño del ADN , Humanos , Poli Adenosina Difosfato Ribosa/genética , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
Type II diabetes (T2D) and specific cancers share many risk factors, however, the molecular mechanisms underlying these connections are often not well-understood. BCDIN3D is an RNA modifying enzyme that methylates specific precursor microRNAs and tRNAHis. In addition to breast cancer, BCDIN3D may also be linked to metabolism, as its gene locus is associated with obesity and T2D. In order to uncover metabolic pathways regulated by BCDIN3D in cancer, we performed an unbiased analysis of the metabolome, transcriptome, and proteome of breast cancer cells depleted for BCDIN3D. Intersection of these analyses showed that BCDIN3D-depleted cells have increased levels of Fructose 1,6 Bisphosphate (F1,6-BP), the last six-carbon glycolytic intermediate accompanied by reduced glycolytic capacity. We further show that elevated F1,6-BP is due to downregulation of Aldolase C (ALDOC), an enzyme that cleaves F1,6-BP mainly in the brain, but whose high expression/amplification is associated with poor prognosis in breast cancer. BCDIN3D regulates ALDOC through a non-canonical mechanism involving the crucial let-7 microRNA family and its target site on the 3'UTR of ALDOC. Overall, our results reveal an important connection between BCDIN3D, let-7 and glycolysis that may be relevant to breast cancer, obesity, and T2D.
Asunto(s)
Neoplasias de la Mama/genética , Diabetes Mellitus Tipo 2/genética , Fructosa-Bifosfato Aldolasa/genética , Metiltransferasas/genética , MicroARNs/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Glucólisis/genética , Xenoinjertos , Humanos , Células MCF-7 , Metaboloma/genética , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Proteoma/genética , Factores de Riesgo , Transcriptoma/genéticaRESUMEN
Type I interferons (IFNs) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. Induction of these ISGs must be regulated for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a role for the RNA base modification N6-methyladenosine (m6A) in the regulation of ISGs. Using ribosome profiling and quantitative mass spectrometry, coupled with m6A-immunoprecipitation and sequencing, we identify a subset of ISGs, including IFITM1, whose translation is enhanced by m6A and the m6A methyltransferase proteins METTL3 and METTL14. We further determine that the m6A reader YTHDF1 increases the expression of IFITM1 in an m6A-binding-dependent manner. Importantly, we find that the m6A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m6A as having a role in post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction.
Asunto(s)
Adenosina/análogos & derivados , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , Transcripción Genética , Estomatitis Vesicular/genética , Vesiculovirus/patogenicidad , Células A549 , Adenosina/metabolismo , Animales , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Antivirales/farmacología , Chlorocebus aethiops , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Interferón beta/farmacología , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Biosíntesis de Proteínas/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transcripción Genética/efectos de los fármacos , Células Vero , Estomatitis Vesicular/metabolismo , Estomatitis Vesicular/virología , Vesiculovirus/crecimiento & desarrollo , Replicación ViralRESUMEN
Nearly 200 distinct chemical modifications of RNAs have been discovered to date. Their analysis via direct methods has been possible in abundant RNA species, such as ribosomal, transfer or viral RNA, since several decades. However, their analysis in less abundant RNAs species, especially cellular messenger RNAs, was rendered possible only recently with the advent of high throughput sequencing techniques. Given the growing biomedical interest of the proteins that write, erase and read RNA modifications, ingenious new methods to enrich and identify RNA modifications at base resolution have been implemented, and more efforts are underway to render them more quantitative. Here, we review several crucial modification-specific (bio)chemical approaches and discuss their advantages and shortcomings for exploring the epitranscriptome.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN , Biología , Secuenciación de Nucleótidos de Alto Rendimiento , ARN/genética , ARN Mensajero/metabolismo , ARN ViralRESUMEN
Stabilization of stalled replication forks is a prominent mechanism of PARP (Poly(ADP-ribose) Polymerase) inhibitor (PARPi) resistance in BRCA-deficient tumors. Epigenetic mechanisms of replication fork stability are emerging but remain poorly understood. Here, we report the histone acetyltransferase PCAF (p300/CBP-associated) as a fork-associated protein that promotes fork degradation in BRCA-deficient cells by acetylating H4K8 at stalled replication forks, which recruits MRE11 and EXO1. A H4K8ac binding domain within MRE11/EXO1 is required for their recruitment to stalled forks. Low PCAF levels, which we identify in a subset of BRCA2-deficient tumors, stabilize stalled forks, resulting in PARPi resistance in BRCA-deficient cells. Furthermore, PCAF activity is tightly regulated by ATR (ataxia telangiectasia and Rad3-related), which phosphorylates PCAF on serine 264 (S264) to limit its association and activity at stalled forks. Our results reveal PCAF and histone acetylation as critical regulators of fork stability and PARPi responses in BRCA-deficient cells, which provides key insights into targeting BRCA-deficient tumors and identifying epigenetic modulators of chemotherapeutic responses.
Asunto(s)
Proteína BRCA1/deficiencia , Proteína BRCA2/deficiencia , Enzimas Reparadoras del ADN/metabolismo , Replicación del ADN , Exodesoxirribonucleasas/metabolismo , Histonas/metabolismo , Proteína Homóloga de MRE11/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación/efectos de los fármacos , Secuencia de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lisina/metabolismo , Modelos Biológicos , Mutación/genética , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Unión Proteica/efectos de los fármacos , Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/genéticaRESUMEN
Cells encounter a multitude of external and internal stress-causing agents that can ultimately lead to DNA damage, mutations and disease. A cascade of signaling events counters these challenges to DNA, which is termed as the DNA damage response (DDR). The DDR preserves genome integrity by engaging appropriate repair pathways, while also coordinating cell cycle and/or apoptotic responses. Although many of the protein components in the DDR are identified, how chemical modifications to DNA impact the DDR is poorly understood. This review focuses on our current understanding of DNA methylation in maintaining genome integrity in mammalian cells. DNA methylation is a reversible epigenetic mark, which has been implicated in DNA damage signaling, repair and replication. Sites of DNA methylation can trigger mutations, which are drivers of human diseases including cancer. Indeed, alterations in DNA methylation are associated with increased susceptibility to tumorigenesis but whether this occurs through effects on the DDR, transcriptional responses or both is not entirely clear. Here, we also highlight epigenetic drugs currently in use as therapeutics that target DNA methylation pathways and discuss their effects in the context of the DDR. Finally, we pose unanswered questions regarding the interplay between DNA methylation, transcription and the DDR, positing the potential coordinated efforts of these pathways in genome integrity. While the impact of DNA methylation on gene regulation is widely understood, how this modification contributes to genome instability and mutations, either directly or indirectly, and the potential therapeutic opportunities in targeting DNA methylation pathways in cancer remain active areas of investigation.
Asunto(s)
Metilación de ADN , Genoma , Animales , HumanosRESUMEN
Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability as well as therapeutic targets in cancer. Here, we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using several cell-based assays and proteomic interaction network analysis. Applying these approaches, we identify 24 of the 42 BRD proteins as promoters of DNA repair and/or genome integrity. We identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allowing direct acetylation by PCAF, and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrids (R-loops) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on topoisomerase II, and BRD2 directly bound and activated topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a framework to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition.
Asunto(s)
Inestabilidad Genómica , Estructuras R-Loop , Reparación del ADN por Recombinación/genética , Factores de Transcripción/genética , Acetilación , Línea Celular , Roturas del ADN de Doble Cadena , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Células HEK293 , Células HeLa , Humanos , Transactivadores/metabolismo , Factores de Transcripción/análisis , Ubiquitinación , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.
Asunto(s)
Pruebas de Enzimas/métodos , Metiltransferasas/metabolismo , ARN/metabolismo , Humanos , Metilación , S-Adenosilmetionina/metabolismoRESUMEN
5' ends are important for determining the fate of RNA molecules. BCDIN3D is an RNA phospho-methyltransferase that methylates the 5' monophosphate of specific RNAs. In order to gain new insights into the molecular function of BCDIN3D, we performed an unbiased analysis of its interacting RNAs by Thermostable Group II Intron Reverse Transcriptase coupled to next generation sequencing (TGIRT-seq). Our analyses showed that BCDIN3D interacts with full-length phospho-methylated tRNAHis and miR-4454. Interestingly, we found that miR-4454 is not synthesized from its annotated genomic locus, which is a primer-binding site for an endogenous retrovirus, but rather by Dicer cleavage of mature tRNAHis. Sequence analysis revealed that miR-4454 is identical to the 3' end of tRNAHis. Moreover, we were able to generate this 'miRNA' in vitro through incubation of mature tRNAHis with Dicer. As found previously for several pre-miRNAs, a 5'P-tRNAHis appears to be a better substrate for Dicer cleavage than a phospho-methylated tRNAHis. Moreover, tRNAHis 3'-fragment/'miR-4454' levels increase in cells depleted for BCDIN3D. Altogether, our results show that in addition to microRNAs, BCDIN3D regulates tRNAHis 3'-fragment processing without negatively affecting tRNAHis's canonical function of aminoacylation.
Asunto(s)
ARN Helicasas DEAD-box/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Metiltransferasas/genética , ARN de Transferencia de Histidina/metabolismo , Ribonucleasa III/genética , Línea Celular , Humanos , MicroARNs/genética , Análisis de Secuencia de ARN , Aminoacilación de ARN de TransferenciaRESUMEN
One-carbon (1C) metabolism is a universal folate-dependent pathway essential for de novo purine and thymidylate synthesis, amino acid interconversion, universal methyl-donor production, and regeneration of redox cofactors. Homozygous deletion of the 1C pathway gene Mthfd1l encoding methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 1-like, which catalyzes mitochondrial formate production from 10-formyltetrahydrofolate, results in 100% penetrant embryonic neural tube defects (NTDs), underscoring the central role of mitochondrially derived formate in embryonic development and providing a mechanistic link between folate and NTDs. However, the specific metabolic processes that are perturbed by Mthfd1l deletion are not known. Here, we performed untargeted metabolomics on whole Mthfd1l-null and wildtype mouse embryos in combination with isotope tracer analysis in mouse embryonic fibroblast (MEF) cell lines to identify Mthfd1l deletion-induced disruptions in 1C metabolism, glycolysis, and the TCA cycle. We found that maternal formate supplementation largely corrects these disruptions in Mthfd1l-null embryos. Serine tracer experiments revealed that Mthfd1l-null MEFs have altered methionine synthesis, indicating that Mthfd1l deletion impairs the methyl cycle. Supplementation of Mthfd1l-null MEFs with formate, hypoxanthine, or combined hypoxanthine and thymidine restored their growth to wildtype levels. Thymidine addition alone was ineffective, suggesting a purine synthesis defect in Mthfd1l-null MEFs. Tracer experiments also revealed lower proportions of labeled hypoxanthine and inosine monophosphate in Mthfd1l-null than in wildtype MEFs, suggesting that Mthfd1l deletion results in increased reliance on the purine salvage pathway. These results indicate that disruptions of mitochondrial 1C metabolism have wide-ranging consequences for many metabolic processes, including those that may not directly interact with 1C metabolism.
Asunto(s)
Aminohidrolasas/genética , Metabolismo Energético , Formiato-Tetrahidrofolato Ligasa/genética , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Redes y Vías Metabólicas , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Mitocondrias/metabolismo , Complejos Multienzimáticos/genética , Defectos del Tubo Neural/genética , Aminohidrolasas/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Ácido Fólico/genética , Ácido Fólico/metabolismo , Formiato-Tetrahidrofolato Ligasa/metabolismo , Formiatos/metabolismo , Glucólisis , Metaboloma , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/patología , Complejos Multienzimáticos/metabolismo , Defectos del Tubo Neural/metabolismo , Defectos del Tubo Neural/patologíaRESUMEN
RNAP II switching from the paused to the productive transcription elongation state is a pivotal regulatory step that requires specific phosphorylations catalyzed by the P-TEFb kinase. Nucleosolic P-TEFb activity is inhibited by its interaction with the ribonuclear protein complex built around the 7SK small nuclear RNA (7SK snRNP). MePCE is the RNA methyltransferase that methylates and stabilizes 7SK in the nucleosol. Here, we report that MePCE also binds chromatin through the histone H4 tail to serve as a P-TEFb activator at specific genes important for cellular identity. Notably, this histone binding abolishes MePCE's RNA methyltransferase activity toward 7SK, which explains why MePCE-bound P-TEFb on chromatin may not be associated with the full 7SK snRNP and is competent for RNAP II activation. Overall, our results suggest that crosstalk between the histone-binding and RNA methylation activities of MePCE regulates P-TEFb activation on chromatin in a 7SK- and Brd4-independent manner.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Histonas/metabolismo , Metiltransferasas/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN/metabolismo , Línea Celular , Cromatina/metabolismo , Humanos , Metilación , Receptor Cross-TalkRESUMEN
Cisplatin derivatives can form various types of DNA lesions (DNA-Pt) and trigger pleiotropic DNA damage responses. Here, we report a strategy to visualize DNA-Pt with high resolution, taking advantage of a novel azide-containing derivative of cisplatin we named APPA, a cellular pre-extraction protocol and the labeling of DNA-Pt by means of click chemistry in cells. Our investigation revealed that pretreating cells with the histone deacetylase (HDAC) inhibitor SAHA led to detectable clusters of DNA-Pt that colocalized with the ubiquitin ligase RAD18 and the replication protein PCNA. Consistent with activation of translesion synthesis (TLS) under these conditions, SAHA and cisplatin cotreatment promoted focal accumulation of the low-fidelity polymerase Polη that also colocalized with PCNA. Remarkably, these cotreatments synergistically triggered mono-ubiquitination of PCNA and apoptosis in a RAD18-dependent manner. Our data provide evidence for a role of chromatin in regulating genome targeting with cisplatin derivatives and associated cellular responses.
Asunto(s)
Antineoplásicos/farmacología , Cromatina/fisiología , Cisplatino/farmacología , Genoma Humano/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/análogos & derivados , Química Clic , ADN/efectos de los fármacos , Daño del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Sondas Moleculares , Antígeno Nuclear de Célula en Proliferación/metabolismo , UbiquitinaciónRESUMEN
Enoxacin is a small molecule that stimulates RNA interference (RNAi) and acts as a growth inhibitor selectively in cancer but not in untransformed cells. Here, we used alkenox, a clickable enoxacin surrogate, coupled with quantitative mass spectrometry, to identify PIWIL3 as a mechanistic target of enoxacin. PIWIL3 is an Argonaute protein of the PIWI subfamily that is mainly expressed in the germline and that mediates RNAi through piRNAs. Our results suggest that cancer cells re-express PIWIL3 to repress RNAi through miRNAs and thus open a new opportunity for cancer-specific targeting.
Asunto(s)
Proteínas Argonautas/análisis , Neoplasias de la Mama/tratamiento farmacológico , Enoxacino/farmacología , Proteínas Argonautas/antagonistas & inhibidores , Proteínas Argonautas/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Enoxacino/química , Femenino , Humanos , Células MCF-7 , Espectrometría de Masas , Estructura MolecularRESUMEN
RNA levels are widely thought to be predictive of RNA function. However, the existence of more than a hundred chemically distinct modifications of RNA alone is a major indication that these moieties may impart distinct functions to subgroups of RNA molecules that share a primary sequence but display distinct RNA "epigenetic" marks. RNAs can be modified on many sites, including 5' and 3' ends, the sugar phosphate backbone, or internal bases, which collectively provide many opportunities for posttranscriptional regulation through a variety of mechanisms. Here, we will focus on how modifications on messenger and microRNAs may affect the process of RNA interference in mammalian cells. We believe that taking RNA modifications into account will not only advance our understanding of this crucial pathway in disease and cancer but will also open the path to exploiting the enzymes that "write" and "erase" them as targets for therapeutic drug development.
Asunto(s)
Interferencia de ARN , Procesamiento Postranscripcional del ARN , Animales , Humanos , Metilación , MicroARNs/fisiología , ARN Mensajero/fisiologíaRESUMEN
Ataxia-telangiectasia mutated (ATM) protein kinase regulates the DNA damage response (DDR) and is associated with cancer suppression. Here we report a cancer-promoting role for ATM. ATM depletion in metastatic cancer cells reduced cell migration and invasion. Transcription analyses identified a gene network, including the chemokine IL-8, regulated by ATM. IL-8 expression required ATM and was regulated by oxidative stress. IL-8 was validated as an ATM target by its ability to rescue cell migration and invasion defects in ATM-depleted cells. Finally, ATM-depletion in human breast cancer cells reduced lung tumors in a mouse xenograft model and clinical data validated IL-8 in lung metastasis. These findings provide insights into how ATM activation by oxidative stress regulates IL-8 to sustain cell migration and invasion in cancer cells to promote metastatic potential. Thus, in addition to well-established roles in tumor suppression, these findings identify a role for ATM in tumor progression.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias de la Mama/metabolismo , Movimiento Celular/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Interleucina-8/metabolismo , Neoplasias Pulmonares/metabolismo , Invasividad Neoplásica/fisiopatología , Estrés Oxidativo/fisiología , Animales , Western Blotting , Fraccionamiento Celular , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Electroforesis en Gel de Poliacrilamida , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiología , Humanos , Luciferasas , Neoplasias Pulmonares/secundario , Ratones , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Due to their short lifespan, rapid division, and ease of genetic manipulation, yeasts are popular model organisms for studying aging in actively dividing cells. To study replicative aging over many cell divisions, individual cells must be continuously separated from their progeny via a laborious manual microdissection procedure. Microfluidics-based soft-lithography devices have recently been used to automate microdissection of the budding yeast Saccharomyces cerevisiae. However, little is known about replicative aging in Schizosaccharomyces pombe, a rod-shaped yeast that divides by binary fission and shares many conserved biological functions with higher eukaryotes. In this report, we develop a versatile multiphoton lithography method that enables rapid fabrication of three-dimensional master structures for polydimethylsiloxane (PDMS)-based microfluidics. We exploit the rapid prototyping capabilities of multiphoton lithography to create and characterize a cell-capture device that is capable of high-resolution microscopic observation of hundreds of individual S. pombe cells. By continuously removing the progeny cells, we demonstrate that cell growth and protein aggregation can be tracked in individual cells for over ~100 h. Thus, the fission yeast lifespan microdissector (FYLM) provides a powerful on-chip microdissection platform that will enable high-throughput studies of aging in rod-shaped cells.