RESUMO
Tumour cell plasticity is a major barrier to the efficacy of targeted cancer therapies but the mechanisms that mediate it are poorly understood. Here, we identify dysregulated RNA splicing as a key driver of tumour cell dedifferentiation in colorectal cancer (CRC). We find that Apc-deficient CRC cells have dysregulated RNA splicing machinery and exhibit global rewiring of RNA splicing. We show that the splicing factor SRSF1 controls the plasticity of tumour cells by controlling Kras splicing and is required for CRC invasion in a mouse model of carcinogenesis. SRSF1 expression maintains stemness in human CRC organoids and correlates with cancer stem cell marker expression in human tumours. Crucially, partial genetic downregulation of Srsf1 does not detrimentally affect normal tissue homeostasis, demonstrating that tumour cell plasticity can be differentially targeted. Thus, our findings link dysregulation of the RNA splicing machinery and control of tumour cell plasticity.
Assuntos
Plasticidade Celular , Neoplasias Colorretais , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Plasticidade Celular/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Camundongos , Splicing de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismoRESUMO
Retrotransposon proliferation poses a threat to germline integrity. While retrotransposons must be activated in developing germ cells in order to survive and propagate, how they are selectively activated in the context of meiosis is unclear. We demonstrate that the transcriptional activation of Ty3/Gypsy retrotransposons and host defense are controlled by master meiotic regulators. We show that budding yeast Ty3/Gypsy co-opts binding sites of the essential meiotic transcription factor Ndt80 upstream of the integration site, thereby tightly linking its transcriptional activation to meiotic progression. We also elucidate how yeast cells thwart Ty3/Gypsy proliferation by blocking translation of the retrotransposon mRNA using amyloid-like assemblies of the RNA-binding protein Rim4. In mammals, several inactive Ty3/Gypsy elements are undergoing domestication. We show that mammals utilize equivalent master meiotic regulators (Stra8, Mybl1, Dazl) to regulate Ty3/Gypsy-derived genes in developing gametes. Our findings inform how genes that are evolving from retrotransposons can build upon existing regulatory networks during domestication.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Germinativas/metabolismo , Meiose/genética , Proteínas de Ligação a RNA/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Retroelementos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Ligação a DNA/genética , Evolução Molecular , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Meiose/fisiologia , Camundongos , Gambás/genética , Gambás/metabolismo , Biossíntese de Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/genética , DNA Polimerase Dirigida por RNA/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
Cilia assembly and disassembly are coupled to actin dynamics, ensuring a coherent cellular response during environmental change. How these processes are integrated remains undefined. The histone lysine demethylase KDM3A plays important roles in organismal homeostasis. Loss-of-function mouse models of Kdm3a phenocopy features associated with human ciliopathies, whereas human somatic mutations correlate with poor cancer prognosis. We demonstrate that absence of KDM3A facilitates ciliogenesis, but these resulting cilia have an abnormally wide range of axonemal lengths, delaying disassembly and accumulating intraflagellar transport (IFT) proteins. KDM3A plays a dual role by regulating actin gene expression and binding to the actin cytoskeleton, creating a responsive "actin gate" that involves ARP2/3 activity and IFT. Promoting actin filament formation rescues KDM3A mutant ciliary defects. Conversely, the simultaneous depolymerization of actin networks and IFT overexpression mimics the abnormal ciliary traits of KDM3A mutants. KDM3A is thus a negative regulator of ciliogenesis required for the controlled recruitment of IFT proteins into cilia through the modulation of actin dynamics.
Assuntos
Actinas/metabolismo , Transporte Biológico/fisiologia , Cílios/fisiologia , Flagelos/fisiologia , Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Animais , Linhagem Celular , Cílios/metabolismo , Flagelos/metabolismo , Expressão Gênica/fisiologia , Humanos , Camundongos , Morfogênese/fisiologia , Mutação/fisiologia , FenótipoRESUMO
BACKGROUND: As with many anti-cancer drugs, the topoisomerase II inhibitor etoposide is considered safe for administration to women in the second and third trimesters of pregnancy, but assessment of effects on the developing fetus have been limited. The purpose of this research was to examine the effect of etoposide on germ cells in the developing ovary. Mouse ovary tissue culture was used as the experimental model, thus allowing us to examine effects of etoposide on all stages of germ cell development in the same way, in vitro. RESULTS: Fetal ovaries from embryonic day 13.5 CD1 mice or neonatal ovaries from postnatal day 0 CD1 mice were cultured with 50-150 ng ml(-1) or 50-200 ng ml(-1) etoposide respectively, concentrations that are low relative to that in patient serum. When fetal ovaries were treated prior to follicle formation, etoposide resulted in dose-dependent damage, with 150 ng ml(-1) inducing a near-complete absence of healthy follicles. In contrast, treatment of neonatal ovaries, after follicle formation, had no effect on follicle numbers and only a minor effect on follicle health, even at 200 ng ml(-1). The sensitivity of female germ cells to etoposide coincided with topoisomerase IIα expression: in the developing ovary of both mouse and human, topoisomerase IIα was expressed in germ cells only prior to follicle formation. CONCLUSIONS: Exposure of pre-follicular ovaries, in which topoisomerase IIα expression was germ cell-specific, resulted in a near-complete elimination of germ cells prior to follicle formation, with the remaining germ cells going on to form unhealthy follicles by the end of culture. In contrast, exposure to follicle-enclosed oocytes, which no longer expressed topoisomerase IIα in the germ cells, had no effect on total follicle numbers or health, the only effect seen specific to transitional follicles. Results indicate the potential for adverse effects on fetal ovarian development if etoposide is administered to pregnant women when germ cells are not yet enclosed within ovarian follicles, a process that starts at approximately 17 weeks gestation and is only complete towards the end of pregnancy.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Diferenciação Celular/efeitos dos fármacos , Etoposídeo/toxicidade , Células Germinativas/patologia , Oócitos/patologia , Folículo Ovariano/patologia , Ovário/patologia , Animais , Células Cultivadas , Feminino , Células Germinativas/efeitos dos fármacos , Camundongos , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacosRESUMO
The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform.
Assuntos
Adenosina Desaminase/metabolismo , Imunidade Inata , Edição de RNA , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Desaminase/genética , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Cruzamentos Genéticos , Citocinas/metabolismo , Perda do Embrião/patologia , Embrião de Mamíferos/patologia , Feminino , Fibroblastos/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Inosina/metabolismo , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação/genética , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/imunologia , Fenótipo , Proteínas de Ligação a RNA/genética , Receptores de Interferon/metabolismo , Análise de Sobrevida , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Uracila/metabolismoRESUMO
The appropriate execution of DNA double-strand break (DSB) repair is critical for genome stability and tumor avoidance. 53BP1 and BRCA1 directly influence DSB repair pathway choice by regulating 5' end resection, but how this is achieved remains uncertain. Here we report that Rif1(-/-) mice are severely compromised for 53BP1-dependent class switch recombination (CSR) and fusion of dysfunctional telomeres. The inappropriate accumulation of RIF1 at DSBs in S phase is antagonized by BRCA1, and deletion of Rif1 suppresses toxic nonhomologous end joining (NHEJ) induced by PARP inhibition in Brca1-deficient cells. Mechanistically, RIF1 is recruited to DSBs via the N-terminal phospho-SQ/TQ domain of 53BP1, and DSBs generated by ionizing radiation or during CSR are hyperresected in the absence of RIF1. Thus, RIF1 and 53BP1 cooperate to block DSB resection to promote NHEJ in G1, which is antagonized by BRCA1 in S phase to ensure a switch of DSB repair mode to homologous recombination.
Assuntos
Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/genética , DNA/metabolismo , Proteínas de Ligação a Telômeros/genética , Animais , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Camundongos , Recombinação Genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded secretory proteins via the ER-associated degradation pathway. This functional diversity of p97 depends on its association with various cofactors, and to further our understanding of p97 function it is important that these cofactors are identified and analyzed. Here, we isolate and characterize the human protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8 is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p97 to the ER membrane for efficient ER-associated degradation.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Melanoma/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Ubiquitina/metabolismo , Adenosina Trifosfatases/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Citoplasma/metabolismo , Degradação Associada com o Retículo Endoplasmático , Eritrócitos/metabolismo , Humanos , Imunoprecipitação , Hibridização In Situ , Melanoma/genética , Ligação Proteica , Dobramento de Proteína , Proteínas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína com ValosinaAssuntos
Meiose , Oócitos/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Quinase do Ponto de Checagem 2 , Proteínas de Ligação a DNA/metabolismo , Feminino , Fase G2 , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
One of the major decisions that germ cells make during their development is whether to differentiate into oocytes or sperm. In mice, the germ cells' decision to develop as male or female depends on sex-determining signalling molecules in the embryonic gonadal environment rather than the sex chromosome constitution of the germ cells themselves. In response to these sex-determining cues, germ cells in female embryos initiate oogenesis and enter meiosis, whereas germ cells in male embryos initiate spermatogenesis and inhibit meiosis until after birth. However, it is not clear whether the signalling molecules that mediate germ cell sex determination act in the developing testis or the developing ovary, or what these signalling molecules might be. Here, we review the evidence for the existence of meiosis-inducing and meiosis-preventing substances in the developing gonad, and more recent studies aimed at identifying these molecules in mice. In addition, we discuss the possibility that some of the reported effects of these factors on germ cell development may be indirect consequences of impairing sexual differentiation of gonadal somatic cells or germ cell survival. Understanding the molecular mechanisms of germ cell sex determination may provide candidate genes for susceptibility to germ cell tumours and infertility in humans.