RESUMO
In eukaryotes, the Suv39 family of proteins tri-methylate lysine 9 of histone H3 (H3K9me) to form constitutive heterochromatin. However, how Suv39 proteins are nucleated at heterochromatin is not fully described. In the fission yeast, current models posit that Argonaute1-associated small RNAs (sRNAs) nucleate the sole H3K9 methyltransferase, Clr4/SUV39H, to centromeres. Here, we show that in the absence of all sRNAs and H3K9me, the Mtl1 and Red1 core (MTREC)/PAXT complex nucleates Clr4/SUV39H at a heterochromatic long noncoding RNA (lncRNA) at which the two H3K9 deacetylases, Sir2 and Clr3, also accumulate by distinct mechanisms. Iterative cycles of H3K9 deacetylation and methylation spread Clr4/SUV39H from the nucleation center in an sRNA-independent manner, generating a basal H3K9me state. This is acted upon by the RNAi machinery to augment and amplify the Clr4/H3K9me signal at centromeres to establish heterochromatin. Overall, our data reveal that lncRNAs and RNA quality control factors can nucleate heterochromatin and function as epigenetic silencers in eukaryotes.
Assuntos
Proteínas de Ciclo Celular , Heterocromatina , Histona-Lisina N-Metiltransferase , Histonas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Metilação , Metiltransferases/metabolismo , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , RNA Fúngico/genética , RNA Interferente Pequeno/genéticaRESUMO
Sneezing is a vital respiratory reflex frequently associated with allergic rhinitis and viral respiratory infections. However, its neural circuit remains largely unknown. A sneeze-evoking region was discovered in both cat and human brainstems, corresponding anatomically to the central recipient zone of nasal sensory neurons. Therefore, we hypothesized that a neuronal population postsynaptic to nasal sensory neurons mediates sneezing in this region. By screening major presynaptic neurotransmitters/neuropeptides released by nasal sensory neurons, we found that neuromedin B (NMB) peptide is essential for signaling sneezing. Ablation of NMB-sensitive postsynaptic neurons in the sneeze-evoking region or deficiency in NMB receptor abolished the sneezing reflex. Remarkably, NMB-sensitive neurons further project to the caudal ventral respiratory group (cVRG). Chemical activation of NMB-sensitive neurons elicits action potentials in cVRG neurons and leads to sneezing behavior. Our study delineates a peptidergic pathway mediating sneezing, providing molecular insights into the sneezing reflex arc.
Assuntos
Tronco Encefálico/fisiopatologia , Neuropeptídeos/metabolismo , Nariz/fisiopatologia , Reflexo/fisiologia , Espirro/fisiologia , Animais , Modelos Animais de Doenças , Hipersensibilidade/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Neurocinina B/análogos & derivados , Neurocinina B/metabolismo , Neurônios/metabolismo , RNA Interferente Pequeno/metabolismo , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPV/metabolismo , Gravação em VídeoRESUMO
Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD+ synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD+ synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 and RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation.
Assuntos
ADP-Ribosilação , Neoplasias Ovarianas/metabolismo , Biossíntese de Proteínas , Proteostase , Ribossomos/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Estresse do Retículo Endoplasmático , Tubas Uterinas/metabolismo , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , NAD/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase , Conformação de Ácido Nucleico , Neoplasias Ovarianas/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Polirribossomos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Ligadas por GPI/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/química , Movimento Celular , Receptor DCC/deficiência , Receptor DCC/genética , Proteínas Ligadas por GPI/química , Cones de Crescimento/fisiologia , Humanos , Ventrículos Laterais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Neurônios/citologia , Neurônios/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with proteins of the PIWI clade of the Argonaute family. First identified in animal germ line cells, piRNAs have essential roles in germ line development. The first function of PIWI-piRNA complexes to be described was the silencing of transposable elements, which is crucial for maintaining the integrity of the germ line genome. Later studies provided new insights into the functions of PIWI-piRNA complexes by demonstrating that they regulate protein-coding genes. Recent studies of piRNA biology, including in new model organisms such as golden hamsters, have deepened our understanding of both piRNA biogenesis and piRNA function. In this Review, we discuss the most recent advances in our understanding of piRNA biogenesis, the molecular mechanisms of piRNA function and the emerging roles of piRNAs in germ line development mainly in flies and mice, and in infertility, cancer and neurological diseases in humans.
Assuntos
Proteínas Argonautas , RNA de Interação com Piwi , Animais , Camundongos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Elementos de DNA Transponíveis , Células Germinativas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Experiences trigger transgenerational small RNA-based responses in C. elegans nematodes. Dedicated machinery ensures that heritable effects are reset, but how the responses segregate in the population is unknown. We show that isogenic individuals differ dramatically in the persistence of transgenerational responses. By examining lineages of more than 20,000 worms, three principles emerge: (1) The silencing each mother initiates is distributed evenly among her descendants; heritable RNAi dissipates but is uniform in every generation. (2) Differences between lineages arise because the mothers that initiate heritable responses stochastically assume different "inheritance states" that determine the progeny's fate. (3) The likelihood that an RNAi response would continue to be inherited increases the more generations it lasts. The inheritance states are determined by HSF-1, which regulates silencing factors and, accordingly, small RNA levels. We found that, based on the parents' inheritance state, the descendants' developmental rate in response to stress can be predicted.
Assuntos
Caenorhabditis elegans/genética , Padrões de Herança/genética , RNA Interferente Pequeno/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Interferência de RNA/fisiologiaRESUMO
Human cerebral cortex size and complexity has increased greatly during evolution. While increased progenitor diversity and enhanced proliferative potential play important roles in human neurogenesis and gray matter expansion, the mechanisms of human oligodendrogenesis and white matter expansion remain largely unknown. Here, we identify EGFR-expressing "Pre-OPCs" that originate from outer radial glial cells (oRGs) and undergo mitotic somal translocation (MST) during division. oRG-derived Pre-OPCs provide an additional source of human cortical oligodendrocyte precursor cells (OPCs) and define a lineage trajectory. We further show that human OPCs undergo consecutive symmetric divisions to exponentially increase the progenitor pool size. Additionally, we find that the OPC-enriched gene, PCDH15, mediates daughter cell repulsion and facilitates proliferation. These findings indicate properties of OPC derivation, proliferation, and dispersion important for human white matter expansion and myelination.
Assuntos
Caderinas/metabolismo , Córtex Cerebral/citologia , Células Ependimogliais/metabolismo , Neurogênese/genética , Células Precursoras de Oligodendrócitos/metabolismo , Proteínas Relacionadas a Caderinas , Caderinas/genética , Proliferação de Células/genética , Células Cultivadas , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Células Ependimogliais/citologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Células Precursoras de Oligodendrócitos/citologia , RNA Interferente Pequeno , RNA-Seq , Análise de Célula Única , Substância Branca/citologia , Substância Branca/embriologia , Substância Branca/metabolismoRESUMO
Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has not been explored. Here, we uncover a function for ribosome collisions in signal transduction. Using translation elongation inhibitors and general cellular stress conditions, including amino acid starvation and UV irradiation, we show that ribosome collisions activate the stress-activated protein kinase (SAPK) and GCN2-mediated stress response pathways. We show that the MAPKKK ZAK functions as the sentinel for ribosome collisions and is required for immediate early activation of both SAPK (p38/JNK) and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that although ZAK generally associates with elongating ribosomes on polysomal mRNAs, it specifically auto-phosphorylates on the minimal unit of colliding ribosomes, the disome. Together, these results provide molecular insights into how perturbation of translational homeostasis regulates cell fate.
Assuntos
Ribossomos/metabolismo , Estresse Fisiológico , Transportadores de Cassetes de Ligação de ATP/metabolismo , Anisomicina/farmacologia , Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Ativação Enzimática , Humanos , MAP Quinase Quinase Quinases/deficiência , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Fosforilação , Polirribossomos/metabolismo , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Raios Ultravioleta , eIF-2 Quinase/metabolismoRESUMO
The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system.
Assuntos
Memória Imunológica/fisiologia , Linfoma Difuso de Grandes Células B/patologia , Proteínas Nucleares/genética , Células Precursoras de Linfócitos B/imunologia , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Cromatina/química , Cromatina/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Histona Desacetilases/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Correpressor 2 de Receptor Nuclear/química , Correpressor 2 de Receptor Nuclear/metabolismo , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
Since the discovery of eukaryotic small RNAs as the main effectors of RNA interference in the late 1990s, diverse types of endogenous small RNAs have been characterized, most notably microRNAs, small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). These small RNAs associate with Argonaute proteins and, through sequence-specific gene regulation, affect almost every major biological process. Intriguing features of small RNAs, such as their mechanisms of amplification, rapid evolution and non-cell-autonomous function, bestow upon them the capacity to function as agents of intercellular communications in development, reproduction and immunity, and even in transgenerational inheritance. Although there are many types of extracellular small RNAs, and despite decades of research, the capacity of these molecules to transmit signals between cells and between organisms is still highly controversial. In this Review, we discuss evidence from different plants and animals that small RNAs can act in a non-cell-autonomous manner and even exchange information between species. We also discuss mechanistic insights into small RNA communications, such as the nature of the mobile agents, small RNA signal amplification during transit, signal perception and small RNA activity at the destination.
Assuntos
Proteínas Argonautas , MicroRNAs , RNA Interferente Pequeno , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Plantas/genética , Plantas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
RNA silencing is a well-established antiviral immunity system in plants, in which small RNAs guide Argonaute proteins to targets in viral RNA or DNA, resulting in virus repression. Virus-encoded suppressors of silencing counteract this defence system. In this Review, we discuss recent findings about antiviral RNA silencing, including the movement of RNA through plasmodesmata and the differentiation between plant self and viral RNAs. We also discuss the emerging role of RNA silencing in plant immunity against non-viral pathogens. This immunity is mediated by transkingdom movement of RNA into and out of the infected plant cells in vesicles or as extracellular nucleoproteins and, like antiviral immunity, is influenced by the silencing suppressors encoded in the pathogens' genomes. Another effect of RNA silencing on general immunity involves host-encoded small RNAs, including microRNAs, that regulate NOD-like receptors and defence signalling pathways in the innate immunity system of plants. These RNA silencing pathways form a network of processes with both positive and negative effects on the immune systems of plants.
Assuntos
MicroRNAs , RNA Viral , Antivirais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Resistência à Doença/genética , MicroRNAs/genética , Proteínas NLR/genética , Proteínas NLR/metabolismo , Doenças das Plantas/genética , Imunidade Vegetal/genética , Plantas/genética , Interferência de RNA , RNA de Plantas , RNA Interferente Pequeno/metabolismoRESUMO
PIWI-interacting small RNAs (piRNAs) establish sequence-specific adaptive restriction of resident genomic parasites to guard genome integrity. In this issue of Cell, Yu, Koppetsch, et al. describe an innate piRNA-response that specifically fragments the viral RNA genome in the germline of recently invaded koalas. This first line of defense might ensure survival until adaptive immunity develops.
Assuntos
Phascolarctidae , Animais , Genoma , Células Germinativas , RNA Interferente PequenoRESUMO
Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.
Assuntos
Gammaretrovirus/genética , Phascolarctidae/genética , RNA Interferente Pequeno/genética , Animais , Elementos de DNA Transponíveis , Gammaretrovirus/metabolismo , Gammaretrovirus/patogenicidade , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Genoma , Células Germinativas/metabolismo , Células Germinativas/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Phascolarctidae/virologia , Splicing de RNA , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Spermiogenesis is a highly orchestrated developmental process during which chromatin condensation decouples transcription from translation. Spermiogenic mRNAs are transcribed earlier and stored in a translationally inert state until needed for translation; however, it remains largely unclear how such repressed mRNAs become activated during spermiogenesis. We previously reported that the MIWI/piRNA machinery is responsible for mRNA elimination during late spermiogenesis in preparation for spermatozoa production. Here we unexpectedly discover that the same machinery is also responsible for activating translation of a subset of spermiogenic mRNAs to coordinate with morphological transformation into spermatozoa. Such action requires specific base-pairing interactions of piRNAs with target mRNAs in their 3' UTRs, which activates translation through coupling with cis-acting AU-rich elements to nucleate the formation of a MIWI/piRNA/eIF3f/HuR super-complex in a developmental stage-specific manner. These findings reveal a critical role of the piRNA system in translation activation, which we show is functionally required for spermatid development.
Assuntos
Proteínas Argonautas/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA Interferente Pequeno/metabolismo , Espermatogênese , Regiões 3' não Traduzidas , Animais , Proteínas Argonautas/genética , Pareamento de Bases , Células Cultivadas , Proteína Semelhante a ELAV 1/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
The RNA-directed DNA methylation (RdDM) pathway in plants controls gene expression via cytosine DNA methylation. The ability to manipulate RdDM would shed light on the mechanisms and applications of DNA methylation to control gene expression. Here, we identified diverse RdDM proteins that are capable of targeting methylation and silencing in Arabidopsis when tethered to an artificial zinc finger (ZF-RdDM). We studied their order of action within the RdDM pathway by testing their ability to target methylation in different mutants. We also evaluated ectopic siRNA biogenesis, RNA polymerase V (Pol V) recruitment, targeted DNA methylation, and gene-expression changes at thousands of ZF-RdDM targets. We found that co-targeting both arms of the RdDM pathway, siRNA biogenesis and Pol V recruitment, dramatically enhanced targeted methylation. This work defines how RdDM components establish DNA methylation and enables new strategies for epigenetic gene regulation via targeted DNA methylation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Metilação de DNA/fisiologia , RNA Polimerases Dirigidas por DNA/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Citosina/metabolismo , DNA/metabolismo , Metilação de DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação da Expressão Gênica de Plantas/genética , RNA Polimerase II/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/metabolismoRESUMO
Epitranscriptomic regulation controls information flow through the central dogma and provides unique opportunities for manipulating cells at the RNA level. However, both fundamental studies and potential translational applications are impeded by a lack of methods to target specific RNAs with effector proteins. Here, we present CRISPR-Cas-inspired RNA targeting system (CIRTS), a protein engineering strategy for constructing programmable RNA control elements. We show that CIRTS is a simple and generalizable approach to deliver a range of effector proteins, including nucleases, degradation machinery, translational activators, and base editors to target transcripts. We further demonstrate that CIRTS is not only smaller than naturally occurring CRISPR-Cas programmable RNA binding systems but can also be built entirely from human protein parts. CIRTS provides a platform to probe fundamental RNA regulatory processes, and the human-derived nature of CIRTS provides a potential strategy to avoid immune issues when applied to epitranscriptome-modulating therapies.
Assuntos
Edição de Genes/métodos , Engenharia de Proteínas/métodos , RNA Guia de Cinetoplastídeos/metabolismo , RNA/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Sistemas CRISPR-Cas/genética , Escherichia coli/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Biossíntese de Proteínas , Proteólise , RNA Interferente Pequeno , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , TransfecçãoRESUMO
PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30 nt piRNAs are processed in the cytoplasm from long non-coding RNAs that often lack RNA processing hallmarks of export-competent transcripts. By studying how these transcripts achieve nuclear export, we uncover an RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1 and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. These findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to export unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Heterocromatina/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas Argonautas/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Helicases DEAD-box/metabolismo , Elementos de DNA Transponíveis , Inativação Gênica , Células Germinativas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Gênica , Proteína Exportina 1RESUMO
Although human genetic studies have implicated many susceptible genes associated with plasma lipid levels, their physiological and molecular functions are not fully characterized. Here we demonstrate that orphan G protein-coupled receptor 146 (GPR146) promotes activity of hepatic sterol regulatory element binding protein 2 (SREBP2) through activation of the extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating hepatic very low-density lipoprotein (VLDL) secretion, and subsequently circulating low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) levels. Remarkably, GPR146 deficiency reduces plasma cholesterol levels substantially in both wild-type and LDL receptor (LDLR)-deficient mice. Finally, aortic atherosclerotic lesions are reduced by 90% and 70%, respectively, in male and female LDLR-deficient mice upon GPR146 depletion. Taken together, these findings outline a regulatory role for the GPR146/ERK axis in systemic cholesterol metabolism and suggest that GPR146 inhibition could be an effective strategy to reduce plasma cholesterol levels and atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Hipercolesterolemia/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Animais , Aterosclerose/sangue , Sequência de Bases , Colesterol/sangue , Dependovirus/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Jejum , Feminino , Hepatócitos/metabolismo , Humanos , Hipercolesterolemia/sangue , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de LDL/metabolismo , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Triglicerídeos/sangue , Regulação para CimaRESUMO
Insulin receptor (IR) signaling is central to normal metabolic control and dysregulated in prevalent chronic diseases. IR binds insulin at the cell surface and transduces rapid signaling via cytoplasmic kinases. However, mechanisms mediating long-term effects of insulin remain unclear. Here, we show that IR associates with RNA polymerase II in the nucleus, with striking enrichment at promoters genome-wide. The target genes were highly enriched for insulin-related functions including lipid metabolism and protein synthesis and diseases including diabetes, neurodegeneration, and cancer. IR chromatin binding was increased by insulin and impaired in an insulin-resistant disease model. Promoter binding by IR was mediated by coregulator host cell factor-1 (HCF-1) and transcription factors, revealing an HCF-1-dependent pathway for gene regulation by insulin. These results show that IR interacts with transcriptional machinery at promoters and identify a pathway regulating genes linked to insulin's effects in physiology and disease.
Assuntos
Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Receptor de Insulina/metabolismo , Animais , Linhagem Celular Tumoral , Cromatina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fator C1 de Célula Hospedeira/antagonistas & inibidores , Fator C1 de Célula Hospedeira/genética , Fator C1 de Célula Hospedeira/metabolismo , Humanos , Insulina/metabolismo , Insulina/farmacologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ligação Proteica , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/metabolismo , Receptor de Insulina/química , Transdução de Sinais/efeitos dos fármacosRESUMO
Chemotherapy is designed to induce cell death. However, at non-lethal doses, cancer cells can choose to remain proliferative or become senescent. The slow development of senescence makes studying this decision challenging. Here, by analyzing single-cell p21 dynamics before, during, and days after drug treatment, we link three distinct patterns of early p21 dynamics to final cell fate. Surprisingly, while high p21 expression is classically associated with senescence, we find the opposite at early times during drug treatment: most senescence-fated cells express much lower p21 levels than proliferation-fated cells. We demonstrate that these dynamics lead to a p21 "Goldilocks zone" for proliferation, in which modest increases of p21 expression can lead to an undesirable increase of cancer cell proliferation. Our study identifies a counter-intuitive role for early p21 dynamics in the cell-fate decision and pinpoints a source of proliferative cancer cells that can emerge after exposure to non-lethal doses of chemotherapy.