RESUMO
Posttranscriptional control of mRNA regulates various biological processes, including inflammatory and immune responses. RNA-binding proteins (RBPs) bind cis-regulatory elements in the 3' untranslated regions (UTRs) of mRNA and regulate mRNA turnover and translation. In particular, eight RBPs (TTP, AUF1, KSRP, TIA-1/TIAR, Roquin, Regnase, HuR, and Arid5a) have been extensively studied and are key posttranscriptional regulators of inflammation and immune responses. These RBPs sometimes collaboratively or competitively bind the same target mRNA to enhance or dampen regulatory activities. These RBPs can also bind their own 3' UTRs to negatively or positively regulate their expression. Both upstream signaling pathways and microRNA regulation shape the interactions between RBPs and target RNA. Dysregulation of RBPs results in chronic inflammation and autoimmunity. Here, we summarize the functional roles of these eight RBPs in immunity and their associated diseases.
Assuntos
MicroRNAs , Estabilidade de RNA , Animais , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The reciprocal coordination between cholesterol absorption in the intestine and de novo cholesterol synthesis in the liver is essential for maintaining cholesterol homeostasis, yet the mechanisms governing the opposing regulation of these processes remain poorly understood. Here, we identify a hormone, Cholesin, which is capable of inhibiting cholesterol synthesis in the liver, leading to a reduction in circulating cholesterol levels. Cholesin is encoded by a gene with a previously unknown function (C7orf50 in humans; 3110082I17Rik in mice). It is secreted from the intestine in response to cholesterol absorption and binds to GPR146, an orphan G-protein-coupled receptor, exerting antagonistic downstream effects by inhibiting PKA signaling and thereby suppressing SREBP2-controlled cholesterol synthesis in the liver. Therefore, our results demonstrate that the Cholesin-GPR146 axis mediates the inhibitory effect of intestinal cholesterol absorption on hepatic cholesterol synthesis. This discovered hormone, Cholesin, holds promise as an effective agent in combating hypercholesterolemia and atherosclerosis.
Assuntos
Colesterol , Hormônios , Proteínas de Ligação a RNA , Animais , Humanos , Camundongos , Colesterol/metabolismo , Hormônios/genética , Hormônios/metabolismo , Hipercolesterolemia/metabolismo , Fígado/metabolismo , Transdução de Sinais , Proteínas de Ligação a RNA/metabolismoRESUMO
Glucose is the main source of energy for cells. In this issue of Cell, a study now shows that glucose has additional non-energetic functions, acting as a biomolecular cue that regulates alternative splicing during epidermal differentiation. As keratinocytes differentiate, glucose associates with RNA-binding protein DDX21 and modulates its interaction properties, which modifies splicing decisions.
Assuntos
Processamento Alternativo , Splicing de RNA , Diferenciação Celular , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , GlucoseRESUMO
Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.
Assuntos
Proteínas de Ligação a RNA , Transativadores , Proteínas de Transporte/metabolismo , Fatores de Alongamento de Peptídeos/genética , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Humanos , Células HeLa , Células HEK293 , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismoRESUMO
Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E. coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.
Assuntos
Escherichia coli , Organelas , Escherichia coli/genética , Organelas/metabolismo , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Solventes/análise , Solventes/metabolismoRESUMO
Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Bases , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Mutação/genética , Motivos de Nucleotídeos/genética , Transição de Fase , Mutação Puntual/genética , Poli A/metabolismo , Ligação Proteica , Multimerização Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de SequênciaRESUMO
The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an in vitro system to silence translation in purified mitochondria. In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.
Assuntos
Regulação da Expressão Gênica , Inativação Gênica , Genes Mitocondriais , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Proteínas Mitocondriais/metabolismo , Oligonucleotídeos/química , Fosforilação Oxidativa , Biossíntese de Proteínas , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Understanding of the RNA virus and its interactions with host proteins could improve therapeutic interventions for COVID-19. By using icSHAPE, we determined the structural landscape of SARS-CoV-2 RNA in infected human cells and from refolded RNAs, as well as the regulatory untranslated regions of SARS-CoV-2 and six other coronaviruses. We validated several structural elements predicted in silico and discovered structural features that affect the translation and abundance of subgenomic viral RNAs in cells. The structural data informed a deep-learning tool to predict 42 host proteins that bind to SARS-CoV-2 RNA. Strikingly, antisense oligonucleotides targeting the structural elements and FDA-approved drugs inhibiting the SARS-CoV-2 RNA binding proteins dramatically reduced SARS-CoV-2 infection in cells derived from human liver and lung tumors. Our findings thus shed light on coronavirus and reveal multiple candidate therapeutics for COVID-19 treatment.
Assuntos
Tratamento Farmacológico da COVID-19 , Reposicionamento de Medicamentos , RNA Viral , Proteínas de Ligação a RNA/antagonistas & inibidores , SARS-CoV-2 , Animais , Linhagem Celular , Chlorocebus aethiops , Aprendizado Profundo , Humanos , Conformação de Ácido Nucleico , RNA Viral/química , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genéticaRESUMO
Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Fosfatase 2/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Fosforilação , Ligação Proteica , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Especificidade por SubstratoRESUMO
SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.
Assuntos
Interações Hospedeiro-Patógeno , RNA Viral/genética , SARS-CoV-2/genética , Animais , COVID-19/virologia , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Genoma Viral , Humanos , Pulmão/virologia , Masculino , Espectrometria de Massas , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/ultraestrutura , Células VeroRESUMO
Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.
Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/química , Microscopia Crioeletrônica , RNA Mensageiro/química , RNA Viral/química , SARS-CoV-2/química , Proteínas do Complexo da Replicase Viral/química , Sequência de Aminoácidos , Coronavirus/química , Coronavirus/classificação , Coronavirus/enzimologia , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Metiltransferases/metabolismo , Modelos Moleculares , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/enzimologia , Alinhamento de Sequência , Transcrição Gênica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.
Assuntos
Núcleo Celular/metabolismo , RNA/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Homólogo 5 da Proteína Cromobox/metabolismo , Cromossomos/metabolismo , DNA/metabolismo , DNA Satélite/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dactinomicina/farmacologia , Feminino , Genoma , Células HEK293 , Heterocromatina/metabolismo , Humanos , Camundongos , Modelos Biológicos , Família Multigênica , RNA Polimerase II/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Estrogen receptor α (ERα) is a hormone receptor and key driver for over 70% of breast cancers that has been studied for decades as a transcription factor. Unexpectedly, we discover that ERα is a potent non-canonical RNA-binding protein. We show that ERα RNA binding function is uncoupled from its activity to bind DNA and critical for breast cancer progression. Employing genome-wide cross-linking immunoprecipitation (CLIP) sequencing and a functional CRISPRi screen, we find that ERα-associated mRNAs sustain cancer cell fitness and elicit cellular responses to stress. Mechanistically, ERα controls different steps of RNA metabolism. In particular, we demonstrate that ERα RNA binding mediates alternative splicing of XBP1 and translation of the eIF4G2 and MCL1 mRNAs, which facilitates survival upon stress conditions and sustains tamoxifen resistance of cancer cells. ERα is therefore a multifaceted RNA-binding protein, and this activity transforms our knowledge of post-transcriptional regulation underlying cancer development and drug response.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/química , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica , Humanos , Camundongos Endogâmicos NOD , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Oncogenes , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Tamoxifeno/farmacologia , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , DNA Helicases/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Esclerose Tuberosa/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/química , Evolução Molecular , Feminino , Humanos , Insulina/farmacologia , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose/química , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/metabolismoRESUMO
Facultative heterochromatin (fHC) concerns the developmentally regulated heterochromatinization of different regions of the genome and, in the case of the mammalian X chromosome and imprinted loci, of only one allele of a homologous pair. The formation of fHC participates in the timely repression of genes, by resisting strong trans activators. In this review, we discuss the molecular mechanisms underlying the establishment and maintenance of fHC in mammals using a mouse model. We focus on X-chromosome inactivation (XCI) as a paradigm for fHC but also relate it to genomic imprinting and homeobox (Hox) gene cluster repression. A vital role for noncoding transcription and/or transcripts emerges as the general principle of triggering XCI and canonical imprinting. However, other types of fHC are established through an unknown mechanism, independent of noncoding transcription (Hox clusters and noncanonical imprinting). We also extensively discuss polycomb-group repressive complexes (PRCs), which frequently play a vital role in fHC maintenance.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Heterocromatina/metabolismo , Proteínas do Grupo Polycomb/genética , Inativação do Cromossomo X , Cromossomo X/metabolismo , Animais , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Feminino , Inativação Gênica , Heterocromatina/química , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espermatozoides/citologia , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/metabolismo , Cromossomo X/químicaRESUMO
Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the spliceosome stabilize the conformation of the snRNA, drive spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.
Assuntos
Fatores de Processamento de RNA/genética , Splicing de RNA , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Spliceossomos/metabolismo , Domínio Catalítico , Sequência Conservada , Éxons , Humanos , Íntrons , Modelos Moleculares , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/genética , Spliceossomos/ultraestruturaRESUMO
Despite tumor-associated macrophages (TAMs) playing a key role in shaping the tumor microenvironment (TME), the mechanisms by which TAMs influence the TME and contribute to cancer progression remain unclear. Here, we show that the N6-methyladenosine reader YTHDF2 regulates the antitumor functions of TAMs. YTHDF2 deficiency in TAMs suppressed tumor growth by reprogramming TAMs toward an antitumoral phenotype and increasing their antigen cross-presentation ability, which in turn enhanced CD8+ T cell-mediated antitumor immunity. YTHDF2 deficiency facilitated the reprogramming of TAMs by targeting interferon-γ-STAT1 signaling. The expression of YTHDF2 in TAMs was regulated by interleukin-10-STAT3 signaling. Selectively targeting YTHDF2 in TAMs using a Toll-like receptor 9 agonist-conjugated small interfering RNA reprogrammed TAMs toward an antitumoral phenotype, restrained tumor growth and enhanced the efficacy of PD-L1 antibody therapy. Collectively, our findings describe the role of YTHDF2 in orchestrating TAMs and suggest that YTHDF2 inhibition is an effective approach to enhance cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Humanos , Macrófagos , Macrófagos Associados a Tumor , Neoplasias/metabolismo , Imunoterapia , Microambiente Tumoral , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
N6-methyladenosine (m6A) is the most abundant mRNA nucleotide modification and regulates critical aspects of cellular physiology and differentiation. m6A is thought to mediate its effects through a complex network of interactions between different m6A sites and three functionally distinct cytoplasmic YTHDF m6A-binding proteins (DF1, DF2, and DF3). In contrast to the prevailing model, we show that DF proteins bind the same m6A-modified mRNAs rather than different mRNAs. Furthermore, we find that DF proteins do not induce translation in HeLa cells. Instead, the DF paralogs act redundantly to mediate mRNA degradation and cellular differentiation. The ability of DF proteins to regulate stability and differentiation becomes evident only when all three DF paralogs are depleted simultaneously. Our study reveals a unified model of m6A function in which all m6A-modified mRNAs are subjected to the combined action of YTHDF proteins in proportion to the number of m6A sites.
Assuntos
Adenosina/análogos & derivados , Proteínas de Ligação a RNA/metabolismo , Adenosina/genética , Adenosina/metabolismo , Diferenciação Celular , Células HeLa , Humanos , Metilação , Metiltransferases/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand ß-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes.
Assuntos
Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Proteínas de Ligação a RNA/ultraestrutura , Fatores de Transcrição/ultraestrutura , Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Citocromos/metabolismo , Transporte de Elétrons/fisiologia , Elétrons , Heme/metabolismo , Complexos Multiproteicos/ultraestrutura , Oxirredução , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Influenza A virus (IAV) is a lytic RNA virus that triggers receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated pathways of apoptosis and mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necroptosis in infected cells. ZBP1 initiates RIPK3-driven cell death by sensing IAV RNA and activating RIPK3. Here, we show that replicating IAV generates Z-RNAs, which activate ZBP1 in the nucleus of infected cells. ZBP1 then initiates RIPK3-mediated MLKL activation in the nucleus, resulting in nuclear envelope disruption, leakage of DNA into the cytosol, and eventual necroptosis. Cell death induced by nuclear MLKL was a potent activator of neutrophils, a cell type known to drive inflammatory pathology in virulent IAV disease. Consequently, MLKL-deficient mice manifest reduced nuclear disruption of lung epithelia, decreased neutrophil recruitment into infected lungs, and increased survival following a lethal dose of IAV. These results implicate Z-RNA as a new pathogen-associated molecular pattern and describe a ZBP1-initiated nucleus-to-plasma membrane "inside-out" death pathway with potentially pathogenic consequences in severe cases of influenza.