RESUMO
GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here, we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology.
RESUMO
Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.
Assuntos
RNA Helicases DEAD-box , Glucose , Queratinócitos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glucose/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , HumanosRESUMO
Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These "glycoRNAs" were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.
Assuntos
Membrana Celular/metabolismo , Polissacarídeos/metabolismo , RNA/metabolismo , Animais , Anticorpos/metabolismo , Sequência de Bases , Vias Biossintéticas , Linhagem Celular , Sobrevivência Celular , Humanos , Espectrometria de Massas , Ácido N-Acetilneuramínico/metabolismo , Poliadenilação , Polissacarídeos/química , RNA/química , RNA/genética , RNA não Traduzido/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Coloração e RotulagemRESUMO
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
During eukaryotic evolution, ribosomes have considerably increased in size, forming a surface-exposed ribosomal RNA (rRNA) shell of unknown function, which may create an interface for yet uncharacterized interacting proteins. To investigate such protein interactions, we establish a ribosome affinity purification method that unexpectedly identifies hundreds of ribosome-associated proteins (RAPs) from categories including metabolism and cell cycle, as well as RNA- and protein-modifying enzymes that functionally diversify mammalian ribosomes. By further characterizing RAPs, we discover the presence of ufmylation, a metazoan-specific post-translational modification (PTM), on ribosomes and define its direct substrates. Moreover, we show that the metabolic enzyme, pyruvate kinase muscle (PKM), interacts with sub-pools of endoplasmic reticulum (ER)-associated ribosomes, exerting a non-canonical function as an RNA-binding protein in the translation of ER-destined mRNAs. Therefore, RAPs interconnect one of life's most ancient molecular machines with diverse cellular processes, providing an additional layer of regulatory potential to protein expression.
Assuntos
Ribossomos/química , Ribossomos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células-Tronco Embrionárias/metabolismo , Retículo Endoplasmático/metabolismo , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Camundongos , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Ribossômicas/metabolismo , Hormônios Tireóideos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ligação a Hormônio da TireoideRESUMO
RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo M , Íntrons , Elementos Nucleotídeos Longos e Dispersos , Splicing de RNA , RNA de Cadeia Dupla , Humanos , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Interferons/metabolismo , Interferons/genética , Animais , Células HEK293 , Camundongos , Transcriptoma , Éxons , Sítios de Splice de RNA , Elementos Alu/genéticaRESUMO
RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.
Assuntos
Ficusina/química , RNA de Cadeia Dupla/química , Animais , Pareamento de Bases , Células HEK293 , Células HeLa , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , RNA Longo não Codificante/químicaRESUMO
The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.
Assuntos
Inativação Gênica , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Transcrição Gênica , Genoma/genética , RNARESUMO
DEAD-box ATPases are major regulators of biomolecular condensates and orchestrate diverse biochemical processes that are critical for the functioning of cells. How DEAD-box proteins are selectively recruited to their respective biomolecular condensates is unknown. We explored this in the context of the nucleolus and DEAD-box protein DDX21. We find that the pH of the nucleolus is intricately linked to the transcriptional activity of the organelle and facilitates the recruitment and condensation of DDX21. We identify an evolutionarily conserved feature of the C terminus of DDX21 responsible for nucleolar localization. This domain is essential for zebrafish development, and its intrinsically disordered and isoelectric properties are necessary and sufficient for the ability of DDX21 to respond to changes in pH and form condensates. Molecularly, the enzymatic activities of poly(ADP-ribose) polymerases contribute to maintaining the nucleolar pH and, consequently, DDX21 recruitment and nucleolar partitioning. These observations reveal an activity-dependent physicochemical mechanism for the selective recruitment of biochemical activities to biomolecular condensates.
Assuntos
RNA Helicases DEAD-box , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/química , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Organelas/metabolismo , Concentração de Íons de HidrogênioRESUMO
Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing.
Assuntos
Espectrometria de Massas/métodos , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/metabolismo , Animais , Cromatina/metabolismo , Feminino , Inativação Gênica , Humanos , Camundongos , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/análiseRESUMO
Discovering the interaction mechanism and location of RNA-binding proteins (RBPs) on RNA is critical for understanding gene expression regulation. Here, we apply selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) on in vivo transcripts compared to protein-absent transcripts in four human cell lines to identify transcriptome-wide footprints (fSHAPE) on RNA. Structural analyses indicate that fSHAPE precisely detects nucleobases that hydrogen bond with protein. We demonstrate that fSHAPE patterns predict binding sites of known RBPs, such as iron response elements in both known loci and previously unknown loci in CDC34, SLC2A4RG, COASY, and H19. Furthermore, by integrating SHAPE and fSHAPE with crosslinking and immunoprecipitation (eCLIP) of desired RBPs, we interrogate specific RNA-protein complexes, such as histone stem-loop elements and their nucleotides that hydrogen bond with stem-loop-binding proteins. Together, these technologies greatly expand our ability to study and understand specific cellular RNA interactions in RNA-protein complexes.
Assuntos
Conformação de Ácido Nucleico , Proteínas de Ligação a RNA/química , RNA/química , Transcriptoma , Células HeLa , Células Hep G2 , Humanos , Ligação de Hidrogênio , Imunoprecipitação , Células K562RESUMO
The subcellular localization of a biopolymer often informs its function. RNA is traditionally confined to the cytosolic and nuclear spaces, where it plays critical and conserved roles across nearly all biochemical processes. Our recent observation of cell surface glycoRNAs may further explain the extracellular role of RNA. While cellular membranes are efficient gatekeepers of charged polymers such as RNAs, a large body of research has demonstrated the accumulation of specific RNA species outside of the cell, termed extracellular RNAs (exRNAs). Across various species and forms of life, protein pores have evolved to transport RNA across membranes, thus providing a mechanistic path for exRNAs to achieve their extracellular topology. Here, we review types of exRNAs and the pores capable of RNA transport to provide a logical and testable path toward understanding the biogenesis and regulation of cell surface glycoRNAs.
Assuntos
Polímeros , RNA , Humanos , RNA/genética , Membrana Celular , Membranas , CitosolRESUMO
The DNA-dependent protein kinase (DNA-PK), which comprises the KU heterodimer and a catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor1. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. KU also binds to RNA, but the relevance of this interaction in mammals is unclear. Here we use mouse models to show that DNA-PK has an unexpected role in the biogenesis of ribosomal RNA (rRNA) and in haematopoiesis. The expression of kinase-dead DNA-PKcs abrogates cNHEJ2. However, most mice that both expressed kinase-dead DNA-PKcs and lacked the tumour suppressor TP53 developed myeloid disease, whereas all other previously characterized mice deficient in both cNHEJ and TP53 expression succumbed to pro-B cell lymphoma3. DNA-PK autophosphorylates DNA-PKcs, which is its best characterized substrate. Blocking the phosphorylation of DNA-PKcs at the T2609 cluster, but not the S2056 cluster, led to KU-dependent defects in 18S rRNA processing, compromised global protein synthesis in haematopoietic cells and caused bone marrow failure in mice. KU drives the assembly of DNA-PKcs on a wide range of cellular RNAs, including the U3 small nucleolar RNA, which is essential for processing of 18S rRNA4. U3 activates purified DNA-PK and triggers phosphorylation of DNA-PKcs at T2609. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit processome. Together our data show that DNA-PK has RNA-dependent, cNHEJ-independent functions during ribosome biogenesis that require the kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Hematopoese/genética , Autoantígeno Ku/metabolismo , Linfoma/enzimologia , Linfoma/fisiopatologia , RNA Ribossômico 18S/metabolismo , Proteínas de Ligação ao Cálcio/genética , Domínio Catalítico/fisiologia , Reparo do DNA/genética , Ativação Enzimática/genética , Células HeLa , Humanos , Linfoma/genética , Modelos Animais , Mutação , Fosforilação , Ligação Proteica , Biossíntese de Proteínas/genética , RNA Ribossômico 18S/genética , RNA Nucleolar Pequeno/metabolismoRESUMO
The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.
Assuntos
COVID-19/virologia , Pulmão/citologia , Modelos Biológicos , Organoides/citologia , Organoides/virologia , SARS-CoV-2/fisiologia , Técnicas de Cultura de Tecidos , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , COVID-19/metabolismo , COVID-19/patologia , Diferenciação Celular , Divisão Celular , Células Clonais/citologia , Células Clonais/metabolismo , Células Clonais/virologia , Humanos , Técnicas In Vitro , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/fisiologia , Integrina alfa6/análise , Integrina beta4/análise , Queratina-5/análise , Organoides/metabolismo , Pneumonia Viral/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2/crescimento & desenvolvimento , Análise de Célula Única , Receptor de TWEAK/análiseRESUMO
Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.
Assuntos
Células-Tronco Neurais , Infecção por Zika virus , Zika virus , Humanos , Encéfalo/metabolismo , Encéfalo/virologia , Células-Tronco Neurais/virologia , RNA Helicases/genética , RNA Helicases/metabolismo , Transativadores/metabolismo , Replicação Viral , Zika virus/fisiologia , Infecção por Zika virus/genéticaRESUMO
Recruitment of the RNA polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA-binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, whereas others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer.
Assuntos
Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Proliferação de Células , Imunoprecipitação da Cromatina , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Fatores de Elongação da TranscriçãoRESUMO
Galectin-3, well characterized as a glycan binding protein, has been identified as a putative RNA binding protein, possibly through participation in pre-mRNA maturation through interactions with splicosomes. Given recent developments with cell surface RNA biology, the putative dual-function nature of galectin-3 evokes a possible non-classical connection between glycobiology and RNA biology. However, with limited functional evidence of a direct RNA interaction, many molecular-level observations rely on affinity reagents and lack appropriate genetic controls. Thus, evidence of a direct interaction remains elusive. We demonstrate that antibodies raised to endogenous human galectin-3 can isolate RNA-protein crosslinks, but this activity remains insensitive to LGALS3 knock-out. Proteomic characterization of anti-galectin-3 IPs revealed enrichment of galectin-3, but high abundance of hnRNPA2B1, an abundant, well-characterized RNA-binding protein with weak homology to the N-terminal domain of galectin-3, in the isolate. Genetic ablation of HNRNPA2B1, but not LGALS3, eliminates the ability of the anti-galectin-3 antibodies to isolate RNA-protein crosslinks, implying either an indirect interaction or cross-reactivity. To address this, we introduced an epitope tag to the endogenous C-terminal locus of LGALS3. Isolation of the tagged galectin-3 failed to reveal any RNA-protein crosslinks. This result suggests that the galectin-3 does not directly interact with RNA and may be misidentified as an RNA-binding protein, at least in HeLa where the putative RNA associations were first identified. We encourage further investigation of this phenomenon employ gene deletions and, when possible, endogenous epitope tags to achieve the specificity required to evaluate potential interactions.
Assuntos
Galectina 3 , RNA , Humanos , Epitopos , Galectina 3/genética , Galectina 3/metabolismo , Galectinas/metabolismo , Proteômica , Proteínas de Ligação a RNARESUMO
Arthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins Sec61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both Sec61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3' end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells.
Assuntos
Culicidae , Dengue , Flavivirus , Animais , Flavivirus/fisiologia , Mamíferos , RNA Viral/genética , RNA Viral/metabolismo , Replicação ViralRESUMO
Despite advances in the field, chronic graft-versus-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. Because treatment options remain limited, we tested efficacy of anticancer, chromatin-modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel enhancer of zeste homolog 2 (EZH2) inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 each improved pulmonary function; impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis; and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells, we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the 2 drugs could lead to the same physiological improvements while targeting unique epigenetic processes, we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5- but not JQ1-treated mice were enriched for proproliferative pathways also seen in GCBs from bone marrow-only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data, these insights led us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO.
Assuntos
Bronquiolite Obliterante , Proteína Potenciadora do Homólogo 2 de Zeste , Centro Germinativo , Doença Enxerto-Hospedeiro , Proteínas , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/patologia , Bronquiolite Obliterante/genética , Bronquiolite Obliterante/metabolismo , Bronquiolite Obliterante/patologia , Doença Crônica , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Inibidores Enzimáticos/farmacologia , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/patologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Humanos , Camundongos , Proteínas/metabolismo , TranscriptomaRESUMO
Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations.