RESUMEN
mRNA vaccines against SARS-CoV-2 have revolutionized vaccine development, but their immunological mechanisms are not fully understood. Here, we investigate injection site responses of mRNA vaccines by generating a comprehensive single-cell transcriptome profile upon lipid nanoparticle (LNP) or LNP-mRNA challenge in female BALB/c mice. We show that LNP-induced stromal pro-inflammatory responses and mRNA-elicited type I interferon responses dominate the initial injection site responses. By tracking the fate of delivered mRNA, we discover that injection site fibroblasts are highly enriched with the delivered mRNA and that they express IFN-ß specifically in response to the mRNA component, not to the LNP component of mRNA vaccines. Moreover, the mRNA-LNP, but not LNP alone, induces migratory dendritic cells highly expressing IFN-stimulated genes (mDC_ISGs) at the injection site and draining lymph nodes. When co-injected with LNP-subunit vaccine, IFN-ß induces mDC_ISGs at the injection site, and importantly, it substantially enhances antigen-specific cellular immune responses. Furthermore, blocking IFN-ß signaling at the injection site significantly decreases mRNA vaccine-induced cellular immune responses. Collectively, these data highlight the importance of injection site fibroblasts and IFN-ß signaling during early immune responses against the mRNA vaccine and provide detailed information on the initial chain of immune reactions elicited by mRNA vaccine injection.
Asunto(s)
Células Dendríticas , Fibroblastos , Inmunidad Celular , Inmunidad Innata , Interferón beta , Ratones Endogámicos BALB C , Nanopartículas , Vacunas de ARNm , Animales , Interferón beta/inmunología , Interferón beta/metabolismo , Femenino , Inmunidad Innata/inmunología , Inmunidad Innata/efectos de los fármacos , Ratones , Vacunas de ARNm/inmunología , Células Dendríticas/inmunología , Nanopartículas/química , Fibroblastos/inmunología , Fibroblastos/metabolismo , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , SARS-CoV-2/inmunología , Lípidos/química , COVID-19/prevención & control , COVID-19/inmunología , ARN Mensajero/metabolismo , ARN Mensajero/genética , Análisis de la Célula Individual , LiposomasRESUMEN
mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.
Asunto(s)
ARN Mensajero , Proteínas de Unión al ARN , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Unión Proteica , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Células HeLa , Factores de Tiempo , Aprendizaje AutomáticoRESUMEN
MicroRNA (miRNA) maturation is critically dependent on structural features of primary transcripts (pri-miRNAs). However, the scarcity of determined pri-miRNA structures has limited our understanding of miRNA maturation. Here, we employed selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), a high-throughput RNA structure probing method, to unravel the secondary structures of 476 high-confidence human pri-miRNAs. Our SHAPE-based structures diverge substantially from those inferred solely from computation, particularly in the apical loop and basal segments, underlining the need for experimental data in RNA structure prediction. By comparing the structures with high-throughput processing data, we determined the optimal structural features of pri-miRNAs. The sequence determinants are influenced substantially by their structural contexts. Moreover, we identified an element termed the bulged GWG motif (bGWG) with a 3' bulge in the lower stem, which promotes processing. Our structure-function mapping better annotates the determinants of pri-miRNA processing and offers practical implications for designing small hairpin RNAs and predicting the impacts of miRNA mutations.
Asunto(s)
MicroARNs , Procesamiento Postranscripcional del ARN , Humanos , MicroARNs/metabolismo , ARN Interferente Pequeño , Ribonucleasa III/genéticaRESUMEN
Shortening of messenger RNA poly(A) tails, or deadenylation, is a rate-limiting step in mRNA decay and is highly regulated during gene expression. The incorporation of non-adenosines in poly(A) tails, or 'mixed tailing', has been observed in vertebrates and viruses. Here, to quantitate the effect of mixed tails, we mathematically modeled deadenylation reactions at single-nucleotide resolution using an in vitro deadenylation system reconstituted with the complete human CCR4-NOT complex. Applying this model, we assessed the disrupting impact of single guanosine, uridine or cytosine to be equivalent to approximately 6, 8 or 11 adenosines, respectively. CCR4-NOT stalls at the 0, -1 and -2 positions relative to the non-adenosine residue. CAF1 and CCR4 enzyme subunits commonly prefer adenosine but exhibit distinct sequence selectivities and stalling positions. Our study provides an analytical framework to monitor deadenylation and reveals the molecular basis of tail sequence-dependent regulation of mRNA stability.
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Poli A , Estabilidad del ARN , ARN Mensajero , Humanos , Cinética , Poli A/metabolismo , Poli A/química , ARN Mensajero/metabolismo , ARN Mensajero/genética , ARN Mensajero/química , Adenosina/metabolismo , Receptores CCR4/metabolismo , Receptores CCR4/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/química , ARN NucleotidiltransferasasRESUMEN
The number of sequenced viral genomes has surged recently, presenting an opportunity to understand viral diversity and uncover unknown regulatory mechanisms. Here, we conducted a screening of 30,367 viral segments from 143 species representing 96 genera and 37 families. Using a library of viral segments in 3' UTR, we identified hundreds of elements impacting RNA abundance, translation, and nucleocytoplasmic distribution. To illustrate the power of this approach, we investigated K5, an element conserved in kobuviruses, and found its potent ability to enhance mRNA stability and translation in various contexts, including adeno-associated viral vectors and synthetic mRNAs. Moreover, we identified a previously uncharacterized protein, ZCCHC2, as a critical host factor for K5. ZCCHC2 recruits the terminal nucleotidyl transferase TENT4 to elongate poly(A) tails with mixed sequences, delaying deadenylation. This study provides a unique resource for virus and RNA research and highlights the potential of the virosphere for biological discoveries.
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ARN , Secuencias Reguladoras de Ácidos Nucleicos , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencia de Bases , Proteínas/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Estabilidad del ARN , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
DROSHA serves as a gatekeeper of the microRNA (miRNA) pathway by processing primary transcripts (pri-miRNAs). While the functions of structured domains of DROSHA have been well documented, the contribution of N-terminal proline-rich disordered domain (PRD) remains elusive. Here we show that the PRD promotes the processing of miRNA hairpins located within introns. We identified a DROSHA isoform (p140) lacking the PRD, which is produced by proteolytic cleavage. Small RNA sequencing revealed that p140 is significantly impaired in the maturation of intronic miRNAs. Consistently, our minigene constructs demonstrated that PRD enhances the processing of intronic hairpins, but not those in exons. Splice site mutations did not affect the PRD's enhancing effect on intronic constructs, suggesting that the PRD acts independently of splicing reaction by interacting with sequences residing within introns. The N-terminal regions from zebrafish and Xenopus DROSHA can replace the human counterpart, indicating functional conservation despite poor sequence alignment. Moreover, we found that rapidly evolving intronic miRNAs are generally more dependent on PRD than conserved ones, suggesting a role of PRD in miRNA evolution. Our study reveals a new layer of miRNA regulation mediated by a low-complexity disordered domain that senses the genomic contexts of miRNA loci.
Asunto(s)
MicroARNs , Ribonucleasa III , Animales , Humanos , Intrones/genética , MicroARNs/genética , MicroARNs/metabolismo , Prolina/genética , Prolina/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Procesamiento Postranscripcional del ARN , Pez CebraRESUMEN
RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs)1,2. However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3' overhang and a terminal loop3-11. Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis-acting element, termed the 'GYM motif' (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified 'ruler'-like counting mechanisms from the 5' and 3' ends of pre-miRNA3-6. Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics.
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ARN Helicasas DEAD-box , MicroARNs , Conformación de Ácido Nucleico , Precursores del ARN , ARN Interferente Pequeño , Ribonucleasa III , Humanos , Emparejamiento Base , ARN Helicasas DEAD-box/metabolismo , MicroARNs/biosíntesis , MicroARNs/genética , MicroARNs/metabolismo , Ribonucleasa III/metabolismo , Interferencia de ARN , ARN Bicatenario/química , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Precursores del ARN/biosíntesis , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , Secuencia de BasesRESUMEN
Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.
Asunto(s)
Microscopía por Crioelectrón , ARN Helicasas DEAD-box , MicroARNs , Precursores del ARN , Ribonucleasa III , Humanos , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/ultraestructura , MicroARNs/biosíntesis , MicroARNs/química , MicroARNs/metabolismo , MicroARNs/ultraestructura , Mutación , Ribonucleasa III/química , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Ribonucleasa III/ultraestructura , Precursores del ARN/química , Precursores del ARN/metabolismo , Precursores del ARN/ultraestructura , ARN Bicatenario/metabolismo , Especificidad por SustratoRESUMEN
Deadenylation generally constitutes the first and pivotal step in eukaryotic messenger RNA decay. Despite its importance in posttranscriptional regulations, the kinetics of deadenylation and its regulation remain largely unexplored. Here we identify La ribonucleoprotein 1, translational regulator (LARP1) as a general decelerator of deadenylation, which acts mainly in the 30-60-nucleotide (nt) poly(A) length window. We measured the steady-state and pulse-chased distribution of poly(A)-tail length, and found that deadenylation slows down in the 30-60-nt range. LARP1 associates preferentially with short tails and its depletion results in accelerated deadenylation specifically in the 30-60-nt range. Consistently, LARP1 knockdown leads to a global reduction of messenger RNA abundance. LARP1 interferes with the CCR4-NOT-mediated deadenylation in vitro by forming a ternary complex with poly(A)-binding protein (PABP) and poly(A). Together, our work reveals a dynamic nature of deadenylation kinetics and a role of LARP1 as a poly(A) length-specific barricade that creates a threshold for deadenylation.
Asunto(s)
Exorribonucleasas , Proteínas de Unión al ARN , Exorribonucleasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión a Poli(A)/genética , Regulación de la Expresión Génica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Poli A/metabolismoRESUMEN
Small, compact genomes confer a selective advantage to viruses, yet human cytomegalovirus (HCMV) expresses the long non-coding RNAs (lncRNAs); RNA1.2, RNA2.7, RNA4.9, and RNA5.0. Little is known about the function of these lncRNAs in the virus life cycle. Here, we dissected the functional and molecular landscape of HCMV lncRNAs. We found that HCMV lncRNAs occupy ~ 30% and 50-60% of total and poly(A)+viral transcriptome, respectively, throughout virus life cycle. RNA1.2, RNA2.7, and RNA4.9, the three abundantly expressed lncRNAs, appear to be essential in all infection states. Among these three lncRNAs, depletion of RNA2.7 and RNA4.9 results in the greatest defect in maintaining latent reservoir and promoting lytic replication, respectively. Moreover, we delineated the global post-transcriptional nature of HCMV lncRNAs by nanopore direct RNA sequencing and interactome analysis. We revealed that the lncRNAs are modified with N6-methyladenosine (m6A) and interact with m6A readers in all infection states. In-depth analysis demonstrated that m6A machineries stabilize HCMV lncRNAs, which could account for the overwhelming abundance of viral lncRNAs. Our study lays the groundwork for understanding the viral lncRNA-mediated regulation of host-virus interaction throughout the HCMV life cycle.
Asunto(s)
Infecciones por Citomegalovirus , ARN Largo no Codificante , Humanos , Citomegalovirus/genética , ARN Largo no Codificante/genética , Células Cultivadas , Transcriptoma , Replicación Viral/genéticaRESUMEN
RNA modifications are a common occurrence across all domains of life. Several chemical modifications, including N6-methyladenosine, have also been found in viral transcripts and viral RNA genomes. Some of the modifications increase the viral replication efficiency while also helping the virus to evade the host immune system. Nonetheless, there are numerous examples in which the host's RNA modification enzymes function as antiviral factors. Although established methods like MeRIP-seq and miCLIP can provide a transcriptome- wide overview of how viral RNA is modified, it is difficult to distinguish between the complex overlapping viral transcript isoforms using the short read-based techniques. Nanopore direct RNA sequencing (DRS) provides both long reads and direct signal readings, which may carry information about the modifications. Here, we describe a refined protocol for analyzing the RNA modifications in viral transcriptomes using nanopore technology.
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Secuenciación de Nanoporos , Nanoporos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Viral/genética , Análisis de Secuencia de ARN/métodos , TranscriptomaRESUMEN
We describe a protocol to conduct a high-throughput in vitro processing assay, using 1,881 human primary microRNAs (pri-miRNAs) and recombinant Microprocessor complex, followed by deep sequencing library generation. This comprehensive approach allows the mapping of cleavage sites and the measurement of processing efficiency of a large number of substrates simultaneously. Our protocol is readily modifiable to investigate the effects of chemicals and regulatory proteins. Moreover, cis-acting elements can be examined by replacing the wild-type pri-miRNAs with mutant variants. For complete details on the use and execution of this profile, please refer to Kim et al. (2021).
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MicroARNs , Humanos , MicroARNs/genética , Microcomputadores , Ribonucleasa III/genéticaRESUMEN
RNA-protein interaction can be captured by crosslinking and enrichment followed by tandem mass spectrometry, but it remains challenging to pinpoint RNA-binding sites (RBSs) or provide direct evidence for RNA-binding. To overcome these limitations, we here developed pRBS-ID, by incorporating the benefits of UVA-based photoactivatable ribonucleoside (PAR; 4-thiouridine and 6-thioguanosine) crosslinking and chemical RNA cleavage. pRBS-ID robustly detects peptides crosslinked to PAR adducts, offering direct RNA-binding evidence and identifying RBSs at single amino acid-resolution with base-specificity (U or G). Using pRBS-ID, we could profile uridine-contacting RBSs globally and discover guanosine-contacting RBSs, which allowed us to characterize the base-specific interactions. We also applied the search pipeline to analyze the datasets from UVC-based RBS-ID experiments, altogether offering a comprehensive list of human RBSs with high coverage (3,077 RBSs in 532 proteins in total). pRBS-ID is a widely applicable platform to investigate the molecular basis of posttranscriptional regulation.
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Sitios de Unión , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Ribonucleósidos/metabolismo , Aminoácidos/metabolismo , Células HeLa , Humanos , Dominios y Motivos de Interacción de Proteínas , Proteómica , Espectrometría de Masas en Tándem , Tiouridina/metabolismoRESUMEN
Argonaute is the primary mediator of metazoan miRNA targeting (MT). Among the currently identified >1,500 human RNA-binding proteins (RBPs), there are only a handful of RBPs known to enhance MT and several others reported to suppress MT, leaving the global impact of RBPs on MT elusive. In this study, we have systematically analyzed transcriptome-wide binding sites for 150 human RBPs and evaluated the quantitative effect of individual RBPs on MT efficacy. In contrast to previous studies, we show that most RBPs significantly affect MT and that all of those MT-regulating RBPs function as MT enhancers rather than suppressors, by making the local secondary structure of the target site accessible to Argonaute. Our findings illuminate the unappreciated regulatory impact of human RBPs on MT, and as these RBPs may play key roles in the gene regulatory network governed by metazoan miRNAs, MT should be understood in the context of co-regulating RBPs.
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MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3'/genética , Sitios de Unión , Evolución Molecular , Células HeLa , Células Hep G2 , Humanos , MicroARNs/genética , Conformación de Ácido Nucleico , Unión Proteica , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
Once inside the host cell, DNA viruses must overcome the physical barrier posed by the nuclear envelope to establish a successful infection. The mechanism underlying this process remains unclear. Here, we show that the herpesvirus exploits the immune adaptor stimulator of interferon genes (STING) to facilitate nuclear import of the viral genome. Following the entry of the viral capsid into the cell, STING binds the viral capsid, mediates capsid docking to the nuclear pore complex via physical interaction, and subsequently enables accumulation of the viral genome in the nucleus. Silencing STING in human cytomegalovirus (HCMV)-susceptible cells inhibited nuclear import of the viral genome and reduced the ensuing viral gene expression. Overexpressing STING increased the host cell's susceptibility to HCMV and herpes simplex virus 1 by improving the nuclear delivery of viral DNA at the early stage of infection. These observations suggest that the proviral activity of STING is conserved and exploited by the herpesvirus family. Intriguingly, in monocytes, which act as latent reservoirs of HCMV, STING deficiency negatively regulated the establishment of HCMV latency and reactivation. Our findings identify STING as a proviral host factor regulating latency and reactivation of herpesviruses.
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Citomegalovirus/fisiología , ADN Viral/metabolismo , Genoma Viral/fisiología , Proteínas de la Membrana/metabolismo , Replicación Viral/fisiología , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , ADN Viral/genética , Regulación Viral de la Expresión Génica , Proteínas de la Membrana/genética , Interferencia de ARN , ARN Interferente Pequeño , Internalización del VirusRESUMEN
Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). More than 1,800 miRNA loci are annotated in humans, but it remains largely unknown whether and at which sites pri-miRNAs are cleaved by DROSHA. Here, we performed in vitro processing on a full set of human pri-miRNAs (miRBase version 21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs on the basis of DROSHA dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing and unproductive cleavage events such as "nick" or "inverse" processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.
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MicroARNs/genética , Procesamiento Postranscripcional del ARN/genética , Ribonucleasa III/genética , Factores de Empalme Serina-Arginina/genética , Sitios de Unión/genética , Genoma Humano/genética , Células HEK293 , Humanos , Interferencia de ARNRESUMEN
SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its abilities to repurpose host RNA-binding proteins (RBPs) and to evade antiviral RBPs. To uncover the SARS-CoV-2 RNA interactome, we here develop a robust ribonucleoprotein (RNP) capture protocol and identify 109 host factors that directly bind to SARS-CoV-2 RNAs. Applying RNP capture on another coronavirus, HCoV-OC43, revealed evolutionarily conserved interactions between coronaviral RNAs and host proteins. Transcriptome analyses and knockdown experiments delineated 17 antiviral RBPs, including ZC3HAV1, TRIM25, PARP12, and SHFL, and 8 proviral RBPs, such as EIF3D and CSDE1, which are responsible for co-opting multiple steps of the mRNA life cycle. This also led to the identification of LARP1, a downstream target of the mTOR signaling pathway, as an antiviral host factor that interacts with the SARS-CoV-2 RNAs. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.
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Autoantígenos/genética , COVID-19/genética , ARN Viral/genética , Ribonucleoproteínas/genética , SARS-CoV-2/genética , COVID-19/virología , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/patogenicidad , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Unión Proteica/genética , Mapas de Interacción de Proteínas/genética , Proteínas de Unión al ARN/genética , SARS-CoV-2/patogenicidad , Serina-Treonina Quinasas TOR/genética , Factores de Transcripción/genética , Transcriptoma/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Replicación Viral/genética , Antígeno SS-BRESUMEN
Cellular senescence is a complex stress response implicated in aging. Autophagy can suppress senescence but is counterintuitively necessary for full senescence. Although its anti-senescence role is well described, to what extent autophagy contributes to senescence establishment and the underlying mechanisms is poorly understood. Here, we show that selective autophagy of multiple regulatory components coordinates the homeostatic state of senescence. We combined a proteomic analysis of autophagy components with protein stability profiling, identifying autophagy substrate proteins involved in several senescence-related processes. Selective autophagy of KEAP1 promoted redox homeostasis during senescence. Furthermore, selective autophagy limited translational machinery components to ameliorate senescence-associated proteotoxic stress. Lastly, selective autophagy of TNIP1 enhanced senescence-associated inflammation. These selective autophagy networks appear to operate in vivo senescence during human osteoarthritis. Our data highlight a caretaker role of autophagy in the stress support network of senescence through regulated protein stability and unravel the intertwined relationship between two important age-related processes.
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Autofagia , Senescencia Celular , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Factor 3 de Iniciación Eucariótica/metabolismo , Células HEK293 , Humanos , Inflamación/patología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Modelos Biológicos , Osteoartritis/metabolismo , Osteoartritis/patología , Estrés Oxidativo , ProteostasisRESUMEN
Adenosine N6-methylation (m6A) is one of the most pervasive mRNA modifications, and yet the physiological significance of m6A removal (demethylation) remains elusive. Here, we report that the m6A demethylase FTO functions as a conserved regulator of motile ciliogenesis. Mechanistically, FTO demethylates and thereby stabilizes the mRNA that encodes the master ciliary transcription factor FOXJ1. Depletion of Fto in Xenopus laevis embryos caused widespread motile cilia defects, and Foxj1 was identified as one of the major phenocritical targets. In primary human airway epithelium, FTO depletion also led to FOXJ1 mRNA destabilization and a severe loss of ciliated cells with an increase of neighboring goblet cells. Consistently, Fto knockout mice showed strong asthma-like phenotypes upon allergen challenge, a result owing to defective ciliated cells in the airway epithelium. Altogether, our study reveals a conserved role of the FTO-FOXJ1 axis in embryonic and homeostatic motile ciliogenesis.