RESUMEN
DEAD- and DExH-box ATPases (DDX/DHXs) are abundant and highly conserved cellular enzymes ubiquitously involved in RNA processing. By remodeling RNA-RNA and RNA-protein interactions, they often function as gatekeepers that control the progression of diverse RNA maturation steps. Intriguingly, most DDX/DHXs localize to membraneless organelles (MLOs) such as nucleoli, nuclear speckles, stress granules, or processing bodies. Recent findings suggest not only that localization to MLOs can promote interaction between DDX/DHXs and their targets but also that DDX/DHXs are key regulators of MLO formation and turnover through their condensation and ATPase activity.In this review, we describe the molecular function of DDX/DHXs in ribosome biogenesis, messenger RNA splicing, export, translation, and storage or decay as well as their association with prominent MLOs. We discuss how the enzymatic function of DDX/DHXs in RNA processing is linked to DDX/DHX condensation, the accumulation of ribonucleoprotein particles and MLO dynamics. Future research will reveal how these processes orchestrate the RNA life cycle in MLO space and DDX/DHX time.
Asunto(s)
ARN Helicasas DEAD-box , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/química , Humanos , Animales , ARN/metabolismo , ARN/genética , ARN/química , Empalme del ARN , Orgánulos/metabolismo , Orgánulos/genética , Ribosomas/metabolismo , Ribosomas/genética , Pliegue del ARN , Procesamiento Postranscripcional del ARN , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
Methylation of RNA nucleotides represents an important layer of gene expression regulation, and perturbation of the RNA methylome is associated with pathophysiology. In cells, RNA methylations are installed by RNA methyltransferases (RNMTs) that are specialized to catalyze particular types of methylation (ribose or different base positions). Furthermore, RNMTs must specifically recognize their appropriate target RNAs within the RNA-dense cellular environment. Some RNMTs are catalytically active alone and achieve target specificity via recognition of sequence motifs and/or RNA structures. Others function together with protein cofactors that can influence stability, S-adenosyl-L-methionine binding, and RNA affinity as well as aiding specific recruitment and catalytic activity. Association of RNMTs with guide RNAs represents an alternative mechanism to direct site-specific methylation by an RNMT that lacks intrinsic specificity. Recently, ribozyme-catalyzed methylation of RNA has been achieved in vitro, and here, we compare these different strategies for RNA methylation from structural and mechanistic perspectives.
Asunto(s)
Conformación de Ácido Nucleico , ARN Catalítico , ARN , ARN Catalítico/metabolismo , ARN Catalítico/química , ARN Catalítico/genética , Metilación , ARN/metabolismo , ARN/genética , ARN/química , Humanos , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , Nucleótidos/metabolismo , Nucleótidos/química , Nucleótidos/genética , ARNt Metiltransferasas/metabolismo , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/química , Especificidad por Sustrato , Animales , Modelos MolecularesRESUMEN
RNAs localizing to the outer cell surface have been recently identified in mammalian cells, including RNAs with glycan modifications known as glycoRNAs. However, the functional significance of cell surface RNAs and their production are poorly known. We report that cell surface RNAs are critical for neutrophil recruitment and that the mammalian homologs of the sid-1 RNA transporter are required for glycoRNA expression. Cell surface RNAs can be readily detected in murine neutrophils, the elimination of which substantially impairs neutrophil recruitment to inflammatory sites in vivo and reduces neutrophils' adhesion to and migration through endothelial cells. Neutrophil glycoRNAs are predominantly on cell surface, important for neutrophil-endothelial interactions, and can be recognized by P-selectin (Selp). Knockdown of the murine Sidt genes abolishes neutrophil glycoRNAs and functionally mimics the loss of cell surface RNAs. Our data demonstrate the biological importance of cell surface glycoRNAs and highlight a noncanonical dimension of RNA-mediated cellular functions.
Asunto(s)
Células Endoteliales , Infiltración Neutrófila , Neutrófilos , ARN , Animales , Ratones , Células Endoteliales/metabolismo , Neutrófilos/metabolismo , ARN/química , ARN/metabolismo , Proteínas de Transporte de Nucleótidos/genética , Proteínas de Transporte de Nucleótidos/metabolismoRESUMEN
Myelin, the insulating sheath that surrounds neuronal axons, is produced by oligodendrocytes in the central nervous system (CNS). This evolutionary innovation, which first appears in jawed vertebrates, enabled rapid transmission of nerve impulses, more complex brains, and greater morphological diversity. Here, we report that RNA-level expression of RNLTR12-int, a retrotransposon of retroviral origin, is essential for myelination. We show that RNLTR12-int-encoded RNA binds to the transcription factor SOX10 to regulate transcription of myelin basic protein (Mbp, the major constituent of myelin) in rodents. RNLTR12-int-like sequences (which we name RetroMyelin) are found in all jawed vertebrates, and we further demonstrate their function in regulating myelination in two different vertebrate classes (zebrafish and frogs). Our study therefore suggests that retroviral endogenization played a prominent role in the emergence of vertebrate myelin.
Asunto(s)
Vaina de Mielina , Retroelementos , Animales , Expresión Génica , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Retroelementos/genética , ARN/metabolismo , Pez Cebra/genética , AnurosRESUMEN
Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.
Asunto(s)
Nucléolo Celular , Proteínas Nucleares , Fuerza Protón-Motriz , Nucléolo Celular/química , Núcleo Celular/química , Proteínas Nucleares/química , ARN/metabolismo , Separación de Fases , Proteínas Intrínsecamente Desordenadas/química , Animales , Xenopus laevis , Oocitos/química , Oocitos/citologíaRESUMEN
Chemical modifications on mRNA represent a critical layer of gene expression regulation. Research in this area has continued to accelerate over the last decade, as more modifications are being characterized with increasing depth and breadth. mRNA modifications have been demonstrated to influence nearly every step from the early phases of transcript synthesis in the nucleus through to their decay in the cytoplasm, but in many cases, the molecular mechanisms involved in these processes remain mysterious. Here, we highlight recent work that has elucidated the roles of mRNA modifications throughout the mRNA life cycle, describe gaps in our understanding and remaining open questions, and offer some forward-looking perspective on future directions in the field.
Asunto(s)
Regulación de la Expresión Génica , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , ARN/genética , ARN/metabolismoRESUMEN
Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.
Asunto(s)
ARN , Retroelementos , ARN/metabolismo , División del ADN , ADN Polimerasa Dirigida por ARN/metabolismo , Transcripción ReversaRESUMEN
In poikilotherms, temperature changes challenge the integration of physiological function. Within the complex nervous systems of the behaviorally sophisticated coleoid cephalopods, these problems are substantial. RNA editing by adenosine deamination is a well-positioned mechanism for environmental acclimation. We report that the neural proteome of Octopus bimaculoides undergoes massive reconfigurations via RNA editing following a temperature challenge. Over 13,000 codons are affected, and many alter proteins that are vital for neural processes. For two highly temperature-sensitive examples, recoding tunes protein function. For synaptotagmin, a key component of Ca2+-dependent neurotransmitter release, crystal structures and supporting experiments show that editing alters Ca2+ binding. For kinesin-1, a motor protein driving axonal transport, editing regulates transport velocity down microtubules. Seasonal sampling of wild-caught specimens indicates that temperature-dependent editing occurs in the field as well. These data show that A-to-I editing tunes neurophysiological function in response to temperature in octopus and most likely other coleoids.
Asunto(s)
Octopodiformes , Proteoma , Animales , Proteoma/metabolismo , Octopodiformes/genética , Edición de ARN , Temperatura , Sistema Nervioso/metabolismo , Adenosina Desaminasa/metabolismo , ARN/metabolismoRESUMEN
A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.
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Técnicas Citológicas , Técnicas Genéticas , ARN , Animales , Transporte Biológico , Mamíferos/metabolismo , ARN/genética , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Virus/genética , Tipificación Molecular , Análisis de Secuencia de ARNRESUMEN
Ribonucleoprotein (RNP) granules are diverse membrane-less organelles that form through multivalent RNA-RNA, RNA-protein, and protein-protein interactions between RNPs. RNP granules are implicated in many aspects of RNA physiology, but in most cases their functions are poorly understood. RNP granules can be described through four key principles. First, RNP granules often arise because of the large size, high localized concentrations, and multivalent interactions of RNPs. Second, cells regulate RNP granule formation by multiple mechanisms including posttranslational modifications, protein chaperones, and RNA chaperones. Third, RNP granules impact cell physiology in multiple manners. Finally, dysregulation of RNP granules contributes to human diseases. Outstanding issues in the field remain, including determining the scale and molecular mechanisms of RNP granule function and how granule dysfunction contributes to human disease.
Asunto(s)
Estructuras del Núcleo Celular , Gránulos Citoplasmáticos , Ribonucleoproteínas , Humanos , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/patología , Gránulos de Ribonucleoproteínas Citoplasmáticas , Procesamiento Proteico-Postraduccional , Ribonucleoproteínas/metabolismo , ARN/metabolismo , Nucléolo Celular/metabolismo , Estructuras del Núcleo Celular/metabolismo , Estructuras del Núcleo Celular/patología , AnimalesRESUMEN
ADP-ribosylation is a ubiquitous modification of biomolecules, including proteins and nucleic acids, that regulates various cellular functions in all kingdoms of life. The recent emergence of new technologies to study ADP-ribosylation has reshaped our understanding of the molecular mechanisms that govern the establishment, removal, and recognition of this modification, as well as its impact on cellular and organismal function. These advances have also revealed the intricate involvement of ADP-ribosylation in human physiology and pathology and the enormous potential that their manipulation holds for therapy. In this review, we present the state-of-the-art findings covering the work in structural biology, biochemistry, cell biology, and clinical aspects of ADP-ribosylation.
Asunto(s)
ADP-Ribosilación , Humanos , Proteínas/metabolismo , ADN/metabolismo , ARN/metabolismo , Animales , Transducción de Señal , Procesamiento Proteico-Postraduccional , ADP Ribosa Transferasas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismoRESUMEN
RNA editing is a widespread epigenetic process that can alter the amino acid sequence of proteins, termed "recoding." In cephalopods, most transcripts are recoded, and recoding is hypothesized to be an adaptive strategy to generate phenotypic plasticity. However, how animals use RNA recoding dynamically is largely unexplored. We investigated the function of cephalopod RNA recoding in the microtubule motor proteins kinesin and dynein. We found that squid rapidly employ RNA recoding in response to changes in ocean temperature, and kinesin variants generated in cold seawater displayed enhanced motile properties in single-molecule experiments conducted in the cold. We also identified tissue-specific recoded squid kinesin variants that displayed distinct motile properties. Finally, we showed that cephalopod recoding sites can guide the discovery of functional substitutions in non-cephalopod kinesin and dynein. Thus, RNA recoding is a dynamic mechanism that generates phenotypic plasticity in cephalopods and can inform the characterization of conserved non-cephalopod proteins.
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Cefalópodos , Dineínas , Animales , Dineínas/genética , Dineínas/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , ARN/metabolismo , Cefalópodos/genética , Cefalópodos/metabolismo , Proteínas/metabolismo , Microtúbulos/metabolismo , Proteínas de Microtúbulos , Miosinas/metabolismoRESUMEN
DEAD-box ATPases constitute a very large protein family present in all cells, often in great abundance. From bacteria to humans, they play critical roles in many aspects of RNA metabolism, and due to their widespread importance in RNA biology, they have been characterized in great detail at both the structural and biochemical levels. DEAD-box proteins function as RNA-dependent ATPases that can unwind short duplexes of RNA, remodel ribonucleoprotein (RNP) complexes, or act as clamps to promote RNP assembly. Yet, it often remains enigmatic how individual DEAD-box proteins mechanistically contribute to specific RNA-processing steps. Here, we review the role of DEAD-box ATPases in the regulation of gene expression and propose that one common function of these enzymes is in the regulation of liquid-liquid phase separation of RNP condensates.
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ARN Helicasas DEAD-box , ARN , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , ARN Helicasas DEAD-box/química , Expresión Génica , Humanos , ARN/metabolismoRESUMEN
In the last decade, the notion that mRNA modifications are involved in regulation of gene expression was demonstrated in thousands of studies. To date, new technologies and methods allow accurate identification, transcriptome-wide mapping, and functional characterization of a growing number of RNA modifications, providing important insights into the biology of these marks. Most of the methods and approaches were developed for studying m6A, the most prevalent internal mRNA modification. However, unique properties of other RNA modifications stimulated the development of additional approaches. In this technical primer, we will discuss the available tools and approaches for detecting and studying different RNA modifications.
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Procesamiento Postranscripcional del ARN , ARN , Epigénesis Genética , ARN/genética , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
Epstein-Barr virus (EBV) is a ubiquitous, oncogenic virus that is associated with a number of different human malignancies as well as autoimmune disorders. The expression of EBV viral proteins and non-coding RNAs contribute to EBV-mediated disease pathologies. The virus establishes life-long latency in the human host and is adept at evading host innate and adaptive immune responses. In this review, we discuss the life cycle of EBV, the various functions of EBV-encoded proteins and RNAs, the ability of the virus to activate and evade immune responses, as well as the neoplastic and autoimmune diseases that are associated with EBV infection in the human population.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Biología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , ARN/metabolismo , Proteínas Virales/metabolismo , Latencia del VirusRESUMEN
The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both in vitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.
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ADN Mitocondrial , Proteínas Mitocondriales , Animales , ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Dimerización , Humanos , Mamíferos/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , ARN/metabolismo , ARN Mitocondrial , ARN Citoplasmático Pequeño , Partícula de Reconocimiento de Señal , Transcripción GenéticaRESUMEN
RNA quality control relies on co-factors and adaptors to identify and prepare substrates for degradation by ribonucleases such as the 3' to 5' ribonucleolytic RNA exosome. Here, we determined cryogenic electron microscopy structures of human nuclear exosome targeting (NEXT) complexes bound to RNA that reveal mechanistic insights to substrate recognition and early steps that precede RNA handover to the exosome. The structures illuminate ZCCHC8 as a scaffold, mediating homodimerization while embracing the MTR4 helicase and flexibly anchoring RBM7 to the helicase core. All three subunits collaborate to bind the RNA, with RBM7 and ZCCHC8 surveying sequences upstream of the 3' end to facilitate RNA capture by MTR4. ZCCHC8 obscures MTR4 surfaces important for RNA binding and extrusion as well as MPP6-dependent recruitment and docking onto the RNA exosome core, interactions that contribute to RNA surveillance by coordinating RNA capture, translocation, and extrusion from the helicase to the exosome for decay.
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Exosomas , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Unión Proteica , ARN/metabolismo , Estabilidad del ARNRESUMEN
Most circular RNAs are produced from the back-splicing of exons of precursor mRNAs. Recent technological advances have in part overcome problems with their circular conformation and sequence overlap with linear cognate mRNAs, allowing a better understanding of their cellular roles. Depending on their localization and specific interactions with DNA, RNA, and proteins, circular RNAs can modulate transcription and splicing, regulate stability and translation of cytoplasmic mRNAs, interfere with signaling pathways, and serve as templates for translation in different biological and pathophysiological contexts. Emerging applications of RNA circles to interfere with cellular processes, modulate immune responses, and direct translation into proteins shed new light on biomedical research. In this review, we discuss approaches used in circular RNA studies and the current understanding of their regulatory roles and potential applications.
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ARN Circular , ARN , Proteínas/metabolismo , ARN/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismoRESUMEN
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.
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Escherichia coli , Orgánulos , Escherichia coli/genética , Orgánulos/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Solventes/análisis , Solventes/metabolismoRESUMEN
Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of client proteins. Genetic modification of scaffold granule proteins or tethering the intrinsically disordered region of human fused in sarcoma (FUS) to oskar mRNA allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate the localization and expression of a maternal RNA that instructs germline formation.