RESUMEN
Over the past decade, genetic engineering has witnessed a revolution with the emergence of a relatively new genetic editing tool based on RNA-guided nucleases: the CRISPR/Cas9 system. Since the first report in 1987 and characterization in 2007 as a bacterial defense mechanism, this system has garnered immense interest and research attention. CRISPR systems provide immunity to bacteria against invading genetic material; however, with specific modifications in sequence and structure, it becomes a precise editing system capable of modifying the genomes of a wide range of organisms. The refinement of these modifications encompasses diverse approaches, including the development of more accurate nucleases, understanding of the cellular context and epigenetic conditions, and the re-designing guide RNAs (gRNAs). Considering the critical importance of the correct performance of CRISPR/Cas9 systems, our scope will emphasize the latter approach. Hence, we present an overview of the past and the most recent guide RNA web-based design tools, highlighting the evolution of their computational architecture and gRNA characteristics over the years. Our study explains computational approaches that use machine learning techniques, neural networks, and gRNA/target interactions data to enable predictions and classifications. This review could open the door to a dynamic community that uses up-to-date algorithms to optimize and create promising gRNAs, suitable for modern CRISPR/Cas9 engineering.
Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Algoritmos , Aprendizaje AutomáticoRESUMEN
Although the study of ribonucleic acid (RNA) therapeutics started decades ago, for many years, this field of research was overshadowed by the growing interest in DNA-based therapies. Nowadays, the role of several types of RNA in cell regulation processes and the development of various diseases have been elucidated, and research in RNA therapeutics is back with force. This short literature review aims to present general aspects of many of the molecules currently used in RNA therapeutics, including in vitro transcribed mRNA (IVT mRNA), antisense oligonucleotides (ASOs), aptamers, small interfering RNAs (siRNAs), and microRNAs (miRNAs). In addition, we describe the state of the art of technologies applied for synthetic RNA manufacture and delivery. Likewise, we detail the RNA-based therapies approved by the FDA so far, as well as the ongoing clinical investigations. As a final point, we highlight the current and potential advantages of working on RNA-based therapeutics and how these could lead to a new era of accessible and personalized healthcare.
Asunto(s)
Aptámeros de Nucleótidos/uso terapéutico , MicroARNs/uso terapéutico , Oligonucleótidos Antisentido , ARN Mensajero/uso terapéutico , ARN Interferente Pequeño/uso terapéutico , Humanos , Oligonucleótidos Antisentido/uso terapéuticoRESUMEN
The RNA exosome is a multisubunit protein complex involved in RNA surveillance of all classes of RNA, and is essential for pre-rRNA processing. The exosome is conserved throughout evolution, present in archaea and eukaryotes from yeast to humans, where it localizes to the nucleus and cytoplasm. The catalytically active subunit Rrp44/Dis3 of the exosome in budding yeast (Saccharomyces cerevisiae) is considered a protein present in these two subcellular compartments, and here we report that it not only localizes mainly to the nucleus, but is concentrated in the nucleolus, where the early pre-rRNA processing reactions take place. Moreover, we show by confocal microscopy analysis that the core exosome subunits Rrp41 and Rrp43 also localize largely to the nucleus and strongly accumulate in the nucleolus. These results shown here shed additional light on the localization of the yeast exosome and have implications regarding the main function of this RNase complex, which seems to be primarily in early pre-rRNA processing and surveillance.
Asunto(s)
Nucléolo Celular/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/metabolismo , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Hongos/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fracciones Subcelulares/metabolismoRESUMEN
The exosome is a conserved multiprotein complex essential for RNA processing and degradation. The nuclear exosome is a key factor for pre-rRNA processing through the activity of its catalytic subunits, Rrp6 and Rrp44. In Saccharomyces cerevisiae, Rrp6 is exclusively nuclear and has been shown to interact with exosome cofactors. With the aim of analyzing proteins associated with the nuclear exosome, in this work, we purified the complex with Rrp6-TAP, identified the co-purified proteins by mass spectrometry, and found karyopherins to be one of the major groups of proteins enriched in the samples. By investigating the biological importance of these protein interactions, we identified Srp1, Kap95, and Sxm1 as the most important karyopherins for Rrp6 nuclear import and the nuclear localization signals recognized by them. Based on the results shown here, we propose a model of multiple pathways for the transport of Rrp6 to the nucleus.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/metabolismo , Carioferinas/metabolismo , Señales de Localización Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Exosomas/enzimología , Eliminación de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Carioferinas/química , Carioferinas/genética , Microscopía Confocal , Microscopía Fluorescente , Señales de Localización Nuclear/química , Señales de Localización Nuclear/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , beta Carioferinas/química , beta Carioferinas/genéticaRESUMEN
Oxindolimine-copper(II) and zinc(II) complexes that previously have shown to induce apoptosis, with DNA and mitochondria as main targets, exhibit here significant inhibition of kinase CDK1/cyclin B protein. Copper species are more active than the corresponding zinc, and the free ligand shows to be less active, indicating a major influence of coordination in the process, and a further modulation by the coordinated ligand. Molecular docking and classical molecular dynamics provide a better understanding of the effectiveness and kinase inhibition mechanism by these compounds, showing that the metal complex provides a stronger interaction than the free ligand with the ATP-binding site. The metal ion introduces charge in the oxindole species, giving it a more rigid conformation that then becomes more effective in its interactions with the protein active site. Analogous experiments resulted in no significant effect regarding phosphatase inhibition. These results can explain the cytotoxicity of these metal complexes towards different tumor cells, in addition to its capability of binding to DNA, and decreasing membrane potential of mitochondria.
Asunto(s)
Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Cobre/química , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Iminas/farmacología , Simulación del Acoplamiento Molecular , Zinc/química , Estabilidad de Medicamentos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Iminas/química , Indoles/química , Indoles/farmacología , Ligandos , Modelos Moleculares , OxindolesRESUMEN
BACKGROUND: Box C/D snoRNPs are responsible for rRNA methylation and processing, and are formed by snoRNAs and four conserved proteins, Nop1, Nop56, Nop58 and Snu13. The snoRNP assembly is a stepwise process, involving other protein complexes, among which the R(2)TP and Hsp90 chaperone. Nop17, also known as Pih1, has been shown to be a constituent of the R(2)TP (Rvb1, Rvb2, Tah1, Pih1) and to participate in box C/D snoRNP assembly by its interaction with Nop58. The molecular function of Nop17, however, has not yet been described. RESULTS: To shed light on the role played by Nop17 in the maturation of snoRNP, here we analyzed the interactions domains of Nop58 - Nop17 - Tah1 and the importance of ATP to the interaction between Nop17 and the ATPase Rvb1/2. CONCLUSIONS: Based on the results shown here, we propose a model for the assembly of box C/D snoRNP, according to which R(2)TP complex is important for reducing the affinity of Nop58 for snoRNA, and for the binding of the other snoRNP subunits.