RESUMEN
Cleavage and polyadenylation factor (CPF/CPSF) is a multi-protein complex essential for formation of eukaryotic mRNA 3' ends. CPF cleaves pre-mRNAs at a specific site and adds a poly(A) tail. The cleavage reaction defines the 3' end of the mature mRNA, and thus the activity of the endonuclease is highly regulated. Here, we show that reconstitution of specific pre-mRNA cleavage with recombinant yeast proteins requires incorporation of the Ysh1 endonuclease into an eight-subunit "CPFcore" complex. Cleavage also requires the accessory cleavage factors IA and IB, which bind substrate pre-mRNAs and CPF, likely facilitating assembly of an active complex. Using X-ray crystallography, electron microscopy, and mass spectrometry, we determine the structure of Ysh1 bound to Mpe1 and the arrangement of subunits within CPFcore. Together, our data suggest that the active mRNA 3' end processing machinery is a dynamic assembly that is licensed to cleave only when all protein factors come together at the polyadenylation site.
Asunto(s)
Endonucleasas/metabolismo , Poliadenilación , Precursores del ARN/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Citocromos c/genética , Citocromos c/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Activación Enzimática , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Simulación del Acoplamiento Molecular , Complejos Multiproteicos , Polinucleotido Adenililtransferasa/genética , Polinucleotido Adenililtransferasa/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Precursores del ARN/genética , ARN de Hongos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , Espectrometría de Masas en Tándem , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
In the version of this article originally published, several co-authors had incorrect affiliation footnote numbers listed in the author list. Tatiana Cañeque and Angelica Mariani should each have affiliation numbers 3, 4 and 5, and Emmanuelle Charafe-Jauffret should have number 6. Additionally, there was an extra space in the name of co-author Robert P. St.Onge. These errors have been corrected in the HTML and PDF versions of the paper and the Supplementary Information PDF.
RESUMEN
Peripheral membrane proteins orchestrate many physiological and pathological processes, making regulation of their activities by small molecules highly desirable. However, they are often refractory to classical competitive inhibition. Here, we demonstrate that potent and selective inhibition of peripheral membrane proteins can be achieved by small molecules that target protein-membrane interactions by a noncompetitive mechanism. We show that the small molecule Bragsin inhibits BRAG2-mediated Arf GTPase activation in vitro in a manner that requires a membrane. In cells, Bragsin affects the trans-Golgi network in a BRAG2- and Arf-dependent manner. The crystal structure of the BRAG2-Bragsin complex and structure-activity relationship analysis reveal that Bragsin binds at the interface between the PH domain of BRAG2 and the lipid bilayer to render BRAG2 unable to activate lipidated Arf. Finally, Bragsin affects tumorsphere formation in breast cancer cell lines. Bragsin thus pioneers a novel class of drugs that function by altering protein-membrane interactions without disruption.
Asunto(s)
Factor 1 de Ribosilacion-ADP/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Factor 1 de Ribosilacion-ADP/metabolismo , Línea Celular Tumoral , GTP Fosfohidrolasas , Proteínas Activadoras de GTPasa , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Células HeLa , Humanos , Membrana Dobles de Lípidos , Glicoproteínas de Membrana/metabolismo , Nucleótidos , Dominios Homólogos a Pleckstrina/fisiología , Unión Proteica , Transducción de Señal , Relación Estructura-Actividad , Sulfotransferasas/metabolismoRESUMEN
Replication of nucleic acids in the absence of genetically encoded enzymes represents a critical process for the emergence of cellular life. Repeated separation of complementary RNA strands is required to achieve multiple cycles of chemical replication, yet thermal denaturation under plausible prebiotic conditions is impaired by the high temperatures required to separate long RNA strands and by concurrent degradation pathways, the latter accelerated by divalent metal ions. Here we show how the melting temperature of oligoribonucleotide duplexes can be tuned by changes in pH, enabling the separation of RNA strands at moderate temperatures. At the same time, the risk of phosphodiester bond cleavage is reduced under the acid denaturation conditions herein described, both in the presence and in the absence of divalent metal ions. Through a combination of ultraviolet and circular dichroism thermal studies and gel electrophoresis, we demonstrate the relevance of geological pH oscillations in the context of the RNA strand separation problem. Our results reveal new insights in the field of prebiotic chemistry, supporting plausible geochemical scenarios in which non-enzymatic RNA replication might have taken place.
Asunto(s)
Replicación del ADN , ADN/química , Prebióticos , ARN/química , Concentración de Iones de Hidrógeno , Conformación de Ácido Nucleico , TemperaturaRESUMEN
Investigations into the chemical origin of life have recently benefitted from a holistic approach in which possible atmospheric, organic, and inorganic systems chemistries are taken into consideration. In this way, we now report that a selective phosphate activating agent, namely methyl isocyanide, could plausibly have been produced from simple prebiotic feedstocks. We show that methyl isocyanide drives the conversion of nucleoside monophosphates to phosphorimidazolides under potentially prebiotic conditions and in excellent yields for the first time. Importantly, this chemistry allows for repeated reactivation cycles, a property long sought in nonenzymatic oligomerization studies. Further, as the isocyanide is released upon irradiation, the possibility of spatially and temporally controlled activation chemistry is thus raised.
Asunto(s)
Nitrilos/química , Nucleótidos/química , Origen de la Vida , Fosfatos/química , Imidazoles/química , Luz , Nucleósidos/química , Procesos FotoquímicosRESUMEN
Non-enzymatic oligomerization of activated ribonucleotides leads to ribonucleic acids that contain a mixture of 2',5'- and 3',5'-linkages, and overcoming this backbone heterogeneity has long been considered a major limitation to the prebiotic emergence of RNA. Herein, we demonstrate non-enzymatic chemistry that progressively converts 2',5'-linkages into 3',5'-linkages through iterative degradation and repair. The energetic costs of this proofreading are met by the hydrolytic turnover of a phosphate activating agent and an acylating agent. With multiple rounds of this energy-dissipative recycling, we show that all-3',5'-linked duplex RNA can emerge from a backbone heterogeneous mixture, thereby delineating a route that could have driven RNA evolution on the early earth.
Asunto(s)
ARN/química , Acetilación , Biopolímeros/química , Cromatografía Líquida de Alta Presión , Hidrólisis , Origen de la Vida , Fosfatos/químicaRESUMEN
Aryl ethynyl anthraquinones have been synthesized by Sonogashira cross-coupling and evaluated as telomeric G-quadruplex ligands, by the FRET melting assay, circular dichroism, the DNA synthesis arrest assay and molecular docking. Both the binding properties and G-quadruplex vs. duplex selectivity are controlled by the structures of the aryl ethynyl moieties.
Asunto(s)
Antraquinonas/química , G-Cuádruplex , Telómero/química , Dicroismo Circular , ADN/química , Fluorescencia , Ligandos , Modelos Moleculares , Polimerasa Taq/metabolismo , Temperatura de TransiciónRESUMEN
Small molecules are central players in chemical biology studies. They promote the perturbation of cellular processes underlying diseases and enable the identification of biological targets that can be validated for therapeutic intervention. Small molecules have been shown to accurately tune a single function of pluripotent proteins in a reversible manner with exceptional temporal resolution. The identification of molecular probes and drugs remains a worthy challenge that can be addressed by the use of biased and unbiased strategies. Hypothesis-driven methodologies employs a known biological target to synthesize complementary hits while discovery-driven strategies offer the additional means of identifying previously unanticipated biological targets. This review article provides a general overview of recent synthetic frameworks that gave rise to an impressive arsenal of biologically active small molecules with unprecedented cellular mechanisms.
Asunto(s)
Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Química Clic , Técnicas Químicas Combinatorias , Humanos , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
It all clicks into place: A potent telomere-targeting small molecule has been identified by using the copper-free 1,3-dipolar cycloaddition of a series of alkyne and azide building blocks catalyzed by a non-Watson-Crick DNA secondary structure (see picture). This method rapidly identifies, otherwise unanticipated, potent small-molecule probes to selectively target a given RNA or DNA.
Asunto(s)
Alquinos/química , Azidas/química , ADN/química , G-Cuádruplex , ARN/química , Química Clic , Fibroblastos/citología , Humanos , Microscopía Fluorescente , Estructura Molecular , Telómero/químicaRESUMEN
The complexity of the simplest conceivable cell suggests that the chemistry of prebiotic mixtures needs to be explored to understand the intricate network of prebiotic reactions that led to the emergence of life. Early cells probably relied upon compatible and interconnected chemistries to link RNA, peptides and membranes. Here we show that several types of vesicles, composed of prebiotically plausible mixtures of amphiphiles, spontaneously form and sustain the methyl isocyanide-mediated activation of amino acids, peptides and nucleotides. Activation chemistry also drives the advantageous conversion of reactive monoacylglycerol phosphates into inert cyclophospholipids, thus supporting their potential role as major constituents of protocells. Moreover, activation of prebiotic building blocks within fatty acid-based vesicles yields lipidated species capable of localising to and functionalising primitive membranes. Our findings describe a potentially prebiotic scenario in which the components of primitive cells undergo activation and provide new species that might have enabled an increase in the functionality of protocells.
RESUMEN
During the transition from prebiotic chemistry to biology, a period of solution-phase, non-enzymatic activation of (oligo)nucleotides must have occurred, and accordingly, a mechanism for phosphate activation must have existed. Herein, we detail results of an investigation into prebiotic phosphate activation chemistry using simple, prebiotically available nitriles whose reactivity is increased by Cu2+ ions. Furthermore, although Cu2+ ions are known to catalyse the hydrolysis of phosphodiester bonds, we found this deleterious activity to be almost completely suppressed by inclusion of amino acids or dipeptides, which may suggest a productive relationship between protein and RNA from the outset.
RESUMEN
The TOP2 poison etoposide has been implicated in the generation of secondary malignancies during cancer treatment. Structural similarities between TOP2 isoforms challenge the rational design of isoform-specific poisons to further delineate these processes. Herein, we describe the synthesis and biological evaluation of a focused library of etoposide analogues, with the identification of two novel small molecules exhibiting TOP2B-dependent toxicity. Our findings pave the way toward studying isoform-specific cellular processes by means of small molecule intervention.