RESUMEN
Covalent modifications to histones are essential for development, establishing distinct and functional chromatin domains from a common genetic sequence. Whereas repressed chromatin is robustly inherited, no mechanism that facilitates inheritance of an activated domain has been described. Here, we report that the Set3C histone deacetylase scaffold Snt1 can act as a prion that drives the emergence and transgenerational inheritance of an activated chromatin state. This prion, which we term [ESI+] for expressed sub-telomeric information, is triggered by transient Snt1 phosphorylation upon cell cycle arrest. Once engaged, the prion reshapes the activity of Snt1 and the Set3C complex, recruiting RNA pol II and interfering with Rap1 binding to activate genes in otherwise repressed sub-telomeric domains. This transcriptional state confers broad resistance to environmental stress, including antifungal drugs. Altogether, our results establish a robust means by which a prion can facilitate inheritance of an activated chromatin state to provide adaptive benefit.
Asunto(s)
Cromatina/genética , Histona Desacetilasas/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/genética , Factores de Transcripción/genética , Puntos de Control del Ciclo Celular/genética , Código de Histonas/genética , Histonas/genética , Fosforilación/genética , Priones/genética , ARN Polimerasa II/genética , Saccharomyces cerevisiae , Complejo Shelterina , Telómero/genética , Transcripción GenéticaRESUMEN
Theory and experiments suggest that organisms would benefit from pre-adaptation to future stressors based on reproducible environmental fluctuations experienced by their ancestors, but the mechanisms driving pre-adaptation remain enigmatic. We report that the [SMAUG+] prion allows yeast to anticipate nutrient repletion after periods of starvation, providing a strong selective advantage. By transforming the landscape of post-transcriptional gene expression, [SMAUG+] regulates the decision between two broad growth and survival strategies: mitotic proliferation or meiotic differentiation into a stress-resistant state. [SMAUG+] is common in laboratory yeast strains, where standard propagation practice produces regular cycles of nutrient scarcity followed by repletion. Distinct [SMAUG+] variants are also widespread in wild yeast isolates from multiple niches, establishing that prion polymorphs can be utilized in natural populations. Our data provide a striking example of how protein-based epigenetic switches, hidden in plain sight, can establish a transgenerational memory that integrates adaptive prediction into developmental decisions.
Asunto(s)
Diferenciación Celular/fisiología , Priones/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Adaptación Fisiológica/fisiología , Proliferación Celular/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Spatiotemporal gene regulation is often driven by RNA-binding proteins that harbor long intrinsically disordered regions in addition to folded RNA-binding domains. We report that the disordered region of the evolutionarily ancient developmental regulator Vts1/Smaug drives self-assembly into gel-like condensates. These proteinaceous particles are not composed of amyloid, yet they are infectious, allowing them to act as a protein-based epigenetic element: a prion [SMAUG+]. In contrast to many amyloid prions, condensation of Vts1 enhances its function in mRNA decay, and its self-assembly properties are conserved over large evolutionary distances. Yeast cells harboring [SMAUG+] downregulate a coherent network of mRNAs and exhibit improved growth under nutrient limitation. Vts1 condensates formed from purified protein can transform naive cells to acquire [SMAUG+]. Our data establish that non-amyloid self-assembly of RNA-binding proteins can drive a form of epigenetics beyond the chromosome, instilling adaptive gene expression programs that are heritable over long biological timescales.
Asunto(s)
Amiloide/genética , Expresión Génica/genética , Priones/genética , Regulación hacia Abajo/genética , Epigénesis Genética/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptome-wide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.
Asunto(s)
ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sitios de Unión , Biología Computacional/métodos , Redes Reguladoras de Genes , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estabilidad del ARN , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN Mensajero/química , Saccharomyces cerevisiae/metabolismoRESUMEN
There are many biological contexts in which DNA damage generates "dirty" breaks with 3'-PO4 (or cyclic-PO4) and 5'-OH ends that cannot be sealed by DNA ligases. Here we show that the Escherichia coli RNA ligase RtcB can splice these dirty DNA ends via a unique chemical mechanism. RtcB transfers GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form a "capped" 3' end structure, DNA3'pp5'G. When a suitable DNA 5'-OH end is available, RtcB catalyzes attack of the 5'-OH on DNA3'pp5'G to form a 3'-5' phosphodiester splice junction. Our findings unveil an enzymatic capacity for DNA 3' capping and the sealing of DNA breaks with 3'-PO4 and 5'-OH termini, with implications for DNA repair and DNA rearrangements.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Roturas del ADN , Reparación del ADN/fisiología , ADN Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genéticaRESUMEN
A cherished tenet of nucleic acid enzymology holds that synthesis of polynucleotide 3'-5' phosphodiesters proceeds via the attack of a 3'-OH on a high-energy 5' phosphoanhydride: either a nucleoside 5'-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A(5')pp(5')N--in the case of polynucleotide ligases. RtcB exemplifies a family of RNA ligases implicated in tRNA splicing and repair. Unlike classic ligases, RtcB seals broken RNAs with 3'-phosphate and 5'-OH ends. Here we show that RtcB executes a three-step ligation pathway entailing (i) reaction of His337 of the enzyme with GTP to form a covalent RtcB-(histidinyl-N)-GMP intermediate; (ii) transfer of guanylate to a polynucleotide 3'-phosphate to form a polynucleotide-(3')pp(5')G intermediate; and (iii) attack of a 5'-OH on the -N(3')pp(5')G end to form the splice junction. RtcB is structurally sui generis, and its chemical mechanism is unique. The wide distribution of RtcB proteins in bacteria, archaea, and metazoa raises the prospect of an alternative enzymology based on covalently activated 3' ends.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/metabolismo , Polinucleótidos/metabolismo , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/genética , Secuencia de Bases , Sitios de Unión , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Histidina/genética , Histidina/metabolismo , Cinética , Datos de Secuencia Molecular , Mutación , Fosfatos/metabolismo , ARN Ligasa (ATP)/genética , ARN Ligasa (ATP)/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismoRESUMEN
The RNA ligase RtcB splices broken RNAs with 5'-OH and either 2',3'-cyclic phosphate or 3'-phosphate ends. The 3'-phosphate ligase activity requires GTP and entails the formation of covalent RtcB-(histidinyl)-GMP and polynucleotide-(3')pp(5')G intermediates. There are currently two models for how RtcB executes the strand sealing step. Scheme 1 holds that the RNA 5'-OH end attacks the 3'-phosphorus of the N(3')pp(5')G end to form a 3',5'-phosphodiester and release GMP. Scheme 2 posits that the N(3')pp(5')G end is converted to a 2',3'-cyclic phosphodiester, which is then attacked directly by the 5'-OH RNA end to form a 3',5'-phosphodiester. Here we show that the sealing of a 2',3'-cyclic phosphate end by RtcB requires GTP, is contingent on formation of the RtcB-GMP adduct, and involves a kinetically valid RNA(3')pp(5')G intermediate. Moreover, we find that RtcB catalyzes the hydrolysis of a 2',3'-cyclic phosphate to a 3'-phosphate at a rate that is at least as fast as the rate of ligation. These results weigh in favor of scheme 1. The cyclic phosphodiesterase activity of RtcB depends on GTP and the formation of the RtcB-GMP adduct, signifying that RtcB guanylylation precedes the cyclic phosphodiesterase and 3'-phosphate ligase steps of the RNA splicing pathway.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Trifosfato/metabolismo , Nucleotidasas/metabolismo , Empalme del ARN , Cinética , Fosfatos/metabolismo , Nucleótidos de Purina/metabolismo , ARN/química , ARN/metabolismoRESUMEN
RNA 3'-phosphate cyclase (RtcA) synthesizes RNA 2',3' cyclic phosphate ends via three steps: reaction with ATP to form a covalent RtcA-(histidinyl-Nε)-AMP intermediate; transfer of adenylate to an RNA 3'-phosphate to form RNA(3')pp(5')A; and attack of the vicinal O2' on the 3'-phosphorus to form a 2',3' cyclic phosphate and release AMP. Here we report the crystal structures of RtcAâ¢ATP, RtcAâ¢ATPâ¢Mn(2+), and RtcAâ¢ATPâ¢Co(2+) substrate complexes and an RtcAâ¢AMP product complex. Together with the structures of RtcA apoenzyme and the covalent RtcA-AMP intermediate, they illuminate the mechanism of nucleotidyl transfer, especially the stereochemical transitions at the AMP phosphate, the critical role of the metal in orienting the PP(i) leaving group of ATP during step 1, and the protein conformational switches that accompany substrate binding and product release. The octahedral metal complex of RtcAâ¢ATPâ¢Mn(2+) includes nonbridging oxygens from each of the ATP phosphates, two waters, and Glu14 as the sole RtcA component. Whereas the RtcA adenylylation step is metal-catalyzed, the subsequent steps in the cyclization pathway are metal-independent.
Asunto(s)
Adenosina Trifosfato/metabolismo , Escherichia coli/enzimología , Ligasas/química , Modelos Moleculares , Conformación Proteica , ARN Nucleotidiltransferasas/química , Catálisis , Cobalto/química , Cobalto/metabolismo , Cristalización , Ligasas/metabolismo , Manganeso/química , Manganeso/metabolismo , Sondas de Oligonucleótidos/genética , ARN Nucleotidiltransferasas/metabolismo , Difracción de Rayos XRESUMEN
tRNA anticodon damage inflicted by the Kluyveromyces lactis γ-toxin underlies an RNA-based innate immune system that distinguishes self from nonself species. γ-toxin arrests the growth of Saccharomyces cerevisiae by incising a single phosphodiester 3' of the wobble base of tRNA(Glu(UUC)) to generate a break with 2',3'-cyclic phosphate and 5'-OH ends. Recombinant γ-toxin cleaves oligonucleotide substrates in vitro that mimic the anticodon stem-loop of tRNA(Glu). A single 2'-deoxy sugar substitution at the wobble nucleoside abolishes anticodon nuclease activity. To gain further insights to γ-toxin's substrate specificity, we tested deoxynucleoside effects at positions other than the site of transesterification. The results attest to a stringent requirement for a ribonucleoside at the uridine 5' of the wobble base. In contrast, every other nonwobble ribonucleoside in the anticodon loop can be replaced by a deoxy without significantly affecting γ-toxin's cleavage activity. Whereas either the 5' half or the 3' half of the anticodon stem can be replaced en bloc with DNA without a major effect, simultaneously replacing both strands with DNA interfered strongly, signifying that γ-toxin requires an A-form helical conformation of the anticodon stem. We purified γ-toxin mutants identified previously as nontoxic in vivo and gauged their anticodon nuclease activities in vitro. The results highlight Glu9 and Arg151 as candidate catalytic residues, along with His209 implicated previously. By analogy to other endoribonucleases, we speculate that γ-toxin drives transesterification by general acid-base catalysis (via His209 and Glu9) and transition-state stabilization (via Arg151).
Asunto(s)
Análisis Mutacional de ADN , Factores Asesinos de Levadura/genética , Factores Asesinos de Levadura/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasas/genética , Sustitución de Aminoácidos , Arginina/genética , Secuencia de Bases , Células Eucariotas/metabolismo , Ácido Glutámico/genética , Kluyveromyces/enzimología , Kluyveromyces/genética , Kluyveromyces/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Mutación Missense/fisiología , Conformación de Ácido Nucleico , Ribonucleasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato/genéticaRESUMEN
RNA 3'-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2',3'-cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP(i); transfer of AMP from Rtc to an RNA 3'-phosphate to form an RNA(3')pp(5')A intermediate; and attack by the terminal nucleoside O2' on the 3'-phosphate to form an RNA 2',3'-cyclic phosphate product and release AMP. The chemical transformations of the cyclase pathway resemble those of RNA and DNA ligases, with the key distinction being that ligases covalently adenylylate 5'-phosphate ends en route to phosphodiester synthesis. Here we show that the catalytic repertoire of RNA cyclase overlaps that of ligases. We report that Escherichia coli RtcA catalyzes adenylylation of 5'-phosphate ends of DNA or RNA strands to form AppDNA and AppRNA products. The polynucleotide 5' modification reaction requires the His(309) nucleophile, signifying that it proceeds through a covalent RtcA-AMP intermediate. We established this point directly by demonstrating transfer of [(32)P]AMP from RtcA to a pDNA strand. RtcA readily adenylylated the 5'-phosphate at a 5'-PO(4)/3'-OH nick in duplex DNA but was unable to covert the nicked DNA-adenylate to a sealed phosphodiester. Our findings raise the prospect that cyclization of RNA 3'-ends might not be the only biochemical pathway in which Rtc enzymes participate; we discuss scenarios in which the 5'-adenylyltransferase of RtcA might play a role.
Asunto(s)
ADN Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Ligasas/metabolismo , ARN Bacteriano/metabolismo , Adenosina Monofosfato/química , Adenosina Monofosfato/genética , Adenosina Monofosfato/metabolismo , Catálisis , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ligasas/química , Ligasas/genética , ARN Bacteriano/química , ARN Bacteriano/genéticaRESUMEN
RtcB enzymes are a newly discovered family of RNA ligases, implicated in tRNA splicing and other RNA repair reactions, that seal broken RNAs with 2',3'-cyclic phosphate and 5'-OH ends. Parsimony and energetics would suggest a one-step mechanism for RtcB sealing via attack by the O5' nucleophile on the cyclic phosphate, with expulsion of the ribose O2' and generation of a 3',5'-phosphodiester at the splice junction. Yet we find that RtcB violates Occam's razor, insofar as (i) it is adept at ligating 3'-monophosphate and 5'-OH ends; (ii) it has an intrinsic 2',3'-cyclic phosphodiesterase activity. The 2',3'-cyclic phosphodiesterase and ligase reactions both require manganese and are abolished by mutation of the RtcB active site. Thus, RtcB executes a unique two-step pathway of strand joining whereby the 2',3'-cyclic phosphodiester end is hydrolyzed to a 3'-monophosphate, which is then linked to the 5'-OH end to form the splice junction. The energy for the 3'-phosphate ligase activity is provided by GTP, which reacts with RtcB in the presence of manganese to form a covalent RtcB-guanylate adduct. This adduct is sensitive to acid and hydroxylamine but resistant to alkali, consistent with a phosphoramidate bond.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Nucleotidasas/metabolismo , ARN/metabolismo , Aminoacil-ARNt Sintetasas/genética , Proteínas de Escherichia coli/genética , Guanosina Trifosfato/metabolismo , Manganeso/metabolismo , Mutagénesis Sitio-Dirigida , Nucleotidasas/genética , Empalme del ARN/genética , Empalme del ARN/fisiologíaRESUMEN
Multicellularity was a watershed development in evolution. However, it also meant that individual cells could escape regulatory mechanisms that restrict proliferation at a severe cost to the organism: cancer. From the standpoint of cellular organization, evolutionary complexity scales to organize different molecules within the intracellular milieu. The recent realization that many biomolecules can "phase-separate" into membraneless organelles, reorganizing cellular biochemistry in space and time, has led to an explosion of research activity in this area. In this review, we explore mechanistic connections between phase separation and cancer-associated processes and emerging examples of how these become deranged in malignancy. SIGNIFICANCE: One of the fundamental functions of phase separation is to rapidly and dynamically respond to environmental perturbations. Importantly, these changes often lead to alterations in cancer-relevant pathways and processes. This review covers recent advances in the field, including emerging principles and mechanisms of phase separation in cancer.
Asunto(s)
Neoplasias , Orgánulos , Humanos , Neoplasias/metabolismo , Orgánulos/metabolismo , InvestigaciónRESUMEN
A central tenet of molecular biology is that heritable information is stored in nucleic acids. However, this paradigm has been overturned by a group of proteins called "prions." Prion proteins, many of which are intrinsically disordered, can adopt multiple conformations, at least one of which has the capacity to self-template. This unusual folding landscape drives a form of extreme epigenetic inheritance that can be stable through both mitotic and meiotic cell divisions. Although the first prion discovered-mammalian PrP-is the causative agent of debilitating neuropathies, many additional prions have now been identified that are not obviously detrimental and can even be adaptive. Intrinsically disordered regions, which endow proteins with the bulk property of "phase-separation," can also be drivers of prion formation. Indeed, many protein domains that promote phase separation have been described as prion-like. In this review, we describe how prions lie at the crossroads of phase separation, epigenetic inheritance, and evolutionary adaptation.
Asunto(s)
Epigénesis Genética , Priones/química , Priones/genética , Animales , Evolución Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Modelos Moleculares , Priones/metabolismo , Dominios Proteicos , Pliegue de Proteína , Levaduras/genética , Levaduras/metabolismoRESUMEN
tRNA restriction by anticodon nucleases underlies cellular stress responses and self-nonself discrimination in a wide range of taxa. Anticodon breakage inhibits protein synthesis, which, in turn, results in growth arrest or cell death. The eukaryal ribotoxin PaT secreted by Pichia acaciae inhibits growth of Saccharomyces cerevisiae via cleavage of tRNA(Gln(UUG)). We find that recombinant PaT incises a synthetic tRNA(Gln(UUG)) stem-loop RNA by transesterification at a single site 3' of the wobble uridine, yielding 2',3'-cyclic phosphate and 5'-OH ends. Incision is suppressed by replacement of the wobble nucleobase with adenine or guanine. The crystal structure of PaT reveals a distinctive fold and active site, essential components of which are demonstrated by mutagenesis. Pichia acaciae evades self-toxicity via a distinctive intracellular immunity protein, ImmPaT, which binds PaT and blocks nuclease activity. Our results highlight the evolutionary diversity of tRNA restriction and immunity systems.