RESUMEN
End resection in homologous recombination (HR) and HR-mediated repair of DNA double-strand breaks (DSBs) removes several kilobases from 5' strands of DSBs, but 3' strands are exempted from degradation. The mechanism by which the 3' overhangs are protected has not been determined. Here, we established that the protection of 3' overhangs is achieved through the transient formation of RNA-DNA hybrids. The DNA strand in the hybrids is the 3' ssDNA overhang, while the RNA strand is newly synthesized. RNA polymerase III (RNAPIII) is responsible for synthesizing the RNA strand. Furthermore, RNAPIII is actively recruited to DSBs by the MRN complex. CtIP and MRN nuclease activity is required for initiating the RNAPIII-mediated RNA synthesis at DSBs. A reduced level of RNAPIII suppressed HR, and genetic loss > 30 bp increased at DSBs. Thus, RNAPIII is an essential HR factor, and the RNA-DNA hybrid is an essential repair intermediate for protecting the 3' overhangs in DSB repair.
Asunto(s)
ARN Polimerasa III/metabolismo , Reparación del ADN por Recombinación , Ciclo Celular , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas/genética , Células HEK293 , Humanos , Proteína Homóloga de MRE11/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Hibridación de Ácido Nucleico , ARN/químicaRESUMEN
RNA polymerase (Pol) III has a specialized role in transcribing the most abundant RNAs in eukaryotic cells, transfer RNAs (tRNAs), along with other ubiquitous small noncoding RNAs, many of which have functions related to the ribosome and protein synthesis. The high energetic cost of producing these RNAs and their central role in protein synthesis underlie the robust regulation of Pol III transcription in response to nutrients and stress by growth regulatory pathways. Downstream of Pol III, signaling impacts posttranscriptional processes affecting tRNA function in translation and tRNA cleavage into smaller fragments that are increasingly attributed with novel cellular activities. In this review, we consider how nutrients and stress control Pol III transcription via its factors and its negative regulator, Maf1. We highlight recent work showing that the composition of the tRNA population and the function of individual tRNAs is dynamically controlled and that unrestrained Pol III transcription can reprogram central metabolic pathways.
Asunto(s)
ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Animales , Humanos , Modelos Biológicos , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación , Conformación Proteica , ARN Polimerasa III/química , Procesamiento Postranscripcional del ARN , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Estrés Fisiológico , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT.
Asunto(s)
Neoplasias de la Mama/patología , Exosomas/patología , ARN no Traducido/metabolismo , Células del Estroma/patología , Microambiente Tumoral , Neoplasias de la Mama/metabolismo , Proteína 58 DEAD Box/metabolismo , Exosomas/metabolismo , Humanos , Factores Reguladores del Interferón/metabolismo , Células MCF-7 , Metástasis de la Neoplasia , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Receptores Inmunológicos , Receptores de Reconocimiento de Patrones/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Células del Estroma/metabolismo , Virosis/metabolismoRESUMEN
Leone et al.1 reveal that Pol III transcription complexes recruit a chaperone, HSP70, to execute cotranscriptional cleavage of precursor tRNA. HSP70 binds to the polymerase and translocates to nascent precursor tRNA and then tRNA. The last complex facilitates Pol III to engage in a new, efficient transcription cycle with another HSP70.
Asunto(s)
ARN de Transferencia , Transcripción Genética , ARN de Transferencia/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , ARN Polimerasa III/metabolismoRESUMEN
Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.
Asunto(s)
Proteínas HSP70 de Choque Térmico , Neoplasias , Humanos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , ARN , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN de Transferencia/genética , ARN no Traducido/genéticaRESUMEN
Transcription of tRNA genes by RNA polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the local chromatin proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII-regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced, and the shutdown of ribosome biogenesis genes was impaired upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and tuning the physiological response to changing metabolic demands.
Asunto(s)
ARN Polimerasa III , Proteínas de Saccharomyces cerevisiae , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Transcripción GenéticaRESUMEN
RNA polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here, we use cryoelectron microscopy (cryo-EM) to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Gene-specific factor TFIIIA interacts with DNA and acts as an adaptor for TFIIIC-promoter interactions. We also visualize DNA binding of TFIIIB subunits, Brf1 and TBP (TATA-box binding protein), which results in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA within the complex undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the transcription initiation complex assembly on the 5S rRNA promoter and allow us to directly compare Pol III and Pol II transcription adaptations.
Asunto(s)
Factores de Transcripción , Transcripción Genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Transcripción TFIIIB/genética , Factor de Transcripción TFIIIB/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Microscopía por Crioelectrón , ADN/metabolismoRESUMEN
TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.
Asunto(s)
Oxidación-Reducción , Factor de Transcripción TFIIIB/química , Factor de Transcripción TFIIIB/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , ARN Polimerasa III/metabolismo , Saccharomyces cerevisiae , Alineación de Secuencia , Transducción de SeñalRESUMEN
Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.
Asunto(s)
Edición Génica , Proteínas de Unión al ARN , Humanos , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Células K562 , Poli U/genética , Poli U/metabolismo , ARN Polimerasa III/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
How repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.
Asunto(s)
Elementos Alu/fisiología , Histonas/metabolismo , Factores de Transcripción TFIII/metabolismo , Acetilación , Elementos Alu/genética , Línea Celular , Cromatina/metabolismo , Cromatina/fisiología , Epigénesis Genética/genética , Regulación de la Expresión Génica/genética , Histonas/genética , Proteínas de Homeodominio/genética , Humanos , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-Postraduccional , ARN Polimerasa III/metabolismo , Factores de Transcripción TFIII/genética , Transcripción Genética/genéticaRESUMEN
Increasing evidence suggests that tRNA levels are dynamically and specifically regulated in response to internal and external cues to modulate the cellular translational program. However, the molecular players and the mechanisms regulating the gene-specific expression of tRNAs are still unknown. Using an inducible auxin-degron system to rapidly deplete RPB1 (the largest subunit of RNA Pol II) in living cells, we identified Pol II as a direct gene-specific regulator of tRNA transcription. Our data suggest that Pol II transcription robustly interferes with Pol III function at specific tRNA genes. This activity was further found to be essential for MAF1-mediated repression of a large set of tRNA genes during serum starvation, indicating that repression of tRNA genes by Pol II is dynamically regulated. Hence, Pol II plays a direct and central role in the gene-specific regulation of tRNA expression.
Asunto(s)
Regulación de la Expresión Génica , ARN Polimerasa III/metabolismo , ARN Polimerasa II/metabolismo , ARN de Transferencia/metabolismo , Proteínas Represoras/metabolismo , Proteínas Celulares de Unión al Retinol/metabolismo , Transcripción Genética , Células HeLa , Humanos , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/genética , ARN Polimerasa III/genética , ARN de Transferencia/genética , Proteínas Represoras/genética , Proteínas Celulares de Unión al Retinol/genéticaRESUMEN
Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5' enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding.
Asunto(s)
ARN Polimerasa III/genética , ARN Polimerasa II/genética , ARN Polimerasa I/genética , ARN de Hongos/química , Saccharomyces cerevisiae/genética , Elongación de la Transcripción Genética , Composición de Base , Secuencia de Bases , Sitios de Unión , Cromatina/química , Cromatina/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Regulación Fúngica de la Expresión Génica , Unión Proteica , Pliegue del ARN , ARN Polimerasa I/metabolismo , ARN Polimerasa II/metabolismo , ARN Polimerasa III/metabolismo , Sitios de Empalme de ARN , Empalme del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , TermodinámicaRESUMEN
RNA polymerase (Pol) III is responsible for transcription of different noncoding genes in eukaryotic cells, whose RNA products have well-defined functions in translation and other biological processes for some, and functions that remain to be defined for others. For all of them, however, new functions are being described. For example, Pol III products have been reported to regulate certain proteins such as protein kinase R (PKR) by direct association, to constitute the source of very short RNAs with regulatory roles in gene expression, or to control microRNA levels by sequestration. Consistent with these many functions, deregulation of Pol III transcribed genes is associated with a large variety of human disorders. Here we review different human diseases that have been linked to defects in the Pol III transcription apparatus or to Pol III products imbalance and discuss the possible underlying mechanisms.
Asunto(s)
Enfermedad/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Neoplasias/genética , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Transcripción Genética , Animales , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/enzimología , Humanos , Mutación , Neoplasias/enzimología , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
Enhancers are the key regulators of other DNA-based processes by virtue of their unique ability to generate nucleosome-depleted regions in a highly regulated manner. Enhancers regulate cell-type-specific transcription of tRNA genes by RNA polymerase III (Pol III). They are also responsible for the binding of the origin replication complex (ORC) to DNA replication origins, thereby regulating origin utilization, replication timing, and replication-dependent chromosome breaks. Additionally, enhancers regulate V(D)J recombination by increasing access of the recombination-activating gene (RAG) recombinase to target sites and by generating non-coding enhancer RNAs and localized regions of trimethylated histone H3-K4 recognized by the RAG2 PHD domain. Thus, enhancers represent the first step in decoding the genome, and hence they regulate biological processes that, unlike RNA polymerase II (Pol II) transcription, do not have dedicated regulatory proteins.
Asunto(s)
Replicación del ADN , Elementos de Facilitación Genéticos , ARN Polimerasa III , Transcripción Genética , Recombinación V(D)J , Animales , Humanos , Replicación del ADN/genética , Regulación de la Expresión Génica/genética , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Transcripción Genética/genética , Recombinación V(D)J/genéticaRESUMEN
The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP-exo/seq)1,2 to define this architecture in Saccharomyces cerevisiae. We identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome-depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI-SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico/genética , Complejos Multiproteicos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Coenzimas/metabolismo , Complejos Multiproteicos/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa I/metabolismo , ARN Polimerasa II/metabolismo , ARN Polimerasa III/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factor de Transcripción TFIID , Factores de Transcripción/metabolismoRESUMEN
Small regulatory RNAs can move between organisms and regulate gene expression in the recipient. Whether the trans-species small RNAs being exported are distinguished from the normal endogenous small RNAs of the source organism is not known. The parasitic plant Cuscuta campestris (dodder) produces many microRNAs that specifically accumulate at the host-parasite interface, several of which have trans-species activity. We found that induction of C. campestris interface-induced microRNAs is similar regardless of host species and occurs in C. campestris haustoria produced in the absence of any host. The loci-encoding C. campestris interface-induced microRNAs are distinguished by a common cis-regulatory element. This element is identical to a conserved upstream sequence element (USE) used by plant small nuclear RNA loci. The properties of the interface-induced microRNA primary transcripts strongly suggest that they are produced via U6-like transcription by RNA polymerase III. The USE promotes accumulation of interface-induced miRNAs (IIMs) in a heterologous system. This promoter element distinguishes C. campestris IIM loci from other plant small RNAs. Our data suggest that C. campestris IIMs are produced in a manner distinct from canonical miRNAs. All confirmed C. campestris microRNAs with documented trans-species activity are interface-induced and possess these features. We speculate that RNA polymerase III transcription of IIMs may allow these miRNAs to be exported to hosts.
Asunto(s)
Cuscuta , MicroARNs , Parásitos , Animales , MicroARNs/genética , MicroARNs/metabolismo , Cuscuta/genética , Cuscuta/metabolismo , Parásitos/genética , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , ARN Polimerasa III/metabolismo , Interacciones Huésped-Parásitos , Plantas/genéticaRESUMEN
Bud27 is a prefoldin-like protein that participates in transcriptional regulation mediated by the three RNA polymerases in Saccharomyces cerevisiae. Lack of Bud27 significantly affects RNA pol III transcription, although the involved mechanisms have not been characterized. Here, we show that Bud27 regulates the phosphorylation state of the RNA pol III transcriptional repressor, Maf1, influences its nuclear localization, and likely its activity. We demonstrate that Bud27 is associated with the Maf1 main phosphatase PP4 in vivo, and that this interaction is required for proper Maf1 dephosphorylation. Lack of Bud27 decreases the interaction among PP4 and Maf1, Maf1 dephosphorylation, and its nuclear entry. Our data uncover a new nuclear function of Bud27, identify PP4 as a novel Bud27 interactor and demonstrate the effect of this prefoldin-like protein on the posttranslational regulation of Maf1. Finally, our data reveal a broader effect of Bud27 on PP4 activity by influencing, at least, the phosphorylation of Rad53.
Asunto(s)
Fosfoproteínas Fosfatasas , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fosforilación , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Regulación Fúngica de la Expresión Génica , Núcleo Celular/metabolismo , ARN Polimerasa III/metabolismo , ARN Polimerasa III/genética , Factores de TranscripciónRESUMEN
In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.
Asunto(s)
Cromatina , ADN-Topoisomerasas de Tipo II , Sustancias Intercalantes , ARN Polimerasa II , Humanos , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/genética , Carbazoles , Cromatina/metabolismo , Dicetopiperazinas , ADN/metabolismo , ADN/química , Daño del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Histonas/metabolismo , Sustancias Intercalantes/farmacología , Sustancias Intercalantes/química , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Polimerasa I/metabolismo , ARN Polimerasa I/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , ARN Polimerasa III/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Transcripción Genética/efectos de los fármacos , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Aclarubicina/farmacologíaRESUMEN
RNA polymerase II (Pol II) small nuclear RNA (snRNA) promoters and type 3 Pol III promoters have highly similar structures; both contain an interchangeable enhancer and "proximal sequence element" (PSE), which recruits the SNAP complex (SNAPc). The main distinguishing feature is the presence, in the type 3 promoters only, of a TATA box, which determines Pol III specificity. To understand the mechanism by which the absence or presence of a TATA box results in specific Pol recruitment, we examined how SNAPc and general transcription factors required for Pol II or Pol III transcription of SNAPc-dependent genes (i.e., TATA-box-binding protein [TBP], TFIIB, and TFIIA for Pol II transcription and TBP and BRF2 for Pol III transcription) assemble to ensure specific Pol recruitment. TFIIB and BRF2 could each, in a mutually exclusive fashion, be recruited to SNAPc. In contrast, TBP-TFIIB and TBP-BRF2 complexes were not recruited unless a TATA box was present, which allowed selective and efficient recruitment of the TBP-BRF2 complex. Thus, TBP both prevented BRF2 recruitment to Pol II promoters and enhanced BRF2 recruitment to Pol III promoters. On Pol II promoters, TBP recruitment was separate from TFIIB recruitment and enhanced by TFIIA. Our results provide a model for specific Pol recruitment at SNAPc-dependent promoters.
Asunto(s)
Regiones Promotoras Genéticas , ARN Polimerasa III/metabolismo , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Células HEK293 , Humanos , Mutación , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , TATA Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Ribosomes are macromolecular machines that are globally required for the translation of all proteins in all cells. Ribosome biogenesis, which is essential for cell growth, proliferation and survival, commences with transcription of a variety of RNAs by RNA Polymerases I and III. RNA Polymerase I (Pol I) transcribes ribosomal RNA (rRNA), while RNA Polymerase III (Pol III) transcribes 5S ribosomal RNA and transfer RNAs (tRNA) in addition to a wide variety of small non-coding RNAs. Interestingly, despite their global importance, disruptions in Pol I and Pol III function result in tissue-specific developmental disorders, with craniofacial anomalies and leukodystrophy/neurodegenerative disease being among the most prevalent. Furthermore, pathogenic variants in genes encoding subunits shared between Pol I and Pol III give rise to distinct syndromes depending on whether Pol I or Pol III function is disrupted. In this review, we discuss the global roles of Pol I and III transcription, the consequences of disruptions in Pol I and III transcription, disorders arising from pathogenic variants in Pol I and Pol III subunits, and mechanisms underpinning their tissue-specific phenotypes.