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1.
Yeast ; 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38874348

RESUMO

The yeast Saccharomyces cerevisiae and most eukaryotes carry two 5' → 3' exoribonuclease paralogs. In yeast, they are called Xrn1, which shuttles between the nucleus and the cytoplasm, and executes major cytoplasmic messenger RNA (mRNA) decay, and Rat1, which carries a strong nuclear localization sequence (NLS) and localizes to the nucleus. Xrn1 is 30% identical to Rat1 but has an extra ~500 amino acids C-terminal extension. In the cytoplasm, Xrn1 can degrade decapped mRNAs during the last round of translation by ribosomes, a process referred to as "cotranslational mRNA decay." The division of labor between the two enzymes is still enigmatic and serves as a paradigm for the subfunctionalization of many other paralogs. Here we show that Rat1 is capable of functioning in cytoplasmic mRNA decay, provided that Rat1 remains cytoplasmic due to its NLS disruption (cRat1). This indicates that the physical segregation of the two paralogs plays roles in their specific functions. However, reversing segregation is not sufficient to fully complement the Xrn1 function. Specifically, cRat1 can partially restore the cell volume, mRNA stability, the proliferation rate, and 5' → 3' decay alterations that characterize xrn1Δ cells. Nevertheless, cotranslational decay is only slightly complemented by cRat1. The use of the AlphaFold prediction for cRat1 and its subsequent docking with the ribosome complex and the sequence conservation between cRat1 and Xrn1 suggest that the tight interaction with the ribosome observed for Xrn1 is not maintained in cRat1. Adding the Xrn1 C-terminal domain to Rat1 does not improve phenotypes, which indicates that lack of the C-terminal is not responsible for partial complementation. Overall, during evolution, it appears that the two paralogs have acquired specific characteristics to make functional partitioning beneficial.

2.
Biochim Biophys Acta Gene Regul Mech ; 1867(1): 194995, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37967810

RESUMO

The tripartite interaction between the chromatin remodeler complex RSC, RNA polymerase subunit Rpb5 and prefoldin-like Bud27 is necessary for proper RNA pol II elongation. Indeed lack of Bud27 alters this association and affects transcription elongation. This work investigates the consequences of lack of Bud27 on the chromatin association of RSC and RNA pol II, and on nucleosome positioning. Our results demonstrate that RSC binds chromatin in gene bodies and lack of Bud27 alters this association, mainly around polyA sites. This alteration impacts chromatin organization and leads to the accumulation of RNA pol II molecules around polyA sites, likely due to pausing or arrest. Our data suggest that RSC is necessary to maintain chromatin organization around those sites, and any alteration of this organization results in the widespread use of alternative polyA sites. Finally, we also find a similar molecular phenotype that occurs upon TOR inhibition with rapamycin, which suggests that alternative polyadenylation observed upon TOR inhibition is likely Bud27-dependent.


Assuntos
Chaperonas Moleculares , Fatores de Iniciação de Peptídeos , Proteínas de Saccharomyces cerevisiae , Cromatina/metabolismo , Nucleossomos/metabolismo , Poliadenilação , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo
3.
Cell Mol Life Sci ; 80(1): 36, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36627412

RESUMO

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27 , Neurogênese , Fatores de Transcrição SOXB1 , Animais , Camundongos , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Expressão Gênica , Neurogênese/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
4.
Nat Commun ; 14(1): 373, 2023 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-36690670

RESUMO

Neural stem cells (NSCs) in the adult murine subependymal zone balance their self-renewal capacity and glial identity with the potential to generate neurons during the lifetime. Adult NSCs exhibit lineage priming via pro-neurogenic fate determinants. However, the protein levels of the neural fate determinants are not sufficient to drive direct differentiation of adult NSCs, which raises the question of how cells along the neurogenic lineage avoid different conflicting fate choices, such as self-renewal and differentiation. Here, we identify RNA-binding protein MEX3A as a post-transcriptional regulator of a set of stemness associated transcripts at critical transitions in the subependymal neurogenic lineage. MEX3A regulates a quiescence-related RNA signature in activated NSCs that is needed for their return to quiescence, playing a role in the long-term maintenance of the NSC pool. Furthermore, it is required for the repression of the same program at the onset of neuronal differentiation. Our data indicate that MEX3A is a pivotal regulator of adult murine neurogenesis acting as a translational remodeller.


Assuntos
Células-Tronco Neurais , Neurogênese , Camundongos , Animais , Neurogênese/genética , Neurônios/fisiologia , Células-Tronco Neurais/metabolismo , Diferenciação Celular/genética , Proteínas de Ligação a RNA/metabolismo
5.
Front Cell Dev Biol ; 10: 912319, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35938168

RESUMO

Stem cells in adult mammalian tissues are held in a reversible resting state, known as quiescence, for prolonged periods of time. Recent studies have greatly increased our understanding of the epigenetic and transcriptional landscapes that underlie stem cell quiescence. However, the transcription factor code that actively maintains the quiescence program remains poorly defined. Similarly, alternative splicing events affecting transcription factors in stem cell quiescence have been overlooked. Here we show that the transcription factor T-cell factor/lymphoid enhancer factor LEF1, a central player in canonical ß-catenin-dependent Wnt signalling, undergoes alternative splicing and switches isoforms in quiescent neural stem cells. We found that active ß-catenin and its partner LEF1 accumulated in quiescent hippocampal neural stem and progenitor cell (Q-NSPC) cultures. Accordingly, Q-NSPCs showed enhanced TCF/LEF1-driven transcription and a basal Wnt activity that conferred a functional advantage to the cultured cells in a Wnt-dependent assay. At a mechanistic level, we found a fine regulation of Lef1 gene expression. The coordinate upregulation of Lef1 transcription and retention of alternative spliced exon 6 (E6) led to the accumulation of a full-length protein isoform (LEF1-FL) that displayed increased stability in the quiescent state. Prospectively isolated GLAST + cells from the postnatal hippocampus also underwent E6 retention at the time quiescence is established in vivo. Interestingly, LEF1 motif was enriched in quiescence-associated enhancers of genes upregulated in Q-NSPCs and quiescence-related NFIX transcription factor motifs flanked the LEF1 binding sites. We further show that LEF1 interacts with NFIX and identify putative LEF1/NFIX targets. Together, our results uncover an unexpected role for LEF1 in gene regulation in quiescent NSPCs, and highlight alternative splicing as a post-transcriptional regulatory mechanism in the transition from stem cell activation to quiescence.

6.
Stem Cells ; 40(10): 949-962, 2022 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-35861517

RESUMO

During an infection, hematopoiesis is altered to increase the output of mature myeloid cells to fight off the pathogen. Despite convincing evidence that hematopoietic stem and progenitor cells (HSPCs) can sense pathogens directly, more mechanistic studies are needed to reveal whether pattern recognition receptor (PRR) signaling initiates myeloid development directly, or indirectly through the production of cytokines by HSPCs that can act in an autocrine/paracrine manner, or by a combination of both direct and indirect mechanisms. In this study, we have used an in vitro model of murine HSPCs to study myeloid differentiation in response to the TLR2 ligand Pam3CSK4 and showed that, besides indirect mechanisms, TLR2 stimulation of HSPCs promotes myelopoiesis directly by initiating a MyD88-dependent signaling. This direct differentiation program involves a combined activation of the transcription factors PU.1, C/EBPß, and IRF7 driven by TBK1 and PI3K/mTOR. Notably, downstream of MyD88, the activated TBK1 kinase can activate mTOR directly and IRF7 induction is mediated by both TBK1 and mTOR. TLR2 signaling also induces NF-κB dependent IL-6 production that may further induce indirect myeloid differentiation. Our results have identified the direct signaling pathways and the transcription factors involved in macrophage development from HSPCs in response to TLR2 engagement, a critical process to trigger a rapid immune response during infection.


Assuntos
Fator 88 de Diferenciação Mieloide , Receptor 2 Toll-Like , Camundongos , Animais , Receptor 2 Toll-Like/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Interleucina-6/metabolismo , Ligantes , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Macrófagos/metabolismo , Citocinas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases
7.
Methods Mol Biol ; 2477: 57-70, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35524111

RESUMO

RNA biogenesis in eukaryotic cells is a tightly regulated multilayered process in which a diverse set of players act in an orchestrated manner via complex molecular interactions to secure the initial flow of gene expression. Transcription from DNA to RNA is the essential first step in RNA biogenesis, and consists of three main phases: initiation, elongation, and termination. In each phase, transcription factors act on RNA polymerases to modulate their passage along the DNA template in a very precise manner, governed by molecular mechanisms, some of which are not yet fully understood. Genome-scale run-on-based methodologies have been developed with the aim of mapping the position of transcriptionally engaged RNA polymerases. Among them, the BioGRO methodology has been instrumental in advancing our understanding of the transcriptional dynamics in yeast. Here we take the previously known BioGRO method further by coupling it with deep sequencing. BioGRO-seq maps elongating RNA polymerases along the genome with strand specificity and single-nucleotide resolution. BioGRO-seq profiling provides insights into the biogenesis and regulation of not just the canonical protein-coding transcriptome, but also into the often more challenging to study noncoding and unstable transcriptome.


Assuntos
Saccharomyces cerevisiae , Transcrição Gênica , DNA , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
8.
Mol Genet Genomics ; 297(2): 463-484, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35187582

RESUMO

BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes mainly associated with transcriptional initiation. They also have a role in alternative splicing, which has been shown for BRM-containing SWI/SNF complexes at a few genes. Here, we have identified a subset of genes which harbour alternative exons that are affected by SWI/SNF ATPases by expressing the ATPases BRG1 and BRM in C33A cells, a BRG1- and BRM-deficient cell line, and analysed the effect on splicing by RNA sequencing. BRG1- and BRM-affected sub-sets of genes favouring both exon inclusion and exon skipping, with only a minor overlap between the ATPase. Some of the changes in alternative splicing induced by BRG1 and BRM expression did not require the ATPase activity. The BRG1-ATPase independent included exons displayed an exon signature of a high GC content. By investigating three genes with exons affected by the BRG-ATPase-deficient variant, we show that these exons accumulated phosphorylated RNA pol II CTD, both serine 2 and serine 5 phosphorylation, without an enrichment of the RNA polymerase II. The ATPases were recruited to the alternative exons, together with both core and signature subunits of SWI/SNF complexes, and promoted the binding of RNA binding factors to chromatin and RNA at the alternative exons. The interaction with the nascent RNP, however, did not reflect the association to chromatin. The hnRNPL, hnRNPU and SAM68 proteins associated with chromatin in cells expressing BRG1 and BRM wild type, but the binding of hnRNPU to the nascent RNP was excluded. This suggests that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and influence their binding to the nascent pre-mRNA particle.


Assuntos
DNA Helicases , Proteínas Nucleares , RNA , Fatores de Transcrição , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Processamento Alternativo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA/genética , RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Front Oncol ; 11: 630482, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33777782

RESUMO

In human glioblastoma (GBM), the presence of a small population of cells with stem cell characteristics, the glioma stem cells (GSCs), has been described. These cells have GBM potential and are responsible for the origin of the tumors. However, whether GSCs originate from normal neural stem cells (NSCs) as a consequence of genetic and epigenetic changes and/or dedifferentiation from somatic cells remains to be investigated. Genomic imprinting is an epigenetic marking process that causes genes to be expressed depending on their parental origin. The dysregulation of the imprinting pattern or the loss of genomic imprinting (LOI) have been described in different tumors including GBM, being one of the earliest and most common events that occurs in human cancers. Here we have gathered the current knowledge of the role of imprinted genes in normal NSCs function and how the imprinting process is altered in human GBM. We also review the changes at particular imprinted loci that might be involved in the development of the tumor. Understanding the mechanistic similarities in the regulation of genomic imprinting between normal NSCs and GBM cells will be helpful to identify molecular players that might be involved in the development of human GBM.

10.
Front Immunol ; 12: 790309, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34975887

RESUMO

More mechanistic studies are needed to reveal the hidden details of in vivo-induced trained immunity. Here, using a Candida albicans live vaccine mouse model we show that vaccination protects mice against a secondary infection and increases the number of bone marrow, and especially, splenic trained monocytes. Moreover, vaccination expands and reprograms hematopoietic stem and progenitor cells (HSPCs) early during infection and mobilize them transiently to the spleen to produce trained macrophages. Trained HSPCs are not only primed for myeloid cell production but also reprogramed to produce a greater amount of proinflammatory cytokines in response to a second challenge. Additionally, their adoptive transfer is sufficient to protect mice against reinfection. Mechanistically, autocrine GM-CSF activation of HSPCs is responsible for the trained phenotype and essential for the vaccine-induced protection. Our findings reveal a fundamental role for HSPCs in the trained immune protective response, opening new avenues for disease prevention and treatment.


Assuntos
Candida albicans/imunologia , Candidíase/prevenção & controle , Vacinas Fúngicas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Reinfecção/prevenção & controle , Vacinação , Animais , Citocinas/biossíntese , Feminino , Células-Tronco Hematopoéticas/fisiologia , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mielopoese
11.
Cell Stem Cell ; 28(2): 285-299.e9, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33207218

RESUMO

Adult stem cells (SCs) transit between the cell cycle and a poorly defined quiescent state. Single neural SCs (NSCs) with quiescent, primed-for-activation, and activated cell transcriptomes have been obtained from the subependymal zone (SEZ), but the functional regulation of these states under homeostasis is not understood. Here, we develop a multilevel strategy to analyze these NSC states with the aim to uncover signals that regulate their level of quiescence/activation. We show that transitions between states occur in vivo and that activated and primed, but not quiescent, states can be captured and studied in culture. We also show that peripherally induced inflammation promotes a transient activation of primed NSCs (pNSCs) mediated by tumor necrosis factor α (TNF-α) acting through its receptor, TNF receptor 2 (TNFR2), and a return to quiescence in a TNF receptor 1 (TNFR1)-dependent manner. Our data identify a signaling pathway promoting NSC alertness and add to the emerging concept that SCs can respond to the systemic milieu.


Assuntos
Células-Tronco Adultas , Células-Tronco Neurais , Humanos , Inflamação , Ventrículos Laterais , Neurogênese , Receptores do Fator de Necrose Tumoral , Receptores Tipo I de Fatores de Necrose Tumoral , Transdução de Sinais , Fator de Necrose Tumoral alfa
12.
RNA Biol ; 18(9): 1310-1323, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33138675

RESUMO

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5' exhibited significant alterations that were compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3' was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.


Assuntos
Cromatina/metabolismo , Exorribonucleases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição/metabolismo , Cromatina/química , Cromatina/genética , Exorribonucleases/genética , Regulação Fúngica da Expressão Gênica , Nucleossomos/genética , Nucleossomos/metabolismo , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética
13.
RNA ; 26(10): 1360-1379, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32503921

RESUMO

Understanding the functional connection that occurs for the three nuclear RNA polymerases to synthesize ribosome components during the ribosome biogenesis process has been the focal point of extensive research. To preserve correct homeostasis on the production of ribosomal components, cells might require the existence of proteins that target a common subunit of these RNA polymerases to impact their respective activities. This work describes how the yeast prefoldin-like Bud27 protein, which physically interacts with the Rpb5 common subunit of the three RNA polymerases, is able to modulate the transcription mediated by the RNA polymerase I, likely by influencing transcription elongation, the transcription of the RNA polymerase III, and the processing of ribosomal RNA. Bud27 also regulates both RNA polymerase II-dependent transcription of ribosomal proteins and ribosome biogenesis regulon genes, likely by occupying their DNA ORFs, and the processing of the corresponding mRNAs. With RNA polymerase II, this association occurs in a transcription rate-dependent manner. Our data also indicate that Bud27 inactivation alters the phosphorylation kinetics of ribosomal protein S6, a readout of TORC1 activity. We conclude that Bud27 impacts the homeostasis of the ribosome biogenesis process by regulating the activity of the three RNA polymerases and, in this way, the synthesis of ribosomal components. This quite likely occurs through a functional connection of Bud27 with the TOR signaling pathway.


Assuntos
Chaperonas Moleculares/genética , Fatores de Iniciação de Peptídeos/genética , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica/genética , Núcleo Celular/genética , RNA Polimerase II/genética , RNA Polimerase III/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/genética
14.
Nucleic Acids Res ; 47(18): 9524-9541, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31392315

RESUMO

Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4-Not complex conditions its post-transcriptional fate. In turn, mRNA degradation factors like Xrn1 are able to influence RNAPII-dependent transcription, making a feedback loop that contributes to mRNA homeostasis. In this work, we have used repressible yeast GAL genes to perform accurate measurements of transcription and mRNA degradation in a set of mutants. This genetic analysis uncovered a link from mRNA decay to transcription elongation. We combined this experimental approach with computational multi-agent modelling and tested different possibilities of Xrn1 and Ccr4 action in gene transcription. This double strategy brought us to conclude that both Xrn1-decaysome and Ccr4-Not regulate RNAPII elongation, and that they do it in parallel. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII. We found that xrn1Δ and ccr4Δ exhibited very different patterns of TFIIS versus RNAPII occupancy, which confirmed their distinct role in controlling transcription elongation. We also found that the relative influence of Xrn1 and Ccr4 is different in the genes encoding ribosomal proteins as compared to the rest of the genome.


Assuntos
Exorribonucleases/genética , RNA Polimerase II/genética , Estabilidade de RNA/genética , Ribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/genética , Impressão Genômica , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética
15.
Nucleic Acids Res ; 47(12): 6250-6268, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31006804

RESUMO

Specialized telomeric proteins have an essential role in maintaining genome stability through chromosome end protection and telomere length regulation. In the yeast Saccharomyces cerevisiae, the evolutionary conserved CST complex, composed of the Cdc13, Stn1 and Ten1 proteins, largely contributes to these functions. Here, we report genetic interactions between TEN1 and several genes coding for transcription regulators. Molecular assays confirmed this novel function of Ten1 and further established that it regulates the occupancies of RNA polymerase II and the Spt5 elongation factor within transcribed genes. Since Ten1, but also Cdc13 and Stn1, were found to physically associate with Spt5, we propose that Spt5 represents the target of CST in transcription regulation. Moreover, CST physically associates with Hmo1, previously shown to mediate the architecture of S-phase transcribed genes. The fact that, genome-wide, the promoters of genes down-regulated in the ten1-31 mutant are prefentially bound by Hmo1, leads us to propose a potential role for CST in synchronizing transcription with replication fork progression following head-on collisions.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Transcrição Gênica , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Quinases Ciclina-Dependentes/genética , Regulação Fúngica da Expressão Gênica , Fase S/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/metabolismo , Quinase Ativadora de Quinase Dependente de Ciclina
16.
Methods ; 159-160: 177-182, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30716396

RESUMO

The biogenesis of RNAs is a multi-layered and highly regulated process that involves a diverse set of players acting in an orchestrated manner throughout the transcription cycle. Transcription initiation, elongation and termination factors act on RNA polymerases to modulate their movement along the DNA template in a very precise manner, more complex than previously anticipated. Genome-scale run-on-based methodologies have been developed to study in detail the position of transcriptionally-engaged RNA polymerases. Genomic run-on (GRO), and its many variants and refinements made over the years, are helping the community to address an increasing amount of scientific questions, spanning an increasing range of organisms and systems. In this review, we aim to summarize the most relevant high throughput methodologies developed to study nascent RNA by run-on methods, compare their main features, advantages and limitations, while putting them in context with alternative ways of studying the transcriptional process.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , RNA/análise , Transcrição Gênica , Animais , Eucariotos/enzimologia , Eucariotos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA/biossíntese , Análise de Sequência de RNA/métodos
17.
Nucleic Acids Res ; 46(16): 8557-8573, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-29860334

RESUMO

SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3' end maturation by facilitating 3' end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3' end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.


Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Processamento de Terminações 3' de RNA , Ribonucleoproteína Nuclear Pequena U1/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Células HeLa , Humanos , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Transativadores/genética , Transativadores/metabolismo
18.
BMC Genomics ; 19(1): 367, 2018 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-29776334

RESUMO

BACKGROUND: Brahma (BRM) is the only catalytic subunit of the SWI/SNF chromatin-remodeling complex of Drosophila melanogaster. The function of SWI/SNF in transcription has long been attributed to its ability to remodel nucleosomes, which requires the ATPase activity of BRM. However, recent studies have provided evidence for a non-catalytic function of BRM in the transcriptional regulation of a few specific genes. RESULTS: Here we have used RNA-seq and ChIP-seq to identify the BRM target genes in S2 cells, and we have used a catalytically inactive BRM mutant (K804R) that is unable to hydrolyze ATP to investigate the magnitude of the non-catalytic function of BRM in transcription regulation. We show that 49% of the BRM target genes in S2 cells are regulated through mechanisms that do not require BRM to have an ATPase activity. We also show that the catalytic and non-catalytic mechanisms of SWI/SNF regulation operate on two subsets of genes that differ in promoter architecture and are linked to different biological processes. CONCLUSIONS: This study shows that the non-catalytic role of SWI/SNF in transcription regulation is far more prevalent than previously anticipated and that the genes that are regulated by SWI/SNF through ATPase-dependent and ATPase-independent mechanisms have specialized roles in different cellular and developmental processes.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Nucleossomos/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Transativadores/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Linhagem Celular , Genômica , Regiões Promotoras Genéticas/genética
19.
Methods Mol Biol ; 1689: 1-7, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29027160

RESUMO

Protein-DNA interactions in vivo can be detected and quantified by chromatin immunoprecipitation (ChIP). ChIP has been instrumental for the advancement of epigenetics and has set the groundwork for the development of a number of ChIP-related techniques that have provided valuable information about the organization and function of genomes. Here, we provide an introduction to ChIP and discuss the applications of ChIP in different research areas. We also review some of the strategies that have been devised to improve ChIP performance.


Assuntos
Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina/métodos , Proteínas de Ligação a DNA , Epigênese Genética , Epigenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/metabolismo , Humanos , Ligação Proteica , Reprodutibilidade dos Testes
20.
Methods Mol Biol ; 1689: 9-28, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29027161

RESUMO

Arguably one of the most valuable techniques to study chromatin organization, ChIP is the method of choice to map the contacts established between proteins and genomic DNA. Ever since its inception, more than 30 years ago, ChIP has been constantly evolving, improving, and expanding its capabilities and reach. Despite its widespread use by many laboratories across a wide variety of disciplines, ChIP assays can be sometimes challenging to design, and are often sensitive to variations in practical implementation.In this chapter, we provide a general overview of the ChIP method and its most common variations, with a special focus on ChIP-seq. We try to address some of the most important aspects that need to be taken into account in order to design and perform experiments that generate the most reproducible, high-quality data. Some of the main topics covered include the use of properly characterized antibodies, alternatives to chromatin preparation, the need for proper controls, and some recommendations about ChIP-seq data analysis.


Assuntos
Imunoprecipitação da Cromatina , Interpretação Estatística de Dados , Sequenciamento de Nucleotídeos em Larga Escala , Projetos de Pesquisa , Animais , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina/métodos , Imunoprecipitação da Cromatina/normas , DNA , Proteínas de Ligação a DNA , Guias como Assunto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Controle de Qualidade , Reprodutibilidade dos Testes , Fluxo de Trabalho
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