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1.
Nucleic Acids Res ; 49(11): 6267-6280, 2021 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-34096575

RESUMEN

Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a cochaperone role during the co-translational folding of actin and tubulin monomers. Additional functions of prefoldin have been described, including a positive contribution to transcription elongation and chromatin dynamics in yeast. Here we show that prefoldin perturbations provoked transcriptional alterations across the human genome. Severe pre-mRNA splicing defects were also detected, particularly after serum stimulation. We found impairment of co-transcriptional splicing during transcription elongation, which explains why the induction of long genes with a high number of introns was affected the most. We detected genome-wide prefoldin binding to transcribed genes and found that it correlated with the negative impact of prefoldin depletion on gene expression. Lack of prefoldin caused global decrease in Ser2 and Ser5 phosphorylation of the RNA polymerase II carboxy-terminal domain. It also reduced the recruitment of the CTD kinase CDK9 to transcribed genes, and the association of splicing factors PRP19 and U2AF65 to chromatin, which is known to depend on CTD phosphorylation. Altogether the reported results indicate that human prefoldin is able to act locally on the genome to modulate gene expression by influencing phosphorylation of elongating RNA polymerase II, and thereby regulating co-transcriptional splicing.


Asunto(s)
Chaperonas Moleculares/fisiología , Empalme del ARN , ARN Mensajero/metabolismo , Transcripción Genética , Línea Celular , Humanos , Intrones , ARN Polimerasa II/metabolismo , Precursores del ARN/metabolismo , Factores de Empalme de ARN/metabolismo , Proteínas Represoras/fisiología , Transcriptoma
2.
EMBO J ; 37(23)2018 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-30373810

RESUMEN

Focal deletions occur frequently in the cancer genome. However, the putative tumor-suppressive genes residing within these regions have been difficult to pinpoint. To robustly identify these genes, we implemented a computational approach based on non-negative matrix factorization, NMF, and interrogated the TCGA dataset. This analysis revealed a metagene signature including a small subset of genes showing pervasive hemizygous deletions, reduced expression in cancer patient samples, and nucleolar function. Amid the genes belonging to this signature, we have identified PNRC1, a nuclear receptor coactivator. We found that PNRC1 interacts with the cytoplasmic DCP1α/DCP2 decapping machinery and hauls it inside the nucleolus. PNRC1-dependent nucleolar translocation of the decapping complex is associated with a decrease in the 5'-capped U3 and U8 snoRNA fractions, hampering ribosomal RNA maturation. As a result, PNRC1 ablates the enhanced proliferation triggered by established oncogenes such as RAS and MYC These observations uncover a previously undescribed mechanism of tumor suppression, whereby the cytoplasmic decapping machinery is hauled within nucleoli, tightly regulating ribosomal RNA maturation.


Asunto(s)
Nucléolo Celular/metabolismo , Proliferación Celular , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , ARN Ribosómico/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Células A549 , Nucléolo Celular/genética , Nucléolo Celular/patología , Bases de Datos de Ácidos Nucleicos , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Células HeLa , Humanos , Células MCF-7 , Neoplasias/genética , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ARN Ribosómico/genética , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Proteínas ras/genética , Proteínas ras/metabolismo
3.
Genome Res ; 29(12): 1974-1984, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31740578

RESUMEN

Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their transcription start site (TSS) usage, chromatin organization, and posttranscriptional consequences in Saccharomyces cerevisiae We show that TSSs of chromatin-sensitive internal cryptic transcripts retain comparable features of canonical TSSs in terms of DNA sequence, directionality, and chromatin accessibility. We define the 5' and 3' boundaries of cryptic transcripts and show that, contrary to RNA degradation-sensitive ones, they often overlap with the end of the gene, thereby using the canonical polyadenylation site, and associate to polyribosomes. We show that chromatin-sensitive cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Finally, we confirm the presence of the predicted polypeptides by reanalyzing N-terminal proteomic data sets. Our work suggests that a fraction of chromatin-sensitive internal cryptic promoters initiates the transcription of alternative truncated mRNA isoforms. The expression of these chromatin-sensitive isoforms is conserved from yeast to human, expanding the functional consequences of cryptic transcription and proteome complexity.


Asunto(s)
Cromatina , Regulación Fúngica de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sitio de Iniciación de la Transcripción , Cromatina/genética , Cromatina/metabolismo , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad del ARN , ARN de Hongos/biosíntesis , ARN de Hongos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Sci Rep ; 11(1): 1820, 2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33469065

RESUMEN

RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.


Asunto(s)
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , SARS-CoV-2/genética , COVID-19/virología , Calor , Humanos , Nasofaringe/virología , ARN Viral/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Juego de Reactivos para Diagnóstico , SARS-CoV-2/aislamiento & purificación , Sensibilidad y Especificidad , Inactivación de Virus
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