BioGRO: un nuveo método de alta resolución para el estudio de la transcripción naciente a escala genómica en levadura

Esta tesis parte de la existencia de una técnica genómica para el estudio de la transcripción naciente en levadura ampliamente utilizada y contrastada: el Genomic run-on, basada en la utilización de macrochips de las ORFs completas del genoma de S. cerevisiae. Debido a la aparición progresiva de nuevas plataformas que permiten interrogar la totalidad de las regiones del genoma, y a una resolución mayor, como los microchips de embaldosado o tiling arrays, el objetivo principal de esta tesis es la puesta a punto de una técnica adaptada a ellas que permita un análisis detallado de la transcripción naciente. Los objetivos concretos que se marcaron fueron: -Desarrollar un nuevo procedimiento de run-on a escala genómica que sustituya el uso de las plataformas basadas en radiactividad y aproveche las plataformas de más resolución, así como de las herramientas bioinformáticas necesarias para el análisis de los datos generados. -Estudiar los perfiles globales de transcripción naciente aprovechando el carácter específico de hebra de los datos para estudiar las dinámicas del transcriptoma global de levadura. Comparar y evaluar la complementariedad de los datos con otras medidas alternativas de tasas de transcripción existentes en la actualidad. -Aplicar la técnica al estudio del efecto de mutantes relacionados con el ciclo de síntesis y degradación del RNA para poder extraer información sobre el funcionamiento de la maquinaria transcripcional y su regulación. -Detectar posibles patrones de actividad de las RNA Polimerasas a lo largo de los transcritos y de las zonas flanqueantes que pudieran obedecer a condicionantes impuestas, tanto por su contexto cromatínico, como por otros factores. -Caracterizar la transcripción naciente producida por las otras RNAP nucleares de levadura. -Desarrollar un protocolo que permita analizar los RNAs nacientes a la máxima resolución mediante secuenciación masiva

A genomic view of mRNA turnover in yeast

The steady-state mRNA level is the result of two opposing processes: transcription and degradation; both of which can provide important points to regulate gene expression. In the model organism yeast Saccharomyces cerevisiae, it is now possible to determine, at the genomic level, the transcription and degradation rates, as well as the mRNA amount, using DNA chip or parallel sequencing technologies. In this way, the contribution of both rates to individual and global gene expressions can be analysed. Here we review the techniques used for the genomic evaluation of the transcription and degradation rates developed for this yeast, and we discuss the integration of the data obtained to fully analyse the expression strategies used by yeast and other eukaryotic cells. Le taux de l"ARNm est maintenu à l"équilibre grâce à deux processus antagonistes : la transcription et la dégradation. Ces deux mécanismes sont cruciaux pour réguler l"expression des gènes. Dans l"organisme modèle Saccharomyces cerevisiae, il est maintenant possible de déterminer, au niveau génomique, les taux respectifs de transcription et de dégradation, ainsi que la quantité d"ARNm présente, en utilisant les puces à ADN ou le séquençage en parallèle. De cette manière, il est possible de connaître la contribution de chacun de ces processus en analysant le niveau d"expression des gènes individuellement et globalement. Nous présentons et comparons dans cet article les techniques utilisées pour évaluer les taux respectifs de transcription et de dégradation des transcrits de cette levure. Nous discutons la possibilité de l"utilisation des données obtenues pour analyser en profondeur les stratégies d"expression employées par la levure ainsi que par d"autres cellules eucaryotes. -------------------------------------------------------------------------------

Cytoplasmic 5'-3' exonuclease Xrn1p is also a genome-wide transcription factor in yeast

The 5' to 3' exoribonuclease Xrn1 is a large protein involved in cytoplasmatic mRNA degradation as a critical component of the major decaysome. Its deletion in the yeast Saccharomyces cerevisiae is not lethal, but it has multiple physiological effects. In a previous study, our group showed that deletion of all tested components of the yeast major decaysome, including XRN1, results in a decrease in the synthetic rate and an increase in half-life of most mRNAs in a compensatory manner. Furthermore, the same study showed that the all tested decaysome components are also nuclear proteins that bind to the 5 region of a number of genes. In the present work, we show that disruption of Xrn1 activity preferentially affects both the synthesis and decay of a distinct subpopulation of mRNAs. The most affected mRNAs are the transcripts of the highly transcribed genes, mainly those encoding ribosome biogenesis and translation factors. Previously, we proposed that synthegradases play a key role in regulating both mRNA synthesis and degradation. Evidently, Xrn1 functions as a synthegradase, whose selectivity might help coordinating the expression of the protein synthetic machinery. We propose to name the most affected genes"Xrn1 synthegrado

El NT-proBNP mantiene su capacidad diagnóstica en los pacientes obesos con insuficiencia cardiaca

Los péptidos natriuréticos son útiles en el diagnóstico de la insuficiencia cardiaca (IC), siendo marcadores independientes de pronóstico. Recientemente, se ha publicado que sexo y edad influyen en los niveles plasmáticos y que, además, la obsidad disminuye el péptido natrurético tipo B (BNP) en individuos sanos. Esto podría afectar a su capacidad diagnóstica y pronóstica en los obesos con IC. Se estudió un total de 31 pacientes con IC y obsesidad en los que analizaron las correlaciones entre niveles de NT-proBNP y parámetros de función ventricular para comprobar si se mantienen en pacientes obsesos

The transcription factor Sfp1 imprints specific classes of mRNAs and links their synthesis and cytoplasmic decay

To properly function as an integrated system, both transcriptional and post-transcriptional machineries must communicate; the underlying mechanisms are poorly understood. Here we focus on ribosomal biosynthetic and ribosomal protein genes, transcription of which is regulated by a promoter-binding transcription factor, Sfp1. We show that Sfp1 also binds their gene bodies, affecting RNA polymerase II (Pol II) configuration, leading to enhanced backtracking and Rpb4 dissociation. Unexpectedly, we discovered that Sfp1 binds a group of those mRNAs encoded by Sfp1-bound genes. Remarkably, Sfp1 regulates deadenylation and decay of its bound mRNAs. The interaction of Sfp1 with its client mRNAs is controlled by their respective promoters and occurs concomitantly with its dissociation from chromatin. Collectively, our data suggest that for a subset of its targets, Sfp1 accompanies Pol II and controls its configuration during elongation, moves to the emerging transcripts co-transcriptionally and regulates their cytoplasmic stability. Thus, Sfp1 co-transcriptional binding imprints mRNA fate and serves as a paradigm for a cross-talk between synthesis and decay of specific mRNAs

Aberrations of Genomic Imprinting in Glioblastoma Formation

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

The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2

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