Genomics of mRNA turnover

Most studies on eukaryotic gene regulation have focused on mature mRNA levels. Nevertheless, the steady-state mRNA level is the result of two opposing biological processes: transcription and degradation, both of which can be important points to regulate gene expression. It is now possible to determine the transcription and degradation rates (TR and DR), as well as the mRNA amount, for each gene using DNA chip technologies. In this way, each individual contribution to gene expression can be analysed. This review will deal with the techniques used for the genomic evaluation of TR and DR developed for the yeast Saccharomyces cerevisiae. They will be described in detail and their potential drawbacks discussed. I will also discuss the integration of the data obtained to fully analyse the expression strategies used by yeast and other eukaryotic cells

Nucleo-cytoplasmic shuttling of RNA-binding factors: mRNA buffering and beyond

Gene expression is a highly regulated process that adapts RNAs and proteins content to the cellular context. Under steady-state conditions, mRNA homeostasis is robustly maintained by tight controls that act on both nuclear transcription and cytoplasmic mRNA stability. In recent years, it has been revealed that several RNA-binding proteins (RBPs) that perform functions in mRNA decay can move to the nucleus and regulate transcription. The RBPs involved in transcription can also travel to the cytoplasm and regulate mRNA degradation and/or translation. The multifaceted functions of these shuttling nucleo-cytoplasm RBPs have raised the possibility that they can act as mRNA metabolism coordinators. In addition, this indicates the existence of crosstalk mechanisms between the enzymatic machineries that drive the different mRNA life-cycle phases. The buffering of the mRNA concentration is the best known consequence of a transcription-degradation crosstalk counteraction, but alternative ways of RBP action can also imply enhanced gene regulation

The culture of conflict in family business

Conflict is part of the human condition. Therefore, it is neither negative nor positive, but rather a natural phenomena. The negative connotations traditionally attributed to conflict are at odds with current theories that explain the use of the term as an engine of change and generator of competitive advantage. The fact that family business is defined by two different systems, the family and the business, can lead to the emergence of many conflicts, but it can also help a business be successful if it can correctly differentiate between the two. To this end, both parties, the family and the business, must keep their own interests at arm's length and focus on common interests and goals.

Antígenos histo-sanguíneos e infección por rotavirus

La infección por rotavirus es la principal causa de gastroenteritis aguda en niños menores de 5 años en todo el mundo. Su incidencia es similar en países desarrollados y en vías de desarrollo, aunque la mayor mortalidad ocurre en estos últimos. Las vacunas frente a rotavirus han demostrado una gran efectividad en los países industrializados, pero su protección es menor en los países del África subsahariana y Asia. Se ha descrito que los antígenos histo-sanguíneos, presentes en la membrana de los enterocitos, actúan como ligandos para distintos genotipos de rotavirus. Esto podría explicar las diferencias que se observan en su distribución geográfica y la distinta eficacia vacunal de unas áreas a otras, puesto que el genotipo P[8] está presente en ambas vacunas. Con el fin de analizar los antígenos histo-sanguíneos de niños con infección aguda por rotavirus en nuestra área geográfica se reclutaron 133 pacientes entre 0 y 5 años de edad, pertenecientes al Departamento de Salud 5 de Valencia, que habían sufrido al menos una infección por rotavirus entre enero de 2013 y diciembre de 2015. Los resultados del análisis se compararon con los obtenidos de un grupo control de 50 niños sanos de edades similares y con los datos obtenidos de donantes de sangre de la Comunidad Valenciana. El diagnóstico de infección por rotavirus se realizó mediante inmunocromatografía o ELISA, y los genotipos G/P se determinaron por transcripción inversa y reacción en cadena de la polimerasa. Los antígenos histo-sanguíneos se analizaron en muestras de saliva. Los antígenos Lewis (Lea y Leb) y los grupos sanguíneos ABO se analizaron por ELISA con anticuerpos específicos. El gen FUT2 se tipificó por PCR-RFLP y fenotípicamente por ELISA con lectina de Ulex europaeus. La edad media de los pacientes fue de 22 meses, con un 84% menores de 3 años de edad. La mayor parte de los pacientes (82%) se infectaron con un solo genotipo de rotavirus, siendo el genotipo G9P[8] el más frecuente (49%). La mayoría de los pacientes infectados (98,5%) resultaron ser secretores (H tipo 1 positivo), homocigotos o heterocigotos para el gen FUT2. La infección fue también más frecuente en los niños con grupos sanguíneos A y AB, en comparación con los niños infectados del grupo O. Los individuos secretores Lewis positivos mostraron de forma significativa (p = 0.000) una mayor predisposición a padecer una infección sintomática por rotavirus. La vacunación frente a rotavirus disminuyó el riesgo de sufrir una infección por rotavirus (p = 0.003), aunque se detectaron fallos vacunales. No se encontró ninguna relación entre la distribución de los genotipos G/P de rotavirus y el estado de vacunación de los pacientes. La diferente expresión de los antígenos histo-sanguíneos (H tipo 1, ABO y antígenos Lewis) en los individuos repercute en la susceptibilidad a la infección por rotavirus, sin poderse establecer diferencias significativas entre los distintos genotipos G/P de rotavirus detectados en nuestro estudio

The transcriptional inhibitor thiolutin blocks mRNA degradation.

Thiolutin is commonly used as a general inhibitor of transcription in yeast. It has been used to calculate mRNA decay rates by stopping the transcription and then determining the relative abundance of individual mRNAs at different times after inhibition. We report here that thiolutin is also an inhibitor of mRNA degradation, and thus its use can lead to miscalculations of mRNA half-lives. The inhibition of mRNA decay seems to affect the mRNA degradation pathway without impeding poly(A) shortening, given that the decay rate of total poly(A) amount is not reduced by thiolutin. Moreover, the thiolutin-dependent inhibition of mRNA degradation has variable effects on different functional groups of genes, suggesting that they use various degradation pathways for their mRNAs

A web application for the unspecific detection of differentially expressed DNA regions in strand-specific expression data

Genomic technologies allow laboratories to produce large-scale data sets, either through the use of next-generation sequencing or microarray platforms. To explore these data sets and obtain máximum value from the data, researchers view their results alongside all the known features of a given reference genome. To study transcriptional changes that occur under a given condition, researchers search for regions of the genome that are differentially expressed between different experimental conditions. In order to identify these regions several algorithms have been developed over the years, along with some bioinformatic platforms that enable their use. However, currently available applications for comparative microarray analysis exclusively focus on changes in gene expression within known transcribed regions of predicted proteincoding genes, the changes that occur in non-predictable genetic elements, such as non-coding RNAs. Here, we present a web application for the visualization of strand-specific tiling microarray or next-generation sequencing data that allows customized detection of differentially expressed regions all along the genome in an unspecific manner, that allows identification of all RNA sequences, predictable or no

An unbiased genetic screen reveals the polygenic nature of the influenza virus anti-interferon response.

Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, interferon (IFN)-dependent upregulation of IFN-stimulated genes (ISGs), and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterized populations and selected mutants of wild-type viruses selected by passage through non-IFN-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that nonsynonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterization induced IFN and ISGs and were unable to counteract the effects of exogenous IFN, yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs but nonetheless replicated to high titers. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in non-IFN-responsive cells allows the virus to relax from these constraints and find a new genome consensus within its sequence space. IMPORTANCE In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses, and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes to interferon evasion have not been well characterized. Here, we examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titers had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine-tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response