The role of ‘DELLA’ proteins in alternative splicing

DELLA proteins, the master repressors of the gibberellin (GA) signaling pathway, are thought to regulate gene expression by mechanisms other than their interaction with bona fide transcription factors. In particular, current work in our lab shows that DELLA and their atypical interactors prefoldins are able to interact with and regulate a number of proteins that are part of the basal transcriptional machinery, regulating the RNA polymerase II activity. Among the novel interactors being studied are proteins involved in mRNA splicing. In this TFM, we have investigated the participation of DELLA and prefoldin in alternative splicing by analyzing protein-protein interaction between these proteins and Sm/LSm, which form heteroheptameric complexes and are an essential part of the spliceosome. We have also advanced in obtaining resources to test genetic interactions between della and sm/lsm mutants. In addition, some alternative splicing events affected by DELLA activity identified in a transcriptomic analysis by RNA-seq, have been validated by semiquantitative RT-PCR. Finally, it has been shown that GA signaling alters the Sm and LSm protein levels.En el laboratorio se está investigando los mecanismos moleculares por los que las proteínas DELLA regulan la expresión génica en plantas. El mecanismo aceptado en la actualidad indica que esta regulación ocurre a través de la interacción de las DELLA con numerosos factores de transcripción. No obstante, resultados recientes del laboratorio apuntan a mecanismos alternativos de regulación génica por parte de estas proteínas. Una de las claves fue la identificación de la interacción de las DELLA con el complejo prefoldina. Este complejo heterohexamérico (PFD1-6) está conservado de levaduras a humanos y se aisló originalmente gracias a su papel en el citoplasma, donde ejerce de co-chaperona ayudando en el plegamiento de tubulinas. En el laboratorio se ha demostrado que las DELLA promueven la acumulación en el núcleo del complejo y los últimos resultados apuntan a que puede tener un papel relevante en la regulación génica. En particular, hemos demostrado que tanto las DELLA como varias subunidades prefoldina interaccionan con varias proteínas Sm y LSM. Las proteínas Sm y LSM forman las 5 snRNP (del inglés, small nuclear ribonucleoprotein particles) del espliceosoma junto con UsnRNA (RNA pequeños nucleares ricos en uridina). Por un lado, las proteínas LSM forman dos tipos de heteroheptámeros (LSM1-7 y LSM2-8), siendo el primero citosólico y participando en el decaimiento de los mRNA mientras que el segundo se localiza en el núcleo estabilizando al snRNA U6. Por otro lado, las otras 4 snRNP, están formadas por un anillo de 7 proteínas Sm (B, D1, D2, D3, E, F y G) y 4 snRNA diferentes (U1, U2, U4 y U5). Además, se ha demostrado que la pérdida de función de PFD provoca alteración en los niveles de U6. Mediante un RNAseq se comprobó que los mutantes pfd4 mostraban fenómenos de procesamiento alternativo de mensajeros (splicing alternativo) diferentes al silvestre tras un tratamiento de frío. Por lo tanto, proponemos que las DELLA pueden tener un rol directo en el procesamiento alternativo. En el contexto de este TFM vamos a evaluar el efecto de DELLA y prefoldinas en el procesamiento alternativo de mensajeros mediante la comprobación de la interacción proteína-proteína entre DELLA y Sm/LSM, y PFD y Sm; tanto por técnicas de doble híbrido como Co-IP. De igual manera, se comprobarán las interacciones genéticas entre mutantes della y sm/lsm. También se confirmarán dianas de splicing alternativo, obtenidas mediante un RNAseq, con PCR cuantitativa o semi-cuantitativa. Finalmente se estudiará si las giberelinas alteran la localización y/o la configuración de los complejos Sm y/o LSM2-8 mediante análisis en el microscopio confocal y cromatografía de exclusión molecular, respectivamente. Los análisis se realizarán en varios fondos mutantes y líneas transgénicas que tienen niveles alterados de DELLA, y en una serie de mutantes pfd preparados en el laboratorio, de manera que podremos diseccionar genéticamente la contribución de las DELLA y PFD.Calleja Cabrera, J. (2018). El papel de las proteínas DELLA en el procesamiento alternativo de mensajeros. http://hdl.handle.net/10251/97802TFG

Prefoldins contribute to maintaining the levels of the spliceosome LSM2–8 complex through Hsp90 in Arabidopsis

Although originally identified as the components of the complex aiding the cytosolic chaperonin CCT in the folding of actins and tubulins in the cytosol, prefoldins (PFDs) are emerging as novel regulators influencing gene expression in the nucleus. Work conducted mainly in yeast and animals showed that PFDs act as transcriptional regulators and participate in the nuclear proteostasis. To investigate new functions of PFDs, we performed a co-expression analysis in Arabidopsis thaliana. Results revealed co-expression between PFD and the Sm-like (LSM) genes, which encode the LSM2–8 spliceosome core complex, in this model organism. Here, we show that PFDs interact with and are required to maintain adequate levels of the LSM2–8 complex. Our data indicate that levels of the LSM8 protein, which defines and confers the functional specificity of the complex, are reduced in pfd mutants and in response to the Hsp90 inhibitor geldanamycin. We provide biochemical evidence showing that LSM8 is a client of Hsp90 and that PFD4 mediates the interaction between both proteins. Consistent with our results and with the role of the LSM2–8 complex in splicing through the stabilization of the U6 snRNA, pfd mutants showed reduced levels of this snRNA and altered pre-mRNA splicing patterns.Fil: Esteve Bruna, David. Universidad Politécnica de Valencia; EspañaFil: Carrasco López, Cristian. Consejo Superior de Investigaciones Científicas; EspañaFil: Blanco Touriñán, Noel. Universidad Politécnica de Valencia; EspañaFil: Iserte, Javier Alonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Calleja Cabrera, Julián. Universidad Politécnica de Valencia; EspañaFil: Perea Resa, Carlos. Consejo Superior de Investigaciones Científicas; EspañaFil: Úrbez, Cristina. Universidad Politécnica de Valencia; EspañaFil: Carrasco, Pedro. Universidad Politécnica de Valencia; EspañaFil: Yanovsky, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Blázquez, Miguel A.. Universidad Politécnica de Valencia; EspañaFil: Salinas, Julio. Consejo Superior de Investigaciones Científicas; EspañaFil: Alabadí, David. Universidad Politécnica de Valencia; Españ

Root Growth Adaptation to Climate Change in Crops

23 Pág.Climate change is threatening crop productivity worldwide and new solutions to adapt crops to these environmental changes are urgently needed. Elevated temperatures driven by climate change affect developmental and physiological plant processes that, ultimately, impact on crop yield and quality. Plant roots are responsible for water and nutrients uptake, but changes in soil temperatures alters this process limiting crop growth. With the predicted variable climatic forecast, the development of an efficient root system better adapted to changing soil and environmental conditions is crucial for enhancing crop productivity. Root traits associated with improved adaptation to rising temperatures are increasingly being analyzed to obtain more suitable crop varieties. In this review, we will summarize the current knowledge about the effect of increasing temperatures on root growth and their impact on crop yield. First, we will describe the main alterations in root architecture that different crops undergo in response to warmer soils. Then, we will outline the main coordinated physiological and metabolic changes taking place in roots and aerial parts that modulate the global response of the plant to increased temperatures. We will discuss on some of the main regulatory mechanisms controlling root adaptation to warmer soils, including the activation of heat and oxidative pathways to prevent damage of root cells and disruption of root growth; the interplay between hormonal regulatory pathways and the global changes on gene expression and protein homeostasis. We will also consider that in the field, increasing temperatures are usually associated with other abiotic and biotic stresses such as drought, salinity, nutrient deficiencies, and pathogen infections. We will present recent advances on how the root system is able to integrate and respond to complex and different stimuli in order to adapt to an increasingly changing environment. Finally, we will discuss the new prospects and challenges in this field as well as the more promising pathways for future research.This work was supported with a grant to LO-S by the Spanish Ministry of Science and Universities (BIO2016-77840-R) and to MP by FP7. FACCE-JPI-ERA-NET+ CLIMATE SMART AGRICULTURE (ERA46-SYBRACLYM). MB was supported by a postdoctoral research fellowship and JC-C by a Ph.D. contract (FPI), both funded by grant SEV-2016-0672 to the CBGP (Centre of Excellence Severo Ochoa Program of the Agencia Estatal de Investigación, Spain).Peer reviewe

An Integrative Approach to Analyze Seed Germination in Brassica napus

International audienceSeed germination is a complex trait determined by the interaction of hormonal, metabolic, genetic, and environmental components. Variability of this trait in crops has a big impact on seedling establishment and yield in the field. Classical studies of this trait in crops have focused mainly on the analyses of one level of regulation in the cascade of events leading to seed germination. We have carried out an integrative and extensive approach to deepen our understanding of seed germination in Brassica napus by generating transcriptomic, metabolic, and hormonal data at different stages upon seed imbibition. Deep phenotyping of different seed germination-associated traits in six winter-type B. napus accessions has revealed that seed germination kinetics, in particular seed germination speed, are major contributors to the variability of this trait. Metabolic profiling of these accessions has allowed us to describe a common pattern of metabolic change and to identify the levels of malate and aspartate metabolites as putative metabolic markers to estimate germination performance. Additionally, analysis of seed content of different hormones suggests that hormonal balance between ABA, GA, and IAA at crucial time points during this process might underlie seed germination differences in these accessions. In this study, we have also defined the major transcriptome changes accompanying the germination process in B. napus. Furthermore, we have observed that earlier activation of key germination regulatory genes seems to generate the differences in germination speed observed between accessions in B. napus. Finally, we have found that protein-protein interactions between some of these key regulator are conserved in B. napus, suggesting a shared regulatory network with other plant species. Altogether, our results provide a comprehensive and detailed picture of seed germination dynamics in oilseed rape. This new framework will be extremely valuable not only to evaluate germination performance of B. napus accessions but also to identify key targets for crop improvement in this important process

Política y sociedad en el exilio republicano

La conmemoración del septuagésimo quinto aniversario de la llegada del exilio republicano español a México, América Latina y el Caribe obliga a generar nuevas formas de investigar e interpretar el fenómeno, desde la obligada mirada de las realidades latinoamericanas marcadas, ciertamente, por una serie de condicionamientos históricos, políticos y sociales intensos y de una gran inestabilidad producto de la implantación de gobiernos de corte dictatorial en la región, que a su vez contaron con el apoyo de los distintos gobiernos estadounidenses. En ese sentido, aun cuando esta obra centra gran parte de su atención en lo sucedido en México no deja de atenderse otros escenarios latinoamericanos como Cuba, Guatemala y Argentina, naciones que recibieron a exiliados de la Guerra Civil española, llegada que tuvo consecuencias en ámbitos como las relaciones internacionales, el seguimiento del conflicto español en la prensa de la región, la opinión pública y la sociedad en general, por ello la pertinencia de denominar a esta obra con el título genérico de Política y sociedad en el exilio republicano español, con el cual se le da continuidad a la colección "Exilio Iberoamericano"Universidad Nacional Autónoma de México/Programas de Apoyo a Proyectos de Investigación e Innovación Tecnológica/"PAPIIT IG 400314"//[“Interacción de los exilios en México e Iberoamérica (siglo XX)"]Libro

Comprehensive analysis and insights gained from long-term experience of the Spanish DILI Registry

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER); Agencia Española del Medicamento; Consejería de Salud de Andalucía.Background & Aims: Prospective drug-induced liver injury (DILI) registries are important sources of information on idiosyncratic DILI. We aimed to present a comprehensive analysis of 843 patients with DILI enrolled into the Spanish DILI Registry over a 20-year time period. Methods: Cases were identified, diagnosed and followed prospectively. Clinical features, drug information and outcome data were collected. Results: A total of 843 patients, with a mean age of 54 years (48% females), were enrolled up to 2018. Hepatocellular injury was associated with younger age (adjusted odds ratio [aOR] per year 0.983; 95% CI 0.974-0.991) and lower platelet count (aOR per unit 0.996; 95% CI 0.994-0.998). Anti-infectives were the most common causative drug class (40%). Liver-related mortality was more frequent in patients with hepatocellular damage aged ≥65 years (p = 0.0083) and in patients with underlying liver disease (p = 0.0221). Independent predictors of liver-related death/transplantation included nR-based hepatocellular injury, female sex, higher onset aspartate aminotransferase (AST) and bilirubin values. nR-based hepatocellular injury was not associated with 6-month overall mortality, for which comorbidity burden played a more important role. The prognostic capacity of Hy's law varied between causative agents. Empirical therapy (corticosteroids, ursodeoxycholic acid and MARS) was prescribed to 20% of patients. Drug-induced autoimmune hepatitis patients (26 cases) were mainly females (62%) with hepatocellular damage (92%), who more frequently received immunosuppressive therapy (58%). Conclusions: AST elevation at onset is a strong predictor of poor outcome and should be routinely assessed in DILI evaluation. Mortality is higher in older patients with hepatocellular damage and patients with underlying hepatic conditions. The Spanish DILI Registry is a valuable tool in the identification of causative drugs, clinical signatures and prognostic risk factors in DILI and can aid physicians in DILI characterisation and management. Lay summary: Clinical information on drug-induced liver injury (DILI) collected from enrolled patients in the Spanish DILI Registry can guide physicians in the decision-making process. We have found that older patients with hepatocellular type liver injury and patients with additional liver conditions are at a higher risk of mortality. The type of liver injury, patient sex and analytical values of aspartate aminotransferase and total bilirubin can also help predict clinical outcomes