Academic literature on the topic 'NSR100'

Abstract Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.

In mammals, different forms of stress, including psychosocial stress, can affect various aspects of human health, promoting mood and anxiety disorders. However, very little is known about the mechanisms underlying the brain physiology of stress response, hindering the development of new therapeutic strategies. We uncover a role for the transcriptional corepressor Lysine Specific Demethylase-1 (LSD1) and its dominant negative splicing isoform neuroLSD1, in the modulation of emotional behavior. In the mouse hippocampus, LSD1 and neuroLSD1 interacting with the transcription factor Serum Response Factor (SRF) and SRFΔ5 participate as molecular transducers of stress stimuli. Likewise LSD1, also SRF is modulated by an alternative splicing isoform without transactivation domain, SRFΔ5. Psychosocial stress acutely reduces the expression of neuroLSD1 through a splicing-based modulation that results in an increase in the amount of LSD1, while the relative ratio between SRF and SRFΔ5 is sensitive both to ASDS and CSDS. Furthermore, SRFΔ5 shows SUS-restricted downregulation that might contribute to shaping psychosocial stress vulnerability, through interfering with homeostatic mechanisms underlying stress resiliency. All these data suggest the involvement of the dual LSD1/neuroLSD1 and SRF/SRFΔ5 in the adaptive response to stress. Alternative splicing is a strategic biological mechanism that allows to create a set of functionally different gene products from a single gene, diversifying gene functions without an increase in the number of genes. neuroLSD1, an activity-dependent splicing isoform that differs from LSD1 for the inclusion of exon 8a, was related to important homeostatic neuronal functions impacting emotional processing. It has recently been published that MALAT1(metastasis associated lung adenocarcinoma transcript 1), a long non-coding RNA, has a crucial role in the alternative splicing mechanism of some genes through the regulation of the splicing factor SRSF1, belonging to the SR protein family. In particular MALAT1 is mainly localized at the level of the nuclear speckles, where it seems to regulate the alternative splicing through the retention of SRSF1 in these nuclear domains and the modulation of their phosphorylation state through an unknown mechanism. We already published that alternative splicing involving LSD1 is positively regulated in trans by two splicing factors NOVA1 and nSR100. In particular, nSR100 is a splicing factor belonging to the SR protein family, as SRSF1, and regulates tissue-specific alternative splicing in a manner dependent on its concentration and its phosphorylation status. We propose MALAT1 as a negative modulator of the neurospecific splicing of LSD1, in particular following ASDS the increased levels of MALAT1 lead to the sequestration of nSR100 at the level of nuclear speckles, making clear the mechanism behind the decrease of the dominant negative neuroLSD1 expression levels following stress We found that following a chronic psychosocial stress the expression levels of MALAT1 seem to be positively regulated only in resilient individuals who manage to maintain physiological expression levels of IEG in the hippocampus. Our hypothesis is that only resilient subjects are still able to modulate maladaptive stress-related transcription, thanks to the increased levels of MALAT1, bringing the system back to basal physiological conditions through the negative regulation of neuroLSD1 formation. All this suggests that MALAT1 could be considered a possible hallmark of resilience.

Positive strand RNA viruses are intracellular parasites, and their genome replication and infection involves complex virus-host interactions. Therefore, identification of host factors and dissection of their functions during virus replication could facilitate our understanding of the mechanism of virus infection. Those host factors may also provide new targets for viral disease control. Tomato bushy stunt virus (TBSV) has recently become one of the model viruses to study positive strand RNA virus replication and hostvirus interactions. To identify host factors involved in TBSV replication we used yeast as a model host. Co-expression of the replication proteins and a replicon RNA (DI RNA) via plasmids in yeast resulted in robust replication of the viral RNA. Previous work using a yeast single gene deletion library (YKO) revealed 96 yeast genes affecting virus replication. The essential yeast genes could not be deleted so we used the Yeast Tet Promoters Hughes Collection (yTHc) where the original promoter was replaced by Tetracyclin-titratable promoter. I tested the 800 essential host genes available in yTHc. In total, we found 30 new host genes whose down-regulated expression either increased or decreased the accumulation of a TBSV repRNA. The identified essential yeast genes fall into different categories on the basis of the cellular processes they are involved in, such as RNA transcription/metabolism, protein metabolism/transport etc. Detailed analysis of the effects of some of the identified yeast genes revealed that they might affect RNA replication by altering (i) the amounts of p33 and p92(pol) viral replication proteins, (ii) the activity of the tombusvirus replicase complex, and (iii) the ratio of plus- versus minus-stranded RNA replication products. Altogether, this and previous YKO screening of yeast led to the identification of 126 host genes (out of ~5,600 genes that represent ~95% of all the known and predicted yeast genes) that affected the accumulation of tombusvirus RNA. In the YKO screening, we found NSR1 (homologous to plant nucleolin) gene, whose deletion led to increased TBSV repRNA accumulation. Nucleolin is an abundant RNA binding protein, which shuttles between the nucleolus, the nucleoplasm and the cytoplasm. This protein is involved in rRNA maturation, ribosome assembly and regulation of cellular RNA metabolism.We found that over-expression of Nsr1p in yeast or nucleolin in Nicotiana benthamiana inhibited the accumulation of tombusvirus RNA by ~10-fold. Temporal regulation of Nsr1p over-expression revealed that the inhibitory effect of Nsr1p was more profound when it was expressed at early stages of viral replication. In vitro binding experiments showed that Nsr1p binds preferably to the RIII in the repRNA (which is derived from 3’ UTR of viral genome). Consistent with its RIII specific binding, over-expression of Nsr1p only reduced 40% of the accumulation of TBSVΔRIII repRNA in yeast. The purified recombinant Nsr1p inhibited the in vitro replication of the viral RNA in a yeast cell-free assay when pre-incubated with the viral RNA before the in vitro replication assay. Our data suggest that Nsr1p/nucleolin inhibits tombusvirus replication by interfering with the recruitment of the viral RNA for replication.

Les cassures double-brins d’ADN (CDBs) constituent une menace pour la viabilité cellulaire et l’intégrité du génome puisque l’absence de la réparation d’une CDB pourrait conduire à la mort cellulaire. En plus de la réparation par jonction d’extrémités nonhomologues (NHEJ) en phase G1 et de la recombinaison homologue (RH) en phase S et G2, les CDBs peuvent être réparées par l’ajout de télomères par l’action de la télomérase; un phénomène qui s’appelle l’ajout de télomères de novo. Ce phénomène pourrait mettre en danger la stabilité génomique parce qu’il engendre, dans la plupart des cas, une perte du bras chromosomique du fragment non-centromérique. En conséquence, ceci engendre soit une perte de l’hétérozygotie (LOH) dans les cellules diploïdes ou la mort cellulaire dans les cellules haploïdes. Dans le but d’empêcher la formation de télomères de novo, la cellule possède des mécanismes et des voies qui préviennent l’action inappropriée de la télomérase à des CDBs. Une des principales questions dans le domaine est de comprendre comment la cellule inhibe l’ajout de télomères de novo par la télomérase en favorisant la réparation des CDBs par les autres voies (NHEJ et la RH).Dans ce projet, nous utilisons la technique d’hybridation in situ en fluorescence (FISH) sur le facteur limitant de la télomérase, l’ARN TLC1 de la levure S. cerevisiae. Nous avons pu montrer que l’ARN TLC1 fait un trafic intranucléaire durant le cycle cellulaire des cellules sauvages. En phase G1/S, l’ARN TLC1 adopte une localisation nucléoplasmique avec les télomères, alors qu’il s’accumule au nucléole en phase G2/M. Nous avons fait l’hypothèse que l’accumulation de l’ARN TLC1 au nucléole en G2/M pourrait réduire la compétition entre la RH, qui est exclusivement nucléoplasmique, et la télomérase pour la réparation des CDBs. Pour tester cette hypothèse, nous avons employé la bléomycine (blm), un composé chimique générant des CDBs, pour traiter des cellules sauvages ou déficientes de la RH par la délétion du gène RAD52. Nous avons observé que l’ARN TLC1 conserve une localisation nucléolaire dans les cellules sauvages traitées par la blm en phase G2/M, alors que dans lescellules délétées de RAD52 exposées à la blm, l’ARN TLC1 se localise maintenant au nucléoplasme et s’associe partiellement aux sites de cassures. De plus, nous avons trouvé que l’accumulation nucléoplasmique de l’ARN TLC1 dans les cellules délétéées de RAD52 traitées à la blm, dépend de la voie de dommage à l’ADN (MRX, ATM/Tel1 et ATR/Mec1) et de la sumoylation par la SUMO E3ligase, Siz1. Plus particulièrement, l’association de la télomérase à des CDBs dépend de son interaction avec Cdc13, une protéine qui recrute la télomérase aux télomères. D’une manière surprenante, nous avons observé une accumulation rapide de Cdc13 à des CDBs en absence de Rad52, bien que nos résultats suggèrent que Rad52 empêche l’accumulation de l’ARN TLC1 au nucléoplasme par l’inhibition de l’accumulation de Cdc13 aux sites de cassures. L’ensemble de nos résultats ont mis en évidence que la télomérase est normalement exclue des sites de la réparation d’ADN. Cependant, en absence d’une voie fonctionnelle de la RH, la télomérase se localise du nucléole au nucléoplasme et s’accumule partiellement à des CDBs d’une manière dépendante de Cdc13 et Siz1.
DNA double-strand breaks (DSB) constitute a threat to genome integrity and cell survival if they are not repaired. In addition to canonical DNA repair systems such as nonhomologous end joining (NHEJ) in G1 and homologous recombination (HR) in S and G2 phases, DSBs can also be repaired by addition of new telomeres by telomerase. This phenomenon is referred to as telomere healing or de novo telomere addition. This process threatens genome stability since it results in chromosome arm loss, which could be lethal in haploid cells and lead to loss of heterozygosity (LOH) in diploid cells. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. One of the questions driving this field is to understand how telomere addition by telomerase is inhibited and DSBs repair can be efficiently performed by canonical DSB repair (NHEJ and HR). In this project, we used fluorescent in situ hybridization (FISH) to detect the endogenous TLC1 RNA, which is the limiting component of telomerase of the budding yeast. Using this technique, we found that TLC1 RNA traffics inside the nucleus during the cell cycle of wild-type cells. In G1 and S phases, TLC1 RNA adopts a nucleoplasmic localization, which is related to its function in telomere elongation, while it accumulates in the nucleolus in G2/M. We hypothesize that the nucleolar accumulation of TLC1 RNA in G2/M may reduce the possibility that telomerase interferes with HR to repair DNA DSB, since HR is excluded from the nucleolus and occurs only in the nucleoplasm. To test this hypothesis, we treated wild-type and rad52 (HR deficient cells) with bleomycin, a radiomimetic agent that generates preferentially DSBs. Our results show that after induction of DSB with bleomycin, TLC1 RNA remains nucleolar in wild-type cells in G2/M, but accumulates in the nucleoplasm and colocalizes partially with DSBs sites in rad52 cells, suggesting that RAD52 inhibits the nucleoplasmic accumulation of TLC1 RNA in the presence of DSBs. Nucleoplasmic accumulation of TLC1 RNA after DSB induction requires the DNA damage pathway (MRX, ATM/Tel1 and ATR/Mec1), and the SUMO ligase E3 Siz1. Interestingly, association of TLC1 RNA with DSBs depends on the single-strand telomeric binding protein Cdc13, which rapidly accumulates at sites of DNA damage, while Rad52 suppresses this process by inhibiting Cdc13 accumulation at DSBs. These results suggest that telomerase is normally excluded from sites of DNA repair. In the absence of functional homologous recombination, telomerase leaves the nucleolus and accumulates partially at DSB in the nucleoplasm in a Cdc13- and Siz1-dependent manner.

El presente proyecto estudia las características espaciales, culturales y arquitectónicas de la ciudad de Girardot. Con el fin de implementar las propiedades al edificio moderno que tiene como objetivo mejorar la atención, accesibilidad y el desarrollo cultural a los ciudadanos girardoteños. Se realizó diferentes encuestas, cálculos estadísticas e investigación de antecedentes sobre la accesibilidad, el desarrollo cultural y la atención al ciudadano que presta las instalaciones del ITUC. Mediante esta investigación se determinó diferentes parámetros espaciales y geométricos para el diseño arquitectónico y estructural. En el documento se evidenciará la investigación cualitativa y cuantitativa ambientada a la atención del ciudadano, el desarrollo cultural y los conocimientos aprendidos en la carrera de ingeniería civil. Este proyecto es una alternativa de diseño, donde se desarrollarán los conocimientos aprendidos de la línea de las estructuras de concreto siguiendo las respectivas normativas para construcciones sismos-resistentes en Colombia (NSR10), se tiene en cuenta los parámetros de diseño que exige esta Norma y el POT del municipio. Los títulos más relevantes para el diseño son el Titulo K y J para el diseño arquitectónico, el titulo A para los parámetros generales del diseño, C para el diseño de los elementos estructurales, el B para la adecuada determinación de cargas sobre la estructura y la NTC 1500 (CÓDIGO COLOMBIANO DE FONTANERÍA) para el diseño de redes internas. Además, en los parámetros espaciales se implementó la energía renovable y espacios eco-sostenibles que desarrollan las diferentes actividades culturales locales. También se desarrolló el proceso constructivo del diseño mediante diferentes softwares y metodologías aplicadas a la ingeniería. Estos softwares determinaron los diferentes procesos, tiempos y materiales del pre-post ejecución del proyecto y así se pudo realizar los diferentes A.P.U (análisis de precios unitarios) del edificio moderno aplicando diferentes metodologías.