RÉSUMÉ
Alternative transcription start site (TSS) usage regulation has been identified as a major means of gene expression regulation in metazoans. However, in fungi, its impact remains elusive as its study has thus far been restricted to model yeasts. Here, we first re-analyzed TSS-seq data to define genuine TSS clusters in 2 species of pathogenic Cryptococcus. We identified 2 types of TSS clusters associated with specific DNA sequence motifs. Our analysis also revealed that alternative TSS usage regulation in response to environmental cues is widespread in Cryptococcus, altering gene expression and protein targeting. Importantly, we performed a forward genetic screen to identify a unique transcription factor (TF) named Tur1, which regulates alternative TSS (altTSS) usage genome-wide when cells switch from exponential phase to stationary phase. ChiP-Seq and DamID-Seq analyses suggest that at some loci, the role of Tur1 might be direct. Tur1 has been previously shown to be essential for virulence in C. neoformans. We demonstrated here that a tur1Δ mutant strain is more sensitive to superoxide stress and phagocytosed more efficiently by macrophages than the wild-type (WT) strain.
Sujet(s)
Protéines fongiques , Régulation de l'expression des gènes fongiques , Génome fongique , Facteurs de transcription , Site d'initiation de la transcription , Protéines fongiques/génétique , Protéines fongiques/métabolisme , Facteurs de transcription/métabolisme , Facteurs de transcription/génétique , Cryptococcus/génétique , Cryptococcus/pathogénicité , Cryptococcus/métabolisme , Cryptococcus neoformans/génétique , Cryptococcus neoformans/pathogénicité , Cryptococcus neoformans/métabolisme , Macrophages/microbiologie , Macrophages/métabolisme , Animaux , Souris , Virulence/génétique , Phagocytose/génétiqueRÉSUMÉ
Drosophila melanogaster imaginal discs are larval internal structures that become the external organs of the adult. They have been used to study numerous developmental processes for more than fifty years. Dissecting these imaginal discs for collection is challenging, as the size of third-instar larvae organs is typically less than 1 mm. Certain experimental applications of the organs require many cells, which requires researchers to spend several hours dissecting them. This paper proposes an alternative to dissection in the form of a mass enrichment protocol. The protocol enables the recovery of many wing imaginal discs by grinding large quantities of third-instar larvae and separating the organs using filtration and a density gradient. The wing imaginal discs collected with this protocol in less than three hours are as well preserved as those collected by dissection. The dissociation and filtration of the extract allow the isolation of a large amount of wing imaginal disc cells.
RÉSUMÉ
Pathogenic fungi require delicate gene regulation mechanisms to adapt to diverse living environments and escape host immune systems. Recent advances in sequencing technology have exposed the complexity of the fungal genome, thus allowing the gradual disentanglement of multiple layers of gene expression control. Alternative transcription start site (aTSS) usage, previously reported to be prominent in mammals and to play important roles in physiopathology, is also present in fungi to fine-tune gene expression. Depending on the alteration in their sequences, RNA isoforms arising from aTSSs acquire different characteristics that significantly alter their stability and translational capacity as well as the properties and biologic functions of the resulting proteins. Disrupted control of aTSS usage has been reported to severely impair growth, virulence, and the infectious capacity of pathogenic fungi. Here, we discuss principle concepts, mechanisms, and the functional implication of aTSS usage in fungi.
RÉSUMÉ
Detecting protein-RNA interactions in vivo is essential for deciphering many important cellular pathways. Several methods have been described for this purpose, among which cross-linking analysis of cDNA, CRAC. This method relies on a first step of UV cross-linking of living yeast cells and several subsequent steps of purification of the protein-RNA complexes, some of which under denaturing condition. Without altering the general principle of the method, we have modified and improved the protocol, with the specific aim of sequencing the nascent RNA isolated from transcription complexes and generate high-resolution and directional transcription maps.
Sujet(s)
Nucléotides , ARN , Réactifs réticulants/métabolisme , ADN complémentaire/génétique , ADN complémentaire/métabolisme , ARN/métabolismeRÉSUMÉ
Apoptosis is associated with numerous phenotypical characteristics, and is thus studied with many tools. In this study, we compared two broadly used apoptotic assays: TUNEL and staining with an antibody targeting the activated form of an effector caspase. To compare them, we developed a protocol based on commonly used tools such as image filtering, z-projection, and thresholding. Even though it is commonly used in image-processing protocols, thresholding remains a recurring problem. Here, we analyzed the impact of processing parameters and readout choice on the accuracy of apoptotic signal quantification. Our results show that TUNEL is quite robust, even if image processing parameters may not always allow to detect subtle differences of the apoptotic rate. On the contrary, images from anti-cleaved caspase staining are more sensitive to handle and necessitate being processed more carefully. We then developed an open-source Fiji macro automatizing most steps of the image processing and quantification protocol. It is noteworthy that the field of application of this macro is wider than apoptosis and it can be used to treat and quantify other kind of images.
Sujet(s)
Apoptose/génétique , Caspases effectrices/génétique , Traitement d'image par ordinateur , Méthode TUNEL/méthodes , Caspases effectrices/composition chimique , Humains , Distribution tissulaire/génétiqueRÉSUMÉ
Transcription termination delimits transcription units but also plays important roles in limiting pervasive transcription. We have previously shown that transcription termination occurs when elongating RNA polymerase II (RNAPII) collides with the DNA-bound general transcription factor Reb1. We demonstrate here that many different DNA-binding proteins can induce termination by a similar roadblock (RB) mechanism. We generated high-resolution transcription maps by the direct detection of RNAPII upon nuclear depletion of two essential RB factors or when the canonical termination pathways for coding and non-coding RNAs are defective. We show that RB termination occurs genomewide and functions independently of (and redundantly with) the main transcription termination pathways. We provide evidence that transcriptional readthrough at canonical terminators is a significant source of pervasive transcription, which is controlled to a large extent by RB termination. Finally, we demonstrate the occurrence of RB termination around centromeres and tRNA genes, which we suggest shields these regions from RNAPII to preserve their functional integrity.
Sujet(s)
Protéines de liaison à l'ADN/métabolisme , RNA polymerase II/métabolisme , Protéines de Saccharomyces cerevisiae/métabolisme , Saccharomyces cerevisiae/génétique , Terminaison de la transcription , Transcription génétique , Protéines de liaison à l'ADN/génétique , Génome fongique , RNA polymerase II/génétique , ARN fongique , Protéines de Saccharomyces cerevisiae/génétiqueRÉSUMÉ
It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human.
Sujet(s)
Apoptose/physiologie , Protéines de Drosophila/métabolisme , Drosophila melanogaster/physiologie , Protéines IAP/métabolisme , Mitochondries/physiologie , Dynamique mitochondriale , Espèces réactives de l'oxygène/métabolisme , Animaux , Apoptose/génétique , Caspases/métabolisme , Cytochromes c/métabolisme , Protéines de Drosophila/génétique , Drosophila melanogaster/génétique , Protéines IAP/génétique , Mitochondries/génétique , Mutation , Transduction du signalRÉSUMÉ
Members of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. We report here the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. We isolated 17 putative modifiers involved in various function or process: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation.
RÉSUMÉ
Widely transcribed compact genomes must cope with the major challenge of frequent overlapping or concurrent transcription events. Efficient and timely transcription termination is crucial to control pervasive transcription and prevent transcriptional interference. In yeast, transcription termination of RNA polymerase II (RNAPII) occurs via two possible pathways that both require recognition of termination signals on nascent RNA by specific factors. We describe here an additional mechanism of transcription termination for RNAPII and demonstrate its biological significance. We show that the transcriptional activator Reb1p bound to DNA is a roadblock for RNAPII, which pauses and is ubiquitinated, thus triggering termination. Reb1p-dependent termination generates a class of cryptic transcripts that are degraded in the nucleus by the exosome. We also observed transcriptional interference between neighboring genes in the absence of Reb1p. This work demonstrates the importance of roadblock termination for controlling pervasive transcription and preventing transcription through gene regulatory regions.
Sujet(s)
Protéines de liaison à l'ADN/métabolisme , RNA polymerase II/métabolisme , Protéines de Saccharomyces cerevisiae/métabolisme , Facteurs de transcription/métabolisme , Transcription génétique , Sites de fixation , Génome fongique , Modèles génétiques , Stabilité de l'ARN , ARN fongique/métabolisme , ARN messager/métabolisme , Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/métabolisme , UbiquitinationRÉSUMÉ
The ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. We then demonstrated that Uba1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of ß-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family.
Sujet(s)
Protéines du cycle cellulaire/métabolisme , Protéines de Drosophila/métabolisme , Drosophila/métabolisme , Protéines membranaires/métabolisme , Proteasome endopeptidase complex/métabolisme , Ubiquitin-protein ligases/métabolisme , Animaux , Apoptose , Protéines du cycle cellulaire/génétique , Drosophila/cytologie , Drosophila/enzymologie , Drosophila/génétique , Protéines de Drosophila/génétique , Protéines membranaires/génétique , Proteasome endopeptidase complex/génétique , Liaison aux protéines , Transport des protéines , Ubiquitin-activating enzymes/génétique , Ubiquitin-activating enzymes/métabolisme , Ubiquitin-protein ligases/génétique , Protéine Bax/génétique , Protéine Bax/métabolismeRÉSUMÉ
Recent studies on yeast transcriptome have revealed the presence of a large set of RNA polymerase II transcripts mapping to intergenic and antisense regions or overlapping canonical genes. Most of these ncRNAs (ncRNAs) are subject to termination by the Nrd1-dependent pathway and rapid degradation by the nuclear exosome and have been dubbed cryptic unstable transcripts (CUTs). CUTs are often considered as by-products of transcriptional noise, but in an increasing number of cases they play a central role in the control of gene expression. Regulatory mechanisms involving expression of a CUT are diverse and include attenuation, transcriptional interference, and alternative transcription start site choice. This review focuses on the impact of cryptic transcription on gene expression, describes the role of the Nrd1-complex as the main actor in preventing nonfunctional and potentially harmful transcription, and details a few systems where expression of a CUT has an essential regulatory function. We also summarize the most recent studies concerning other types of ncRNAs and their possible role in regulation.
Sujet(s)
Cathétérisme veineux central/soins infirmiers , Prévention des infections/méthodes , Unités de soins intensifs néonatals , Sepsie/soins infirmiers , Sepsie/prévention et contrôle , Spécialités en soins infirmiers/méthodes , Cathétérisme veineux central/effets indésirables , Cathétérisme veineux central/méthodes , Soins infirmiers factuels/méthodes , Hôpitaux communautaires , Humains , Nouveau-né , Prévention des infections/normes , Équipe infirmiers/méthodes , Politique organisationnelle , Évaluation de programme , Spécialités en soins infirmiers/normesRÉSUMÉ
Individuals who have maladaptive patterns of drinking alcohol fall into the category of vulnerable research participants for many reasons, not the least of which includes the stigma often placed on individuals who abuse alcohol. Vulnerable subgroups within the population of people who abuse alcohol include women; older adults; incarcerated, socioeconomically disadvantaged, and mentally ill individuals; as well as people from racial minorities. Thus, as research participants, individuals who abuse alcohol can be labeled a doubly vulnerable population. Belonging to more than one population simultaneously can lead to a compromised ability to protect one's own interests or greater susceptibility to harm related to participation in research studies. Arguments against including people who abuse alcohol as research participants will be presented, followed by the argument for including theses individuals, which is suggested as the more ethically sound of the two points of view.
Sujet(s)
Alcoolisme , Éthique de la recherche , Expérimentation humaine/éthique , Troubles mentaux , Défense du patient/éthique , Populations vulnérables , Sujet âgé , Alcoolisme/complications , Diagnostic mixte (psychiatrie) , Désaccords et litiges , Recommandations comme sujet , Humains , Troubles mentaux/complications , Minorités , Prejugé , Prisonniers , Soins infirmiers en psychiatrie/éthique , StéréotypesRÉSUMÉ
The Institute of Medicine (IOM) has recommended that more minorities be represented in the health care workforce and that cross-cultural education be included in all health care programs (Betancourt, J.R., & Maina, A.W., 2004). Although nurse educators have embraced the need of inclusion of diversity in the curriculum, there remains a gap between the desire to educate about diversity and the application of this concept into the curriculum. Common barriers include difficulty fitting it into an already full curriculum and a lack of experience in the area of diversity. The conceptual framework of James Banks may be considered when implementing multiculturalism into an undergraduate nursing program, an associate degree program, or over a semester as a freestanding course on diversity. The Banks model identifies five dimensions of multicultural education (content integration, knowledge construction, prejudice reduction, equitable pedagogy, and empowering school culture) to facilitate the process of conceptualization and implementation of multicultural education. These dimensions build upon each other and can be leveled throughout a program curriculum or across a semester. Examples of assignments for each dimension and level are provided, as well as a discussion of the faculty role in implementing multiculturalism.
Sujet(s)
Formation au diplôme infirmier (USA)/méthodes , Modèles éducatifs , Modèles de soins infirmiers , Soins infirmiers transculturels/enseignement et éducation , Soins infirmiers transculturels/méthodes , Programme d'études , Corps enseignant et administratif de l'école d'infirmières , Humains , Recherche en enseignement des soins infirmiersRÉSUMÉ
Initiator caspases are activated within specialized complexes, one of which is the apoptosome. The apoptosome is always constituted by at least an initiator caspase and a caspase activator. Apoptosome activation enables maturation of the associated caspase and constitutes a key step for cell fate. This activating complex is found throughout metazoans but its composition and regulation seem slightly different from one species to another. This review focuses on the composition and activation of the apoptosome in different species and details the role of mitochondrial factors and Bcl-2 family members in this activation.
Sujet(s)
Apoptose , Mitochondries/métabolisme , Protéines proto-oncogènes c-bcl-2/métabolisme , Animaux , Caenorhabditis elegans , Caspases/métabolisme , Drosophila melanogaster , Activation enzymatique , HumainsRÉSUMÉ
The purpose of this study was to determine the effects of an osteoporosis educational intervention on knowledge, health beliefs, and self-efficacy in college-age Puerto Rican women. The Health Belief Model and Purnell Model formed the theoretical framework for the study. Three hypotheses were tested with a convenience, randomized sample of 51 experimental and 54 control subjects, ages 18-25. The findings support the hypotheses that educational intervention improved osteoporosis knowledge, health beliefs, but had no effect on self-efficacy. Results and directions for future research are discussed.
Sujet(s)
Éducation pour la santé/organisation et administration , Connaissances, attitudes et pratiques en santé , Hispanique ou Latino/enseignement et éducation , Ostéoporose post-ménopausique/prévention et contrôle , Auto-efficacité , Femmes/enseignement et éducation , Adolescent , Adulte , Attitude envers la santé/ethnologie , Caraïbe , Femelle , Hispanique ou Latino/ethnologie , Humains , Modèles psychologiques , Analyse multifactorielle , Ostéoporose post-ménopausique/ethnologie , Évaluation de programme , Porto Rico/ethnologie , Étudiants/psychologie , Enquêtes et questionnaires , Universités , Femmes/psychologie , Jeune adulteRÉSUMÉ
Hidden transcription in eukaryotes carries a large potential of regulatory functions that are only recently beginning to emerge. Cryptic unstable transcripts (CUTs) are generated by RNA polymerase II (Pol II) and rapidly degraded after transcription in wild-type yeast cells. Whether CUTs or the act of transcription without RNA production have a function is presently unclear. We describe here a nonconventional mechanism of transcriptional regulation that relies on the selection of alternative transcription start sites to generate CUTs or mRNAs. Transcription from TATA box proximal start sites generates unstable transcripts and downregulates expression of the URA2 gene under repressing conditions. Uracil deprivation activates selection of distal start sites, leading to the production of stable mRNAs. We describe the elements that govern degradation of the CUT and activation of mRNA production by downstream transcription initiation. Importantly, we show that a similar mechanism applies to other genes in the nucleotides biogenesis pathway.
Sujet(s)
Aspartate carbamoyltransferase/génétique , Carbamoyl-phosphate synthase (glutamine-hydrolyzing)/génétique , Régulation de l'expression des gènes fongiques , Nucléotides/métabolisme , Protéines de Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae , Recyclage du substrat/physiologie , Transcription génétique , Région 5' flanquante , Aspartate carbamoyltransferase/métabolisme , Séquence nucléotidique , Carbamoyl-phosphate synthase (glutamine-hydrolyzing)/métabolisme , Analyse de mutations d'ADN , Données de séquences moléculaires , Phénotype , Régions promotrices (génétique) , ARN messager/génétique , ARN messager/métabolisme , Séquences d'acides nucléiques régulatrices , Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/métabolisme , Protéines de Saccharomyces cerevisiae/métabolisme , Régions terminatrices (génétique)RÉSUMÉ
Despite public health campaigns and safer sex messages, many men who have sex with men (MSM) continue to participate in high-risk sexual behaviors, which may make them vulnerable to HIV infection and sexually transmitted infections. The purpose of this study was to determine the relationship of depressive symptoms, self-esteem, and sexual behaviors in a predominantly Hispanic sample of MSM. This correlational study sampled 205 MSM (M = 37 years of age, SD = +/-8) representing the diverse ethnic composition of South Florida. This sample consisted of ethnic minorities (79%) with a large number of foreign-born men (69%). Participants completed measures of depressive symptoms, self-esteem, and sexual behaviors. Results indicated that higher levels of depressive symptoms and higher levels of self-esteem had a statistically significant relationship to lower levels of safer sexual behaviors. Lower income, lower educational level, and preference for Spanish language were associated with higher levels of depressive symptoms; lower income was associated with lower levels of self-esteem; and foreign birth and a preference for Spanish language were associated with lower levels of safer sex behaviors. Higher levels of depressive symptoms and higher levels of self-esteem were associated with high-risk sexual behaviors in this sample of MSM. Further research needs to be directed at culturally specific mental health and HIV prevention strategies for these vulnerable MSM.
Sujet(s)
Dépression/ethnologie , Homosexualité masculine/statistiques et données numériques , Concept du soi , Comportement sexuel/ethnologie , Maladies sexuellement transmissibles/épidémiologie , Adulte , Facteurs âges , Études transversales , Dépression/diagnostic , Études de suivi , Hispanique ou Latino/statistiques et données numériques , Humains , Incidence , Mâle , Adulte d'âge moyen , Prise de risque , Partenaire sexuel , Maladies sexuellement transmissibles/diagnostic , Enquêtes et questionnaires , Rapports sexuels non protégés/prévention et contrôle , Rapports sexuels non protégés/statistiques et données numériques , Jeune adulteRÉSUMÉ
Cotranscriptional loading of proteins onto nascent transcripts contributes to the formation of messenger ribonucleoprotein particles (mRNPs) competent for nuclear export. The transcription machinery is believed to play a pivotal role in mRNP assembly, which is at least partially linked to the function of the THO/TREX complex and the mRNA termination/polyadenylation apparatus. Here we demonstrate a prominent role for the rate of transcription in the production of export-competent mRNPs. We show that a transcription-defective allele of the Rad3p helicase, a component of the TFIIH transcription initiation factor, suppresses several phenotypes associated with defective mRNA processing and export. Strikingly, the effects of compromised Rad3p activity can be phenocopied by a transcription elongation drug as well as by other mutations affecting transcription. Our results suggest that efficient mRNP assembly is under a kinetic control that is influenced by the rate of transcription.