Requirement for highly efficient pre-mRNA splicing during Drosophila early embryonic development

Drosophila syncytial nuclear divisions limit transcription unit size of early zygotic genes. As mitosis inhibits not only transcription, but also pre-mRNA splicing, we reasoned that constraints on splicing were likely to exist in the early embryo, being splicing avoidance a possible explanation why most early zygotic genes are intronless. We isolated two mutant alleles for a subunit of the NTC/Prp19 complexes, which specifically impaired pre-mRNA splicing of early zygotic but not maternally encoded transcripts. We hypothesized that the requirements for pre-mRNA splicing efficiency were likely to vary during development. Ectopic maternal expression of an early zygotic pre-mRNA was sufficient to suppress its splicing defects in the mutant background. Furthermore, a small early zygotic transcript with multiple introns was poorly spliced in wild-type embryos. Our findings demonstrate for the first time the existence of a developmental pre-requisite for highly efficient splicing during Drosophila early embryonic development and suggest in highly proliferative tissues a need for coordination between cell cycle and gene architecture to ensure correct gene expression and avoid abnormally processed transcripts.FCT-Fundacao para a Ciencia e Tecnologia (Portugal) [PTDC/SAU-BID/111796/2009, PTDC/BIA-BCM/111822/ 2009, PTDC/BBB-BQB/0712/2012, PEst-OE/EQB/LA0023/2013, SFRH/BPD/47957/2008, SFRH/BPD/63869/2009]info:eu-repo/semantics/publishedVersio

Absence of the spindle assembly checkpoint restores mitotic fidelity upon loss of sister chromatid cohesion

The fidelity of mitosis depends on cohesive forces that keep sister chromatids together. This is mediated by cohesin that embraces sister chromatid fibers from the time of their replication until the subsequent mitosis [1-3]. Cleavage of cohesin marks anaphase onset, where single chromatids are dragged to the poles by the mitotic spindle [4-6]. Cohesin cleavage should only occur when all chromosomes are properly bio-oriented to ensure equal genome distribution and prevent random chromosome segregation. Unscheduled loss of sister chromatid cohesion is prevented by a safeguard mechanism known as the spindle assembly checkpoint (SAC) [7, 8]. To identify specific conditions capable of restoring defects associated with cohesion loss, we screened for genes whose depletion modulates Drosophila wing development when sister chromatid cohesion is impaired. Cohesion deficiency was induced by knockdown of the acetyltransferase separation anxiety (San)/Naa50, a cohesin complex stabilizer [9-12]. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key SAC proteins, Mad2 and Mpsl, suppressed developmental defects associated with San depletion. SAC impairment upon cohesin removal, triggered by San depletion or artificial removal of the cohesin complex, prevented extensive genome shuffling, reduced segregation defects, and restored cell survival. This counterintuitive phenotypic suppression was caused by an intrinsic bias for efficient chromosome biorientation at mitotic entry, coupled with slow engagement of error-correction reactions. Thus, in contrast to SAC's role as a safeguard mechanism for mitotic fidelity, removal of this checkpoint alleviates mitotic errors when sister chromatid cohesion is compromised.Lisboa Regional Operational Programme (Lisboa 2020) through the European Regional Development Fund (FEDER); Fundacao para a Ciencia e a Tecnologia (FCT; Portugal); FCT [SFRH/BPD/87482/2012, SFRH /BD/52438/2013, PD/BD/52428/2013, PD/00117/2012, CRM: 0027030, PTDC/BEX-BID/0395/2014, UID/BIM/04773/2013 CBMR 1334, IF/00851/2012/CP0185/CT0004]; Association for International Cancer Research [AICR 10-0553]; EMBO Installation Grant [IG2778]; European Research Council Starting Grant [ERC-2014-STG-638917]; [PPBI-POCI-01-0145-FEDER-022122]; [LISBOA-01-0145-FEDER-022170

Dilemas éticos en el “buen morir”: Cuando los tiempos subjetivos no acompañan los tiempos cronológicos de la enfermedad

Throughout this text, we will analyze the concept of time and how this time could determine or limit what we understand in palliative care as "dying well". To understand these concepts, we use the bibliographic review as a work methodology, we search for authors from various disciplines, who suggest thinking about dying well, the subjective and chronological time. The review consists of an approach to the concepts, ideas, theories that motivate us to raise some questions regarding our practice, and thus, produce this writing. The following questions will guide this analysis: what happens when the chronological time of the disease is not the necessary time to accompany the speed with which death presents itself in the subjectivity of the patient and their family?, is there an ideal death?, should we all go through an ideal death?, what happens when chronological time does not allow us to subjectively elaborate the idea of dying?, can the loving and sympathetic care of the diseased body, by emphasizing the inherent dignity of the human being, have an impact in the perception of chronological time?.A lo largo del presente texto, será el concepto del tiempo quien guíe el recorrido y cómo este tiempo podría condicionar o limitar lo que en cuidados paliativos entendemos como “el buen morir”. Para acercarnos a dichos conceptos, utilizamos como metodología de trabajo, la revisión bibliográfica, realizamos una exploración de autores de diversas disciplinas, que plantean e invitan a pensar el buen morir, los tiempos subjetivos y cronológicos. La revisión consta de un acercamiento a los conceptos, ideas, teorías, que nos impulsan a generar algunas preguntas respecto a nuestra práctica, y así producir dicho escrito. Orientarán este recorrido los siguientes interrogantes: ¿qué sucede cuando los tiempos cronológicos de la enfermedad no son los tiempos necesarios para acompañar la velocidad con que la muerte se nos presenta en la subjetividad de un paciente y en el seno de una familia?,  ¿hay una muerte ideal?, ¿todos deberíamos atravesar por una muerte ideal?, ¿qué sucede cuando el tiempo cronológico no nos permite elaborar subjetivamente la idea del morir?, ¿puede el cuidado amoroso y empático del cuerpo enfermo, al hacer hincapié en la dignidad inherente al ser humano, tener algún  impacto en la percepción del tiempo cronológico?

A dual-function SNF2 protein drives chromatid resolution and nascent transcripts removal in mitosis

Mitotic chromatin is largely assumed incompatible with transcription due to changes in the transcription machinery and chromosome architecture. However, the mechanisms of mitotic transcriptional inactivation and their interplay with chromosome assembly remain largely unknown. By monitoring ongoing transcription in Drosophila early embryos, we reveal that eviction of nascent mRNAs from mitotic chromatin occurs after substantial chromosome compaction and is not promoted by condensin I. Instead, we show that the timely removal of transcripts from mitotic chromatin is driven by the SNF2 helicase-like protein Lodestar (Lds), identified here as a modulator of sister chromatid cohesion defects. In addition to the eviction of nascent transcripts, we uncover that Lds cooperates with Topoisomerase 2 to ensure efficient sister chromatid resolution and mitotic fidelity. We conclude that the removal of nascent transcripts upon mitotic entry is not a passive consequence of cell cycle progression and/or chromosome compaction but occurs via dedicated mechanisms with functional parallelisms to sister chromatid resolution.info:eu-repo/semantics/publishedVersio

Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation

Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here, we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells

Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in Drosophila

Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of beta-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4. C8 and 02) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a beta-strand connected by a short loop to an alpha-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as,a potential model organism for MADD. (C) 2012 Elsevier B.V. All rights reserved.Fundacao para a Ciencia e Tecnologia (FCT/MCTES, Portugal) [PTDC/SAU-GMG/70033/2006, PTDC/QUI-BIQ/113027/2009, PTDC/BIA-BCM/111822/2009, PTDC/SAU-BID/111796/2009, SFRH/BPD/41609/2007, SFRH/BPD/74475/2010, SFRH/BPD/34763/2007]; CLIMB UK; [PEst-OE/EQB/LA0004/2011]info:eu-repo/semantics/publishedVersio

Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers.

Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. Here we test this assumption in three types of Drosophila epithelium; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells seems to be driven by lateral adhesion, which pulls cells born outside the epithelial layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions.The authors are grateful to R. Nieuwburg, the St Johnston group, and other Gurdon Institute members for suggestions. We thank the Bloomington Stock Center, J. Knoblich, and the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for fly stocks. We thank N. Lowe for technical assistance. This work was supported by a Wellcome Trust Principal Fellowship to D.St.J. (080007), and by core support from the Wellcome Trust (092096) and Cancer Research UK (A14492). D.T.B. was supported by a Marie Curie Fellowship and the Wellcome Trust. H.E.L. was supported by a Herchel Smith Studentship.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb324

The Gonadotropin-Inhibitory Hormone: What We Know and What We Still Have to Learn From Fish

Gonadotropin-inhibitory hormone, GnIH, is named because of its function in birds and mammals; however, in other vertebrates this function is not yet clearly established. More than half of the vertebrate species are teleosts. This group is characterized by the 3R whole genome duplication, a fact that could have been responsible for the great phenotypic complexity and great variability in reproductive strategies and sexual behavior. In this context, we revise GnIH cell bodies and fibers distribution in adult brains of teleosts, discuss its relationship with GnRH variants and summarize the few reports available about the ontogeny of the GnIH system. Considering all the information presented in this review, we propose that in teleosts, GnIH could have other functions beyond reproduction or act as an integrative signal in the reproductive process. However, further studies are required in order to clarify the role of GnIH in this group including its involvement in development, a key stage that strongly impacts on adult life

Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodelling

Oocytes are arrested for long periods of time in the prophase of the first meiotic division (prophase I). As chromosome condensation poses significant constraints to gene expression, the mechanisms regulating transcriptional activity in the prophase I-arrested oocyte are still not entirely understood. We hypothesized that gene expression during the prophase I arrest is primarily epigenetically regulated. Here we comprehensively define the Drosophila female germ line epigenome throughout oogenesis and show that the oocyte has a unique, dynamic and remarkably diversified epigenome characterized by the presence of both euchromatic and heterochromatic marks. We observed that the perturbation of the oocyte's epigenome in early oogenesis, through depletion of the dKDM5 histone demethylase, results in the temporal deregulation of meiotic transcription and affects female fertility. Taken together, our results indicate that the early programming of the oocyte epigenome primes meiotic chromatin for subsequent functions in late prophase I