Percorrer por autor "Prudencio, Pedro"
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- Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organismsPublication . Rathore, Om; Faustino, Alexandra; Prudencio, Pedro; Van Damme, Petra; Cox, C. J.; Martinho, Rui GoncaloProtein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes.
- Analysis of mammalian native elongating transcript sequencing (mNET-seq) high-throughput dataPublication . Prudencio, Pedro; Rebelo, Kenny; Grosso, Ana Rita; Martinho, Rui Gonçalo; Carmo-Fonseca, MariaMammalian Native Elongating Transcript sequencing (mNET-seq) is a recently developed technique that generates genome-wide profiles of nascent transcripts associated with RNA polymerase II (Pol II) elongation complexes. The ternary transcription complexes formed by Pol II, DNA template and nascent RNA are first isolated, without crosslinking, by immunoprecipitation with antibodies that specifically recognize the different phosphorylation states of the polymerase large subunit C-terminal domain (CTD). The coordinate of the 3' end of the RNA in the complexes is then identified by high-throughput sequencing. The main advantage of mNET-seq is that it provides global, bidirectional maps of Pol II CTD phosphorylation-specific nascent transcripts and coupled RNA processing at single nucleotide resolution. Here we describe the general pipeline to prepare and analyse high-throughput data from mNET-seq experiments.
- Drosophila aPKC is required for mitotic spindle orientation during symmetric division of epithelial cellsPublication . Guilgur, Leonardo G.; Prudencio, Pedro; Ferreira, Tania; Pimenta-Marques, Ana Rita; Goncalo Martinho, RuiEpithelial cells mostly orient the spindle along the plane of the epithelium (planar orientation) for mitosis to produce two identical daughter cells. The correct orientation of the spindle relies on the interaction between cortical polarity components and astral microtubules. Recent studies in mammalian tissue culture cells suggest that the apically localised atypical protein kinase C (aPKC) is important for the planar orientation of the mitotic spindle in dividing epithelial cells. Yet, in chicken neuroepithelial cells, aPKC is not required in vivo for spindle orientation, and it has been proposed that the polarization cues vary between different epithelial cell types and/or developmental processes. In order to investigate whether Drosophila aPKC is required for spindle orientation during symmetric division of epithelial cells, we took advantage of a previously isolated temperature-sensitive allele of aPKC. We showed that Drosophila aPKC is required in vivo for spindle planar orientation and apical exclusion of Pins (Raps). This suggests that the cortical cues necessary for spindle orientation are not only conserved between Drosophila and mammalian cells, but are also similar to those required for spindle apicobasal orientation during asymmetric cell division.
- Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesisPublication . Ferreira, Tania; Prudencio, Pedro; Martinho, Rui GoncaloNurse cell dumping is an actin-myosin based process, where 15 nurse cells of a given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. We isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis we observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. Our work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. We show that Pkn negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. We propose Pim provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase. (C) 2014 Elsevier Inc. All rights reserved.
- Early programming of the oocyte epigenome temporally controls late prophase I transcription and chromatin remodellingPublication . Navarro-Costa, Paulo; McCarthy, Alicia; Prudencio, Pedro; Greer, Christina; Guilgur, Leonardo Gastón; Becker, Jorg D.; Secombe, Julie; Rangan, Prashanth; Martinho, Rui GoncaloOocytes 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.
- Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in DrosophilaPublication . J. Gonçalves, Emanuel; Henriques, Barbara J.; Rodrigues, Joao V.; Prudencio, Pedro; Rocha, Hugo; Vilarinho, Laura; Martinho, Rui Goncalo; Gomes, Claudio M.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.
- NineTeen Complex-subunit Salsa is required for efficient splicing of a subset of introns and dorsal-ventral patterningPublication . Rathore, Om; Silva, Rui D.; Ascensao-Ferreira, Mariana; Matos, Ricardo; Carvalho, Celia; Marques, Bruno; Tiago, Margarida N.; Prudencio, Pedro; Andrade, Raquel P.; Roignant, Jean-Yves; Barbosa-Morais, Nuno; Martinho, Rui GoncaloThe NineTeen Complex (NTC), also known as pre-mRNA-processing factor 19 (Prp19) complex, regulates distinct spliceosome conformational changes necessary for splicing. During Drosophila midblastula transition, splicing is particularly sensitive to mutations in NTC-subunit Fandango, which suggests differential requirements of NTC during development. We show that NTC-subunit Salsa, the Drosophila ortholog of human RNA helicase Aquarius, is rate-limiting for splicing of a subset of small first introns during oogenesis, including the first intron of gurken. Germline depletion of Salsa and splice site mutations within gurken first intron impair both adult female fertility and oocyte dorsal-ventral patterning, due to an abnormal expression of Gurken. Supporting causality, the fertility and dorsal-ventral patterning defects observed after Salsa depletion could be suppressed by the expression of a gurken construct without its first intron. Altogether, our results suggest that one of the key rate-limiting functions of Salsa during oogenesis is to ensure the correct expression and efficient splicing of the first intron of gurken mRNA. Retention of gurken first intron compromises the function of this gene most likely because it undermines the correct structure and function of the transcript 5'UTR.
- Requirement for highly efficient pre-mRNA splicing during Drosophila early embryonic developmentPublication . Guilgur, Leonardo Gastón; Prudencio, Pedro; Sobral, Daniel; Liszekova, Denisa; Rosa, Andre; Martinho, Rui GoncaloDrosophila 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.