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- 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.
- 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.
- 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.
- 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.