Signalling Pathways Involved in Adult Heart Formation Revealed by Gene Expression Profiling in Drosophila

Drosophila provides a powerful system for defining the complex genetic programs that drive organogenesis. Under control of the steroid hormone ecdysone, the adult heart in Drosophila forms during metamorphosis by a remodelling of the larval cardiac organ. Here, we evaluated the extent to which transcriptional signatures revealed by genomic approaches can provide new insights into the molecular pathways that underlie heart organogenesis. Whole-genome expression profiling at eight successive time-points covering adult heart formation revealed a highly dynamic temporal map of gene expression through 13 transcript clusters with distinct expression kinetics. A functional atlas of the transcriptome profile strikingly points to the genomic transcriptional response of the ecdysone cascade, and a sharp regulation of key components belonging to a few evolutionarily conserved signalling pathways. A reverse genetic analysis provided evidence that these specific signalling pathways are involved in discrete steps of adult heart formation. In particular, the Wnt signalling pathway is shown to participate in inflow tract and cardiomyocyte differentiation, while activation of the PDGF-VEGF pathway is required for cardiac valve formation. Thus, a detailed temporal map of gene expression can reveal signalling pathways responsible for specific developmental programs and provides here substantial grasp into heart formation

Sci Rep

Mosquito- and tick-borne pathogens including Chikungunya, Dengue, Japanese encephalitis, West Nile, Yellow fever and Zika virus, represent a new economic and public health challenge. In the absence of effective vaccines and specific therapies, only supportive regimens are administrated for most of these infections. Thus, the development of a targeted therapy is mandatory to stop the rapid progression of these pathogens and preoccupant associated burdens such as Guillain-Barre syndrome, microcephaly. For this, it is essential to develop biochemical tools to help study and target key viral enzymes involved in replication such as helicase complexes, methyl-transferases and RNA-dependent RNA polymerases. Here, we show that a highly purified ZIKV polymerase domain is active in vitro. Importantly, we show that this isolated domain is capable of de novo synthesis of the viral genome and efficient elongation without terminal nucleotide transferase activity. Altogether, this isolated polymerase domain will be a precious tool to screen and optimize specific nucleoside and non-nucleoside inhibitors to fight against Zika infections

Discovery of Novel Integrase Inhibitors Acting outside the Active Site Through High-Throughput Screening

International audienc

The <i>mat r-nad1e-nad5c</i> cotranscript from wheat mitochondria.

<p>(A) Scheme of the <i>mat r-nad1e-nad5c</i> RNA and the <i>trans</i>-splicing counterparts. Gray squares represent the fifth and the third exons of <i>nad1</i> and <i>nad5</i> respectively on the same molecule associated to the maturase-reverse transcriptase ORF (<i>mat-r</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052644#pone.0052644-Chapdelaine1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052644#pone.0052644-Farr1" target="_blank">[24]</a>. Blue arrowheads indicate the residues edited in the coding regions of the transcript. Red arrowheads indicate the edited residues in non-coding regions. Dotted lines indicate the splicing junctions of <i>nad1e</i> and <i>nad5c</i> exons with <i>nad1d</i> and <i>nad5b</i> exons. (B) The domain 6 of both, <i>nad1-I4</i> and <i>nad5-I2,</i> group II <i>trans</i>-introns is depicted as stem-loop structure. The most stable conformations are presented for the non-edited and edited mRNA. A red arrow indicates the edited residue.</p

Expression levels of the chimeric <i>nad1e</i> mutants.

<p>(A) Putative hairpin structure of D6 domain from the <i>nad1e trans</i>-intron in the constructs used in these experiments. The edited version of the D6 region (SDxwt) and the SDxA, SDxRv and SDxDb mutants are presented. The arrow indicates the position of the editing target C residue in the precursor transcript. Red letters indicate the residues modified in the constructs. (B) Agarose gel electrophoresis of PCR products from <i>trans</i>-spliced <i>nad1</i>. The introduced gene contains the <i>nad1e</i> exon and the 3′-half intron under the control of the mitochondrial <i>cox2</i> promoter (for details see Materials and methods). The upper panel shows the PCR product from precursor cDNA molecules. The middle panel shows the <i>trans</i>-spliced <i>nad1c</i>/<i>nad1d</i> exons, linked to the recombinant <i>nad1e</i> exon. The lower panel shows the PCR products of the endogenous <i>atp9</i> transcripts as a control for RNA preparations from electroporated mitochondria. Only RNA samples where PCR analysis showed no DNA contamination were used for further analysis. No amplification products were observed on cDNA from untransformed mitochondria using the primers specific for the <i>nad1</i> trans-splicing analysis. The primers used are detailed in File S1. The signal in gels corresponds to one (<i>atp9</i>), two (precursor <i>nad1e</i>) or three (<i>trans</i>-spliced product) rounds of 20 PCR cycles. The sequence of precursor and spliced PCR products were determined after purification from the gel.</p

RNA editing levels within the spliced <i>nad1</i> chimeric transcript.

<p>The SDx construct was electroporated into isolated mitochondria as described in Materials and Methods. After 18 hr incubation, the mtRNA was isolated and the chimeric spliced products were obtained by RT-PCR using primers located in the exon <i>nad1b</i> and the terminator <i>cob</i>. The PCR product inserted into the pGEM-T vector was cloned and sequenced. Editing levels of C-targets on exons <i>nad1b</i> to <i>nad1e</i> represent the average of 29 independent clones (gray bars). The positions of C targets in the mature <i>nad1</i> transcript, considering the first nucleotide the beginning ATG codon from exon <i>nad1a,</i> are indicated below the bars.</p

RNA editing in trans-splicing <i>nad5c</i> branched molecules.

<p>(A) Scheme of the <i>nad5b</i> and <i>mat-r-nad1e</i>-<i>nad5c</i> transcripts. The dimension of boxes do not represents the actual size of the exons. The primers used for branched splicing intermediate analysis are indicated by arrowheads. (B) Agarose gel electrophoresis analysis of RT-PCR products from the <i>nad5c</i> branched structure. cDNA was synthesized with primer P1b, followed by PCR amplification using primers P2b and P4 (lane 1) or primers P2b and P5 (lane 2). The primer P4 is specific of the maturase Dx domain and P5 is specific of exon <i>nad1e</i>. M, molecular weight markers. (C) Selected parts of the electropherogram of a 1.5 kbp cloned PCR product are shown. The sequence starting from the maturase domain Dx and the beginning of the 3′ half of the <i>nad1-I4</i> intron (upper panel), the end of the 3′ half of the <i>nad1e</i> intron and the <i>nad1e</i> exon (middle panel) and the <i>nad5c</i> branched splicing intermediate (lower panel) are shown. Red arrowheads show the position of edited residues.</p

In organello <i>trans</i>-splicing after electroporation of <i>nad1e</i> recombinant vectors.

<p>(A) The four transcription units required to assemble the <i>nad1</i> mRNA in wheat mitochondria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052644#pone.0052644-Chapdelaine1" target="_blank">[21]</a> are indicated in the upper part of panel A (<i>nad1</i> exons a, b, c, d, and e are represented by boxes). The four chimeric <i>nad1e</i> transgene constructs, <i>Mat1</i> containing all the 3′-half intron 4 (<i>3</i>′ <i>nad1-I4</i>) and the <i>nad1e</i> exon, <i>Dx1</i> containing only the maturase domain (Dx) from the <i>mat-r</i> ORF were linked to the inverted repeat (double stem-loop) from the non coding region of wheat apocytochrome b gene (<i>Ir-cob)</i>. These constructs were fused to the cytochrome oxidase subunit 2 (<i>cox2</i>) promoter to obtain <i>coxMat1</i> and <i>cox2Dx1</i> recombinant vectors. The arrows signal the specific exon and <i>cob</i> primers used in electroporation <i>trans</i>-splicing analyses. (B) Agarose gel electrophoresis of RT-PCR products obtained with the primer <i>cob</i> combined with primers located either in exons <i>nad1b</i> (Pb) <i>or nad1c</i> (Pc). The position of the PCR primers is indicated by arrows. M, molecular weight marker. The different constructs differ on upstream sequences from <i>nad1e</i> exon.</p