Tau excess impairs mitosis and kinesin-5 function, leading to aneuploidy and cell death

International audienceIn neurodegenerative diseases such as Alzheimer's disease (AD), cell cycle defects and associated aneuploidy have been described. However, the importance of these defects in the physiopathology of AD and the underlying mechanistic processes are largely unknown, in particular with respect to the microtubule (MT)-binding protein Tau, which is found in excess in the brain and cerebrospinal fluid of affected individuals. Although it has long been known that Tau is phosphorylated during mitosis to generate a lower affinity for MTs, there is, to our knowledge, no indication that an excess of this protein could affect mitosis. Here, we studied the effect of an excess of human Tau (hTau) protein on cell mitosis in vivo. Using the Drosophila developing wing disc epithelium as a model, we show that an excess of hTau induces a mitotic arrest, with the presence of monopolar spindles. This mitotic defect leads to aneuploidy and apoptotic cell death. We studied the mechanism of action of hTau and found that the MT-binding domain of hTau is responsible for these defects. We also demonstrate that the effects of hTau occur via the inhibition of the function of the kinesin Klp61F, the Drosophila homologue of kinesin-5 (also called Eg5 or KIF11). We finally show that this deleterious effect of hTau is also found in other Drosophila cell types (neuroblasts) and tissues (the developing eye disc), as well as in human HeLa cells. By demonstrating that MT-bound Tau inhibits the Eg5 kinesin and cell mitosis, our work provides a new framework to consider the role of Tau in neurodegenerative diseases

Etude fonctionnelle de la Ténectine, un nouveau ligand des intégrines chez Drosophila melanogaster

Les intégrines sont des récepteurs de surface cellulaire permettant de relier le cytosquelette à la matrice extracellulaire présents chez tous les animaux et impliqués dans des processus clés au cours du développement comme la prolifération, la migration, la différenciation et la mort cellulaire. ténectine (tnc) est un nouveau gène codant potentiellement un ligand des intégrines alphaPS2betaPS chez la drosophile. Au cours de l embryogenèse, tnc s exprime dans de nombreux tissus d origine neuro-ectodermique comme les intestins antérieur et postérieur, les trachées et le système nerveux central. Afin de déterminer son rôle, nous avons généré des mutants par les techniques d interférence ARN et de remobilisation d un élément P. L analyse de leurs phénotypes et les résultats d expériences biochimiques et génétiques ont démontré que Tnc est un nouveau ligand des intégrines alphaPS2betaPS, impliqué dans l adhésion des épithélia alaires, la fasciculation axonal et la morphogenèse synaptique. Les résultats préliminaires suggèrent également que Tnc jouerait un rôle indépendant des intégrines dans la morphogenèse de l intestin postérieur et des trachées. Par ailleurs, plusieurs données nous indiquent que la 20 hydroxyecdysone et l hormone juvénile pourraient intervenir dans la régulation de l expression de tnc.Integrins are cell surface receptors of the extracellular matrix present in all animals and involved in key developmental processes such as cell migration, proliferation, differentiation and survival. tenectin (tnc) is a novel gene encoding a putative ligand of Drosophila alphaPS2betaPS integrin. In situ hybridization experiments revealed that tnc is expressed in many neuroectodermic tissues such as foregut, hingut, tracheas and central nervous system during embryonic development. In order to understand the role of tenectin during development we have established mutant lines, using RNA interference (RNAi) and P element remobilization processes. Phenotype analysis as well as biochemical and genetical experiments have demonstrated that Tenectin is a novel ligand of the Drosophila alphaPS2betaPS integrin and that Tnc is implied in wing epithelial attachment, axon fasciculation and synaptogenesis. Preliminary results suggest that tenectin plays although integrin-independant functions in trachea and hindgut morphogenesis. Moreover, some data suggest that tnc could be hormonally regulated by 20 hydroxyecdysone and juvenile hormone.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF

Tenectin, a novel extracellular matrix protein expressed during Drosophila melanogaster embryonic development

1567-133X (Print) Journal Article Research Support, Non-U.S. Gov'tDuring Drosophila embryonic development, various morphogenetic processes require the remodeling of the extracellular matrix. In a previous study, we have identified and characterized a cDNA encoding a novel putative extracellular matrix protein named tenebrin, in the beetle Tenebrio molitor. Here, we examine the expression of the Drosophila ortholog, referred to as Tenectin (Tnc), during embryonic development. Tnc is expressed in the majority of tissues of neuroectodermic origin such as hindgut, foregut, tracheal system, anal plate, and CNS. In the CNS, the Tnc transcript is restricted to a few cells, whereas the protein is located in the dorsal part of the axonal tracts. In the hindgut and the trachea, Tnc protein is expressed on the apical pole of the cells. Tnc is an extracellular matrix protein secreted in a polarized way in different organs of Drosophila embryos

RNA-Seq Analysis to Detect Abnormal Fusion Transcripts Linked to Chromothripsis

International audienceRNA-Seq approach enables the detection and characterization of fusion or chimeric transcript associated to complex genome rearrangement. Until now, these events are classically identified at DNA level.Here we describe a complete procedure including a novel way of analyzing reads that combines genomic locations and local coverage to directly infer chimeric junctions with a high sensitivity and specificity, allowing identification of different classes of chimeric RNA events. We also recommend the best practices for the bioinformatics analysis and describe the experimental process for RNA validation using real-time PCR and sequencing

piRNAs and epigenetic conversion in Drosophila

International audienceTransposable element (TE) activity is repressed in the Drosophila germline by Piwi-Interacting RNAs (piRNAs), a class of small non-coding RNAs. These piRNAs are produced by discrete genomic loci containing TE fragments. In a recent publication, we tested for the existence of a strict epigenetic induction of piRNA production capacity by a locus in the D. melanogaster genome. We used 2 lines carrying a transgenic 7-copy tandem cluster (P-lacZ-white) at the same genomic site. This cluster generates in both lines a local heterochromatic sector. One line (T-1) produces high levels of ovarian piRNAs homologous to the P-lacZ-white transgenes and shows a strong capacity to repress homologous sequences in trans, whereas the other line (BX2) is devoid of both of these capacities. The properties of these 2 lines are perfectly stable over generations. We have shown that the maternal transmission of a cytoplasm carrying piRNAs from the first line can confer to the inert transgenic locus of the second, a totally de novo capacity to produce high levels of piRNAs as well as the ability to induce homology-dependent silencing in trans. These new properties are stably inherited over generations (n > 50). Furthermore, the converted locus has itself become able to convert an inert transgenic locus via cytoplasmic maternal inheritance. This results in a stable epigenetic conversion process, which can be performed recurrently-a phenomenon termed paramutation and discovered in Maize 60 y ago. Paramutation in Drosophila corresponds to the first stable paramutation in animals and provides a model system to investigate the epigenetically induced emergence of a piRNA-producing locus, a crucial step in epigenome shaping. In this Extra View, we discuss some additional functional aspects and the possible molecular mechanism of this piRNA-linked paramutation

Inheritance of paramutation in plants and Drosophila

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Important neuronal toxicity of microtubule-bound Tau in vivo in Drosophila

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Targeted RNA-Seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle

International audienceWe have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) NB transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3' splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications

Kmerator Suite: design of specific k -mer signatures and automatic metadata discovery in large RNA-seq datasets

International audienceThe huge body of publicly available RNA-sequencing (RNA-seq) libraries is a treasure of functional information allowing to quantify the expression of known or novel transcripts in tissues. However, transcript quantification commonly relies on alignment methods requiring a lot of computational resources and processing time, which does not scale easily to large datasets. K-mer decomposition constitutes a new way to process RNA-seq data for the identification of transcriptional signatures, as k-mers can be used to quantify accurately gene expression in a less resource-consuming way. We present the Kmerator Suite, a set of three tools designed to extract specific k-mer signatures, quantify these k-mers into RNA-seq datasets and quickly visualize large dataset characteristics. The core tool, Kmerator, produces specific k-mers for 97% of human genes, enabling the measure of gene expression with high accuracy in simulated datasets. KmerExploR, a direct application of Kmerator, uses a set of predictor gene-specific k-mers to infer metadata including library protocol, sample features or contaminations from RNA-seq datasets. KmerExploR results are visualized through a user-friendly interface. Moreover, we demonstrate that the Kmerator Suite can be used for advanced queries targeting known or new biomarkers such as mutations, gene fusions or long non-coding RNAs for human health applications

Genome Alert!: A standardized procedure for genomic variant reinterpretation and automated gene–phenotype reassessment in clinical routine

International audiencePurpose: Retrospective interpretation of sequenced data in light of the current literature is a major concern of the field. Such reinterpretation is manual and both human resources and variable operating procedures are the main bottlenecks.Methods: Genome Alert! method automatically reports changes with potential clinical significance in variant classification between releases of the ClinVar database. Using ClinVar submissions across time, this method assigns validity category to gene-disease associations.Results: Between July 2017 and December 2019, the retrospective analysis of ClinVar submissions revealed a monthly median of 1247 changes in variant classification with potential clinical significance and 23 new gene-disease associations. Re-examination of 4929 targeted sequencing files highlighted 45 changes in variant classification, and of these classifications, 89% were expert validated, leading to 4 additional diagnoses. Genome Alert! gene-disease association catalog provided 75 high-confidence associations not available in the OMIM morbid list; of which, 20% became available in OMIM morbid list For more than 356 negative exome sequencing data that were reannotated for variants in these 75 genes, this elective approach led to a new diagnosis.Conclusion: Genome Alert! (https://genomealert.univ-grenoble-alpes.fr/) enables systematic and reproducible reinterpretation of acquired sequencing data in a clinical routine with limited human resource effect