A paramutation is an epigenetic interaction between two alleles of a locus, through which one allele induces a heritable modification in the other allele without modifying the DNA sequence. The paramutated allele itself becomes paramutagenic, that is, capable of epigenetically converting a new paramutable allele. Here we describe a case of paramutation in animals showing long-term transmission over generations. We previously characterized a homology-dependent silencing mechanism referred to as the trans-silencing effect (TSE), involved in P-transposable-element repression in the germ line. We now show that clusters of P-element-derived transgenes that induce strong TSE can convert other homologous transgene clusters incapable of TSE into strong silencers, which transmit the acquired silencing capacity through 50 generations. The paramutation occurs without any need for chromosome pairing between the paramutagenic and the paramutated loci, and is mediated by maternal inheritance of cytoplasm carrying Piwi-interacting RNAs (piRNAs) homologous to the transgenes. The repression capacity of the paramutated locus is abolished by a loss-of-function mutation of the aubergine gene involved in piRNA biogenesis, but not by a loss-of-function mutation of the Dicer-2 gene involved in siRNA production. The paramutated cluster, previously producing barely detectable levels of piRNAs, is converted into a stable, strong piRNA-producing locus by the paramutation and becomes fully paramutagenic itself. Our work provides a genetic model for the emergence of piRNA loci, as well as for RNA-mediated trans-generational repression of transposable elements.

A new class of small RNAs (endo-siRNAs) produced from endogenous double-stranded RNA (dsRNA) precursors was recently shown to mediate transposable element (TE) silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation, as has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, we used the viral suppressors of RNA silencing B2 and P19. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. We hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3-9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. Our findings provide evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila.RNAi ͉ nucleus ͉ viruses R ecent deep sequencing efforts have provided critical information on Drosophila small RNA repertoires in various tissues and during distinct developmental stages (1-7). Four classes of small RNAs mediate posttranscriptional gene silencing in Drosophila: i) Ϸ22-nt miRNAs are processed from stem-loop precursors by Dicer-1 and repress mRNA expression; ii) Ϸ25-nt piRNAs are produced from transposable element (TE) transcripts in gonads where they silence TEs through a feedback regulatory mechanism involving the PIWI subfamily of Argonautes (2, 3, 8-11); iii) 21-nt siRNAs are processed from long dsRNAs by Dicer-2 and trigger RNAi, for instance in response to viral infection (12-14); and iv) recently discovered 21-nt endo-siRNAs are processed from endogenous dsRNA precursors by Dicer-2 and silence TEs, and possibly endogenous mRNA in somatic tissues (1,(5)(6)(7)15).In S. pombe, siRNAs produced from repetitive sequences in chromosome pericentromeres direct heterochromatin formation and transcriptional gene silencing. As in S. pombe (16), Drosophila heterochromatin is prominent in pericentromeric regions, mostly comprised of short satellite repeats and TEs, and is associated with histone H3 methylation on lysine 9 (H3K9) by the histone methylase Su(var)3-9 (Clr4 in S pombe). This allows recruitment of the Heterochromatin Protein HP1 (SWI6 in S. pombe) to maintain and spread heterochromatin to nearby genes (17). Despite these analogies, the evidence supporting a role of small RNAs in heterochromatin formation and transcriptional gene silencing in Drosophila remain indirect (18,19). Mutants for the ...

The microtubule-associated protein Tau is found in large amount in axons of neurons and is involved in human neurodegenerative diseases called tauopathies, which include Alzheimer's disease. In these diseases, the Tau protein is abnormally hyperphosphorylated and one therapeutic strategy currently under consideration consists in inhibiting Tau phosphorylation. However, the consequences of an excess of hypophosphorylated Tau onto neuronal physiology have not been investigated in vivo. Here we studied how important is Tau phosphorylation for axonal transport and neurohormone release in vivo, using the Drosophila model. Surprisingly, our results demonstrate a stronger toxicity of hypophosphorylated Tau for neuronal function, when compared with normal or pseudophosphorylated Tau. This reveals a potential limit of the current therapeutic strategy aimed at inhibiting Tau phosphorylation.

An important step in epithelial organ development is size maturation of the organ lumen to attain correct dimensions. Here we show that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein.

In 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.

Morphogenesis of the adult structures of holometabolous insects is regulated by ecdysteroids and juvenile hormones and involves cell-cell interactions mediated in part by the cell surface integrin receptors and their extracellular matrix (ECM) ligands. These adhesion molecules and their regulation by hormones are not well characterized. We describe the gene structure of a newly described ECM molecule, tenectin, and demonstrate that it is a hormonally regulated ECM protein required for proper morphogenesis of the adult wing and male genitalia. Tenectin’s function as a new ligand of the PS2 integrins is demonstrated by both genetic interactions in the fly and by cell spreading and cell adhesion assays in cultured cells. Its interaction with the PS2 integrins is dependent on RGD and RGD-like motifs. Tenectin’s function in looping morphogenesis in the development of the male genitalia led to experiments that demonstrate a role for PS integrins in the execution of left-right asymmetry.

We 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.

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