Emilie Brasset

Transcriptional properties and splicing of the flamenco piRNA cluster.

In Drosophila, the piRNA cluster, flamenco, produces most of the piRNAs (PIWI‐interacting RNAs) that silence transposable elements in the somatic follicle cells during oogenesis. These piRNAs are thought to be processed from a long single‐stranded precursor transcript. Here, we demonstrate that flamenco transcription is initiated from an RNA polymerase II promoter containing an initiator motif (Inr) and downstream promoter element (DPE) and requires the transcription factor, Cubitus interruptus. We show that the flamenco precursor transcript undergoes differential alternative splicing to generate diverse RNA precursors that are processed to piRNAs. Our data reveal dynamic processing steps giving rise to piRNA cluster precursors.

Spatio-temporal requirements for transposable element piRNA-mediated silencing during Drosophila oogenesis

During Drosophila oogenesis, transposable element (TE) repression involves the Piwi-interacting RNA (piRNA) pathway which ensures genome integrity for the next generation. We developed a transgenic model to study repression of the Idefix retrotransposon in the germline. Using a candidate gene KD-approach, we identified differences in the spatio-temporal requirements of the piRNA pathway components for piRNA-mediated silencing. Some of them (Aub, Vasa, Spn-E) are necessary in very early stages of oogenesis within the germarium and appear to be less important for efficient TE silencing thereafter. Others (Piwi, Ago3, Mael) are required at all stages of oogenesis. Moreover, during early oogenesis, in the dividing cysts within the germarium, Idefix anti-sense transgenes escape host control, and this is associated with very low piwi expression. Silencing of P-element-based transgenes is also strongly weakened in these cysts. This region, termed the ‘Piwiless pocket’ or Pilp, may ensure that new TE insertions occur and are transmitted to the next generation, thereby contributing to genome dynamics. In contrast, piRNA-mediated silencing is strong in germline stem cells in which TE mobilization is tightly repressed ensuring the continued production of viable germline cysts.

Dot COM, a nuclear transit center for the primary piRNA pathway in Drosophila.

The piRNA pathway protects genomes by silencing mobile elements. Despite advances in understanding the processing events that generate piRNAs for silencing, little is known about how primary transcripts are transported from their genomic clusters to their processing centers. Using a model of the Drosophila COM/flamenco locus in ovarian somatic cells, we identified a prominent nuclear structure called Dot COM, which is enriched in long transcripts from piRNA clusters but located far from their transcription sites. Remarkably, transcripts from multiple clusters accumulate at Dot COM, which is often juxtaposed with Yb-bodies, the cytoplasmic processing centers for cluster transcripts. Genetic evidence suggests that the accumulation of precursor transcripts at Dot COM represents one of the most upstream events in the piRNA pathway. Our results provide new insights into the initial steps of the piRNA pathway, and open up a new research area important for a complete understanding of this conserved pathway.

NucBase, an easy to use read mapper for small RNAs

BackgroundHigh-throughput deep-sequencing technology has generated an unprecedented number of expressed sequence reads that offer the opportunity to get insight into biological systems. Several databases report the sequence of small regulatory RNAs which play a prominent role in the control of transposable elements (TE). However, the huge amount of data reported in these databases remains mostly unexplored because the available tools are hard for biologists to use.ResultsHere we report NucBase, a new program designed to make an exhaustive search for sequence matches and to align short sequence reads from large nucleic acid databases to genomes or input sequences. NucBase includes a graphical interface which allows biologists to align sequences with ease and immediately visualize matched sequences, their number and their genomic position. NucBase identifies nucleic motives with strict identity to input sequences, and it capably finds candidates with one or several mismatches. It offers the opportunity to identify “core sequences” comprised of a chosen number of consecutive matching nucleotides. This software can be run locally on any Windows, Linux or Mac OS computer with 32-bit architecture compatibility.ConclusionsSince this software is easy to use and can detect reads that were undetected by other software, we believe that it will be useful for biologists involved in the field of TE silencing by small non-coding RNAs. We hope NucBase will be useful for a larger community of researchers, since it makes exploration of small nucleic sequences in any organism much easier.

Microsporidian Genomes Harbor a Diverse Array of Transposable Elements that Demonstrate an Ancestry of Horizontal Exchange with Metazoans.

Microsporidian genomes are the leading models to understand the streamlining in response to a pathogenic lifestyle; they are gene-poor and often possess small genomes. In this study, we show a feature of microsporidian genomes that contrasts this pattern of genome reduction. Specifically, genome investigations targeted at Anncaliia algerae, a human pathogen with a genome size of 23 Mb, revealed the presence of a hitherto undetected diversity in transposable elements (TEs). A total of 240 TE families per genome were identified, exceeding that found in many free-living fungi, and searches of microsporidian species revealed that these mobile elements represent a significant portion of their coding repertoire. Their phylogenetic analysis revealed that many cases of ancestry involve recent and bidirectional horizontal transfers with metazoans. The abundance and horizontal transfer origin of microsporidian TEs highlight a novel dimension of genome evolution in these intracellular pathogens, demonstrating that factors beyond reduction are at play in their diversification.

Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila

Combining genome sequence analysis and functional analysis, we show that some full‐length copies of tirant are present in heterochromatic regions in Drosophila simulons and that when tested in vitro, these copies have a functional promoter. However, when inserted in heterochromatic regions, tirant copies are inactive in vivo, and only transcription of euchromatic copies can be detected. Thus, our data indicate that the localization of the element is a hallmark of its activity in vivo and raise the question of genomic invasions by transposable elements and the importance of their genomic integration sites.— Fablet, M., Lerat, E., Rebollo, R., Horard, B., Burlet, N., Martinez, S., Brasset, E., Gilson, E., Vaury, C., Vieira, C. Genomic environment influences the dynamics of the tirant LTR retrotransposon in Drosophila. FASEBJ. 23, 1482–1489 (2009)

Distinct features of the piRNA pathway in somatic and germ cells: from piRNA cluster transcription to piRNA processing and amplification

Transposable elements (TEs) are major components of genomes. Their mobilization may affect genomic expression and be a threat to genetic stability. This is why they have to be tightly regulated by a dedicated system. In the reproductive tissues of a large range of organisms, they are repressed by a subclass of small interfering RNAs called piRNAs (PIWI interacting RNAs). In Drosophila melanogaster, piRNAs are produced both in the ovarian germline cells and in their surrounding somatic cells. Accumulating evidence suggests that germinal and somatic piRNA pathways are far more different than previously thought. Here we review the current knowledge on piRNA production in both these cell types, and explore their similarities and differences.

History of the discovery of a master locus producing piRNAs: the flamenco/COM locus in Drosophila melanogaster

The discovery of transposable elements (TEs) in the 1950s by B. McClintock implied the existence of cellular regulatory systems controlling TE activity. The discovery of flamenco (flam) an heterochromatic locus from Drosophila melanogaster and its ability to survey several TEs such as gypsy, ZAM, and Idefix contributed to peer deeply into the mechanisms of the genetic and epigenetic regulation of TEs. flam was the first cluster producing small RNAs to be discovered long before RNAi pathways were identified in 1998. As a result of the detailed genetic analyses performed by certain laboratories and of the sophisticated genetic tools they developed, this locus has played a major role in our understanding of piRNA mediated TE repression in animals. Here we review the first discovery of this locus and retrace decades of studies that led to our current understanding of the relationship between genomes and their TE targets.

Polycomb group-dependent, heterochromatin protein 1-independent, chromatin structures silence retrotransposons in somatic tissues outside ovaries.

Somatic cells are equipped with different silencing mechanisms that protect the genome against retrotransposons. In Drosophila melanogaster, a silencing pathway implicating the argonaute protein PIWI represses retrotransposons in cells surrounding the oocyte, whereas a PIWI-independent pathway is involved in other somatic tissues. Here, we show that these two silencing mechanisms result in distinct chromatin structures. Using sensor transgenes, we found that, in somatic tissues outside of the ovaries, these transgenes adopt a heterochromatic configuration implicating hypermethylation of H3K9 and K27. We identified the Polycomb repressive complexes (PRC1 and 2), but not heterochromatin protein 1 to be necessary factors for silencing. Once established, the compact structure is stably maintained through cell divisions. By contrast, in cells where the silencing is PIWI-dependent, the transgenes display an open and labile chromatin structure. Our data suggest that a post-transcriptional gene silencing (PTGS) mechanism is responsible for the repression in the ovarian somatic cells, whereas a mechanism that couples PTGS to transcriptional gene silencing operates to silence retrotransposons in the other somatic tissues.

Export of piRNA precursors by EJC triggers assembly of cytoplasmic Yb-body in Drosophila

PIWI-interacting RNAs (piRNAs) are effectors of transposable element (TE) silencing in the reproductive apparatus. In Drosophila ovarian somatic cells, piRNAs arise from longer single-stranded RNA precursors that are processed in the cytoplasm presumably within the Yb-bodies. piRNA precursors encoded by the flamenco (flam) piRNA cluster accumulate in a single focus away from their sites of transcription. In this study, we identify the exportin complex containing Nxf1 and Nxt1 as required for flam precursor nuclear export. Together with components of the exon junction complex (EJC), it is necessary for the efficient transfer of flam precursors away from their site of transcription. Indeed, depletion of these components greatly affects flam intra-nuclear transit. Moreover, we show that Yb-body assembly is dependent on the nucleo-cytoplasmic export of flam transcripts. These results suggest that somatic piRNA precursors are thus required for the assembly of the cytoplasmic transposon silencing machinery.