Dual-strand Clusters Research Articles

Regulation of Uni-Strand and Dual-Strand piRNA Clusters in Germ and Somatic Tissues in <i>Drosophila melanogaster</i> under Control of <i>rhino</i>

Drosophila melanogaster is a common genetic object for research of RNA-interference pathways and mobile elements regulation. Nowadays taking a part in control of retrotransposon expression the system of piRNA-interfecence well studied in ovary tissues. It is strongly believed that D. melanogaster piRNA-interference is used for retrotransposon suppression only in gonads, and two distinct pathways of piRNA biogenesis exist. Both mechanisms use transcripts of piRNA-clusters (accumulations of truncated and defect mobile elements copies): from unstrand clusters in the first case and from dualstrand clusters in the second, transcribed with one or both DNA chains correspondingly. It is well-known that proper dualstrand clusters function depends on the gene rhino, while unistrand clusters are transcribed rhino-independent and transcripts are spliced. In this paper we show that rhino participates in unistrand flamenco transcripts splicing and the piRNA-interference significance for regulation of several retrotransposons not only in gonads, but in other organs.

Maternally inherited siRNAs initiate piRNA cluster formation

PIWI-interacting RNAs (piRNAs) guide transposable element repression in animal germ lines. In Drosophila, piRNAs are produced from heterochromatic loci, called piRNA clusters, which act as information repositories about genome invaders. piRNA generation by dual-strand clusters depends on the chromatin-bound Rhino-Deadlock-Cutoff (RDC) complex, which is deposited on clusters guided by piRNAs, forming a positive feedback loop in which piRNAs promote their own biogenesis. However, how piRNA clusters are formed before cognate piRNAs are present remains unknown. Here, we report spontaneous de novo piRNA cluster formation from repetitive transgenic sequences. Cluster formation occurs over several generations and requires continuous trans-generational maternal transmission of small RNAs. We discovered that maternally supplied small interfering RNAs (siRNAs) trigger de novo cluster activation in progeny. In contrast, siRNAs are dispensable for cluster function after its establishment. These results reveal an unexpected interplay between the siRNA and piRNA pathways and suggest a mechanism for de novo piRNA cluster formation triggered by siRNAs.

Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export.

The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.

Maelstrom Represses Canonical Polymerase II Transcription within Bi-directional piRNA Clusters in Drosophila melanogaster

In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. Here, we report that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. We propose that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins.

A novel model of THO/TREX loading onto target RNAs in metazoan gene expression.

The THO/TREX complex consists of several conserved subunits and is required for mRNA export. In metazoans, THO/TREX binds a subset of mRNAs during RNA splicing, and facilitates their nuclear export. How THO/TREX selects RNA targets is, however, incompletely understood. In our recent study, we reported that THO is loaded onto Piwi-interacting RNA (piRNA) precursor transcripts independent of splicing, and facilitates convergent transcription in Drosophila ovary. The precursors are later processed into mature piRNAs, small noncoding RNAs that silence transposable elements (TEs). We observed that piRNAs originating from dual-strand clusters, where precursors are transcribed from both strands, were specifically affected by THO mutation. Analysis of THO-bound RNAs showed enrichment of dual-strand cluster transcripts. Interestingly, THO loading onto piRNA precursors was dependent on Cutoff (Cuff), which comprises the Rhino-Deadlock-Cutoff (RDC) complex that is recruited to dual-strand clusters by recognizing H3K9me3 and licenses convergent transcription from he cluster. We also found that THO mutation affected transcription from dual-strand clusters. Therefore, we concluded that THO/TREX is recruited to dual-strand piRNA clusters, independent of splicing events, via multi-protein interactions with chromatin structure. Then, it facilitates transcription likely by suppressing premature termination to ensure adequate expression of piRNA precursors. [BMB Reports 2016; 49(7): 355-356]

The Drosophila HP1 Homolog Rhino Is Required for Transposon Silencing and piRNA Production by Dual-Strand Clusters

Piwi-interacting RNAs (piRNAs) silence transposons and maintain genome integrity during germline development. In Drosophila, transposon-rich heterochromatic clusters encode piRNAs either on both genomic strands (dual-strand clusters) or predominantly one genomic strand (uni-strand clusters). Primary piRNAs derived from these clusters are proposed to drive a ping-pong amplification cycle catalyzed by proteins that localize to the perinuclear nuage. We show that the HP1 homolog Rhino is required for nuage organization, transposon silencing, and ping-pong amplification of piRNAs. rhi mutations virtually eliminate piRNAs from the dual-strand clusters and block production of putative precursor RNAs from both strands of the major 42AB dual-strand cluster, but not of transcripts or piRNAs from the uni-strand clusters. Furthermore, Rhino protein associates with the 42AB dual-strand cluster,but does not bind to uni-strand cluster 2 or flamenco. Rhino thus appears to promote transcription of dual-strand clusters, leading to production of piRNAs that drive the ping-pong amplification cycle.