Endogenous siRNAs Derived from Transposons and mRNAs in Drosophila Somatic Cells

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Science (Impact Factor: 33.61). 06/2008; 320(5879):1077-81. DOI: 10.1126/science.1157396
Source: PubMed


Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from
exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs),
21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary
regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires
the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated
by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line.

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Available from: Megha Ghildiyal, May 16, 2014
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    • "Reviewing previous studies on Drosophila sRNAs we hypothesize that our conclusions drawn on the basis of the tupaia data could also be valid for sRNA silencing pathways in Drosophila. In Drosophila, siRNAs were assumed to silence TEs mainly in the soma, displaying a more or less separation of the siRNA-and piRNA-pathway into soma and germline (Ghildiyal et al. 2008). However, TE-derived si-as well as piRNAs were sequenced from the Drosophila ovary somatic sheet cell line (Lau et al. 2009). "
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    ABSTRACT: Argonaute proteins comprising Piwi-like and Argonaute-like proteins and their guiding small RNAs combat mobile DNA on the transcriptional and post-transcriptional level. While Piwi-like proteins and associated piRNAs are generally restricted to the germline, Argonaute-like proteins and siRNAs have been linked with transposon control in the germline as well as in the soma. Intriguingly, evolution has realized distinct Argonaute subfunctionalization patterns in different species but our knowledge about mammalian RNA interference pathways relies mainly on findings from the mouse model. However, mice differ from other mammals by absence of functional Piwil3 and expression of an oocyte-specific Dicer isoform. Thus, studies beyond the mouse model are required for a thorough understanding of function and evolution of mammalian RNA interference pathways. We high-throughput sequenced small RNAs from the male Tupaia belangeri germline, which represents a close outgroup to primates, hence phylogenetically links mice with humans. We identified transposon-derived piRNAs as well as siRNAs clearly contrasting the separation of piRNA- and siRNA-pathways into male and female germline as seen in mice. Genome-wide analysis of tree shrew transposons reveal that putative siRNAs map to transposon sites that form foldback secondary structures thus representing suitable Dicer substrates. In contrast piRNAs target transposon sites that remain accessible. With this we provide a basic mechanistic explanation how secondary structure of transposon transcripts influences piRNA- and siRNA-pathway utilization. Finally, our analyses of tree shrew piRNA clusters indicate A-Myb and the testis-expressed transcription factor RFX4 to be involved in the transcriptional regulation of mammalian piRNA clusters. © 2015 Rosenkranz et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
    RNA 03/2015; 21(5). DOI:10.1261/rna.048603.114 · 4.94 Impact Factor
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    • "A number of transposition repressive mechanisms, regulating Tc1-mariner elements, have been discovered to date, starting from self-regulation (overexpression inhibition [47-49], post-translational modifications of the transposase [50,51], self-encoded repressors [52,53]) to the cell-developed control systems (siRNA [54] and piRNA [55] pathways, chromatin-level transcriptional repression [56]), or simply stochastic accumulation of detrimental mutations in the transposase-coding gene [57]. Some of these control mechanisms have been demonstrated for Bari-like elements [58-60]. "
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    ABSTRACT: Background Bari-like transposons belong to the Tc1-mariner superfamily, and they have been identified in several genomes of the Drosophila genus. This transposon’s family has been used as paradigm to investigate the complex dynamics underlying the persistence and structural evolution of transposable elements (TEs) within a genome. Three structural Bari variants have been identified so far and can be distinguished based on the organization of their terminal inverted repeats. Bari3 is the last discovered member of this family identified in Drosophila mojavensis, a recently emerged species of the Repleta group of the genus Drosophila. Results We studied the insertion pattern of Bari3 in different D. mojavensis populations and found evidence of recent transposition activity. Analysis of the transposase domains unveiled the presence of a functional nuclear localization signal, as well as a functional binding domain. Using luciferase-based assays, we investigated the promoter activity of Bari3 as well as the interaction of its transposase with its left terminus. The results suggest that Bari3 is transposition-competent. Finally we demonstrated transposase transcript processing when the transposase gene is overexpressed in vivo and in vitro. Conclusions Bari3 displays very similar structural and functional features with its close relative, Bari1. Our results strongly suggest that Bari3 is an independent element that has generated genomic diversity in D. mojavensis. It can autonomously transcribe its transposase gene, which in turn can localize in the nucleus and bind the terminal inverted repeats of the transposon. Nevertheless, the identification of an unpredicted spliced form of the Bari3 transposase transcript allows us to hypothesize a control mechanism of its mobility based on mRNA processing. These results will aid the studies on the Bari family of transposons, which is intriguing for its widespread diffusion in Drosophilids coupled with a structural diversity generated during the evolution of Bari-like elements in their host genomes.
    Mobile DNA 07/2014; 5(1):21. DOI:10.1186/1759-8753-5-21 · 2.11 Impact Factor
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    • "As the negative class of ''non-cis-NAT siRNA loci'' is not well defined, we built a one-class predictive model (Karatzoglou et al. 2004) that uses the positive class as the training set and classifies the testing set based on how similar they are to the training set. The model was trained on the previously published cis-NAT siRNA loci from our and other's laboratories (Czech et al. 2008; Ghildiyal et al. 2008; Okamura et al. 2008a), using the above features, and applied to predict cis-NAT-siRNAs on all segments genome-wide, separately for each library and for a set combined from all libraries. The overall characteristics of these loci were as follows: minimum expression of 21-nt reads $1 RPM for both sense and antisense strands (5th–95th percentile range was 2.6–56.6 "
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    ABSTRACT: We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.
    Genome Research 07/2014; 24(7):1236-50. DOI:10.1101/gr.161554.113 · 14.63 Impact Factor
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