Endogenous siRNAs Derived from Transposons and mRNAs in Drosophila Somatic Cells (original) (raw)
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Endogenous RNA Interference Provides a Somatic Defense against Drosophila Transposons
Current Biology, 2008
Background-Because of the mutagenic consequences of mobile genetic elements, elaborate defenses have evolved to restrict their activity. A major system that controls the activity of transposable elements (TEs) in flies and vertebrates is mediated by Piwi-interacting RNAs (piRNAs), which are ~24-30 nucleotide RNAs that are bound by Piwi-class effectors. The piRNA system is thought to provide primarily a germline defense against TE activity.
In vitro analysis of RNA interference in Drosophila melanogaster
Methods, 2003
Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21-to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.
Small RNAs and the control of transposons and viruses in Drosophila
Trends in Microbiology, 2009
RNA interference (RNAi) -post-transcriptional gene silencing guided by small interfering RNA (siRNA) -is an important antiviral defense mechanism in insects and plants. Several recent studies in Drosophila identified endogenous siRNAs corresponding to transposons, to structured cellular transcripts and to overlapping convergent transcripts. In addition, one of these studies detected a large pool of Argonaute-2 associated siRNAs that mapped to the genome of flock house virus, a (+) RNA virus. Our bioinformatic analyses indicate that these viral siRNAs mapped in roughly equal proportions to both (+) and (-) viral RNA strands. These reports attribute an important function to RNAi in the defense against parasitic nucleic acids (viruses and transposable elements) and provide a novel mechanism for RNAibased regulation of cellular gene expression. Furthermore, the detection of viral siRNAs of both (+) and (-) polarity implicates double-stranded RNA replication intermediates as the Dicer substrates that mediate antiviral defense.
An endogenous small interfering RNA pathway in Drosophila
Nature, 2008
Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of 22 nucleotides in length, which arise from structured precursors through the action of Drosha-Pasha and Dicer-1-Loquacious complexes 1-7 . These join Argonaute-1 to regulate gene expression . A second endogenous small RNA class, the Piwiinteracting RNAs, bind Piwi proteins and suppress transposons 10,11 . Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of singlestranded RNAs 12,13 . Here we show that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious 1,4,5 rather than the canonical Dicer-2 partner, R2D2 (ref. 14). Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles.
Viral Suppressors of RNA Silencing Hinder Exogenous and Endogenous Small RNA Pathways in Drosophila
PLoS ONE, 2009
Background: In plants and insects, RNA interference (RNAi) is the main responder against viruses and shapes the basis of antiviral immunity. Viruses counter this defense by expressing viral suppressors of RNAi (VSRs). While VSRs in Drosophila melanogaster were shown to inhibit RNAi through different modes of action, whether they act on other silencing pathways remained unexplored. Methodology/Principal Findings: Here we show that expression of various plant and insect VSRs in transgenic flies does not perturb the Drosophila microRNA (miRNA) pathway; but in contrast, inhibits antiviral RNAi and the RNA silencing response triggered by inverted repeat transcripts, and injection of dsRNA or siRNA. Strikingly, these VSRs also suppressed transposon silencing by endogenous siRNAs (endo-siRNAs). Conclusions/Significance: Our findings identify VSRs as tools to unravel small RNA pathways in insects and suggest a cosuppression of antiviral RNAi and endo-siRNA silencing by viruses during fly infections.
RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster
Cellular Microbiology, 2006
Drosophila melanogaster has a robust and efficient innate immune system, which reacts to infections ranging from bacteria to fungi and, as discovered recently, viruses as well. The known Drosophila immune responses rely on humoral and cellular activities, similar to those found in the innate immune system of other animals. Recently, RNAi or 'RNA silencing' has arisen as a possible means by which Drosophila can react to a specific pathogens, transposons and retroviral elements, in a fashion similar to that of a traditional mammalian adaptive immune system instead of in a more generalized and genome encoded innate immune-based response. RNAi is a highly conserved regulation and defence mechanism, which suppresses gene expression via targeted RNA degradation directed by either exogenous dsRNA (cleaved into siRNAs) or endogenous miRNAs. In plants, RNAi has been found to act as an antiviral immune response system. Here we show that RNAi is an antiviral response used by Drosophila to combat infection by Drosophila X Virus, a birnavirus, as well. Additionally, we identify multiple core RNAi pathway genes, including piwi, vasa intronic gene ( vig ), aubergine ( aub ), armitage ( armi ), Rm62 , r2d2 and Argonaute2 ( AGO2 ) as having vital roles in this response in whole organisms. Our findings establish Drosophila as an ideal model for the study of antiviral RNAi responses in animals.
Antiviral immunity in Drosophila requires systemic RNA interference spread
Nature, 2009
Multicellular organisms evolved sophisticated defense systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations 1-4. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity 5-7. However, the antiviral RNAi defense in flies is thought to be a local, cell-autonomous process, since flies are considered unable to generate a systemic RNAi response 8. Here we show that a recently defined doublestranded RNA (dsRNA) uptake pathway 9 is essential for effective antiviral RNAi immunity in adult flies. Mutant flies defective in this dsRNA uptake pathway were hypersensitive to infection with Drosophila C virus (DCV) and Sindbis virus. Mortality in dsRNA-uptake defective flies was accompanied by 100-to 10 5-fold increases in viral titers and higher levels of viral RNA. Furthermore, inoculating naked dsRNA into flies elicited a sequence specific antiviral immune response that required an intact dsRNA uptake pathway. These findings suggest that spread of dsRNA to uninfected sites is essential for effective antiviral immunity. Strikingly, infection with Sindbis-GFP suppressed expression of host-encoded GFP at a distal site. Thus, similar to proteinbased immunity in vertebrates, the antiviral RNAi-response in flies also relies on the systemic spread of a virus-specific immunity signal. Based on the recent identification of a dsRNA uptake pathway in flies 9, 10 , we hypothesized that dsRNA produced and released from infected cells can be taken up locally, and perhaps at distal sites, to establish systemic pre-existing immunity in uninfected cells (Fig. 1). We thus examined whether naked dsRNA can mediate systemic RNAi spread by inoculating flies with dsRNA corresponding to two different regions of the Sindbis virus genome (dsSin1 and dsSin2, Supplementary Fig. S1a and Fig.2a). Two days after dsRNA Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
RNA, 2003
RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21–23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in Drosophila. DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of o...