Unknown,Transcriptomics,Genomics,Proteomics

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Functional Specialization of the Small Interfering RNA Pathway in Response to Virus Infection

ABSTRACT: In Drosophila, the siRNA pathway is initiated when exogenous or endogenous double stranded RNA (dsRNA) is processed into siRNAs by Dicer-2 (Dcr-2) and a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs). The siRNAs are then loaded onto Argonaute-2 (Ago2) protein by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to the siRNA. Dcr-2, R2D2, and Ago2 have also been shown to be required for innate antiviral defense in Drosophila. However, the biogenesis of virus-derived siRNAs (vsiRNAs) and their targets in virus-infected cells remain unclear. Here, we analyzed the antiviral response in Drosophila by monitoring the replication of different RNA viruses and deep sequencing of small RNAs in infected animals. We show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and transcription. These vsiRNAs then directly target viral transcripts but not genomes, to inhibit viral replication. The biogenesis of vsiRNAs was virtually independent of Loqs and R2D2. R2D2, however, was essential for sorting and loading of vsiRNAs onto Ago2 and effective silencing of viral RNA expression. Loqs was completely dispensable for silencing of viruses in contrast to its role in silencing of endogenous targets. Our results suggest the existence of a specific siRNA pathway triggered by viral infection independent of conserved dsRBP cofactors and separate from the endogenous pathway. Inhibition of virus replication resulting from direct injection of viral RNA into Drosophila embryos was also not dependent on Loqs, suggesting the distinction of the two pathways is not related to the mode of entry but recognition of intrinsic features of viral RNA or its mode of replication. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response We analyzed the small RNA reponse to viral infection by deep sequencing of small RNA libraries from wild type and RNAi mutant adult flies infected with Sindbis birus and Vesicular Stomatatis virus.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Richard Carthew

PROVIDER: E-GEOD-36449 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing mechanisms that repress TEs expression: endogenous siRNAs (esiRNAs or endo-siRNAs) and Piwi-interacting small RNAs (piRNAs). The biogenesis of endo-siRNAs involves Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2 that primarily helps to direct siRNAs for loading into Ago2. We provide deep sequencing evidence consistent with the idea that R2D2 and Loqs-PD can function in part redundantly. Certain transposons display a preference for either dsRBD-protein for production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in the germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs) that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway. small RNA sampling experiment; small RNAs were prepared from head & thorax as well as dissected ovaries of Adult female Drosophila melanogaster. We used homozygous mutants of the dsRBD proteins Loqs and r2d2 to determine their contribution to the biogenesis of transposon-derived small RNAs. Heterozygous mutant animals served as control. For each RNA sample, we performed one deep-sequencing run without any treatment, and in parallel one sequencing run after periodate oxidation and beta-elimination. After this treatment, only Ago2, Piwi, Aub and Ago3-loaded small RNAs remain as they carry a 2'-O-methyl modification at their 3'-end. This helps to determine the loading status of the small RNAs detected. In total 8 different RNA samples were prepared and 16 libraries were sequenced.

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Functional Specialization of the Small Interfering RNA Pathway in Response to Virus Infection

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