Abstract
RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi) is mediated by small interfering RNAs (siRNAs), which are liberated from double-stranded (ds)RNA precursors by Dicer and guide the RNA-induced silencing complex (RISC) to targets. Although principles governing small RNA sorting into RISC have been uncovered, the spectrum of RNA species that can be targeted by Dicer proteins, particularly the viral RNAs present during an infection, are poorly understood. Dicer-2 potently restricts viral infection in insects by generating virus-derived siRNAs from viral RNA. To better characterize the substrates of Dicer-2, we examined the virus-derived siRNAs produced during the Drosophila antiviral RNAi response to four different viruses using high-throughput sequencing. We found that each virus was uniquely targeted by the RNAi pathway; dicing substrates included dsRNA replication intermediates and intramolecular RNA stem loops. For instance, a putative intergenic RNA hairpin encoded by Rift Valley Fever virus generates abundant small RNAs in both Drosophila and mosquito cells, while repetitive sequences within the genomic termini of Vaccinia virus, which give rise to abundant small RNAs in Drosophila, were found to be transcribed in both insect and mammalian cells. Moreover, we provide evidence that the RNA species targeted by Dicer-2 can be modulated by the presence of a viral suppressor of RNAi. This study uncovered several novel, heavily targeted features within viral genomes, offering insight into viral replication, viral immune evasion strategies, and the mechanism of antiviral RNAi.
Highlights
RNA silencing pathways are critical regulators of gene expression in organisms ranging from yeast to humans
We found that the majority of Vesicular Stomatitis virus (VSV) virus-derived siRNAs (vsiRNAs) are 21 nt in length (Figure 1B), which is the expected size of Dcr-2 cleavage products that are active in an Argonaute 2 (Ago2)-containing RNA-induced silencing complex (RISC)
We reasoned that in the absence of infection, synthetic dsRNAs bearing viral sequences would be processed by the RNA interference (RNAi) pathway to yield small interfering RNAs (siRNAs) that are distributed between the positive and negative strands, We examined the siRNAs produced in Drosophila cells bathed with an in vitro synthesized GFP dsRNA, and compared them to the vsiRNAs generated from VSV-encoded GFP, which is expressed off of a subgenomic transcript during infection
Summary
RNA silencing pathways are critical regulators of gene expression in organisms ranging from yeast to humans. RNA silencing is initiated by the action of RNase III enzymes, which target RNAs with dsRNA character to generate small RNA duplexes [1]. SiRNA biogenesis is carried out by Dicer-2 (Dcr-2), which generates predominantly 21 nucleotide (nt) products from several types of precursors [3]. Endogenous siRNAs (esiRNAs) are generated from long hairpins, retrotransposons or convergent transcription units and regulate endogenous gene expression, while exogenous siRNAs (exo-siRNAs) are processed from long dsRNAs, a process commonly manipulated in molecular biology to achieve targeted gene silencing [1]. The miRNA pathway generates mature miRNAs from stem-loop structures within pri-miRNA transcripts through a series of RNase III cleavage events; Drosha first liberates the pre-miRNA from the primary transcript, Dicer-1 (Dcr-1) generates the mature product, generally 22–23 nt in length [1]. The piRNA pathway, active predominantly in the germline, targets transposon-derived RNAs to produce 24–29 nt piRNAs, the enzymatic processing and secondary structure of piRNA pathway substrates is poorly understood [1,2]
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