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Role of small RNAs in antiviral immunity and systemic RNA interference of insects

Book - Dissertation

RNA(i) interference is a post-transcriptional gene silencing mechanism that is guided by non-coding small (s)RNA molecules. These sRNAs can be classified, based on their role in distinct biological processes, in three main cell-autonomous pathways: miRNAs regulate endogenous gene expression, piRNAs control retrotransposon activity and siRNAs reduce virus accumulation. In insects, the latter is activated by dsRNA molecules (i.e. products of viral replication), which are then cleaved into small interfering (si)RNAs and used by an Argonaute2 (Ago2) protein to target and degrade complementary RNA. In addition, this pathway can also be induced by artificial long dsRNA and has therefore been widely used as a reverse genetics research tool. Due to its strong gene-silencing capacity and specificity, RNAi holds great potential to contribute to the development of novel ways to control pest insects and combat viral infections in beneficial and disease-vectoring insects. Nonetheless, obtaining a potent RNAi response in the entire organism upon uptake of exogenous dsRNA requires spreading of this response to different cells and tissues, a process designated as systemic (sys)RNAi. However, as with every technique, RNAi also has some limitations, including the refractory response that several insect species present to dsRNA administered at an extracellular level. In this context, understanding the mechanism by which the RNAi signal is transferred intercellularly could be of major importance. In addition, despite the fact that it is widely accepted that the spread of RNAi is crucial for antiviral immunity in insects, the mechanisms involved in this systemic immune response remain to be elucidated. Moreover, it is also not clear if artificial dsRNA molecules and viral replication intermediates are processed and systemically spread in the same manner. Therefore, this thesis focuses on the identification of sRNA species (16-36 nt) upon dsRNA treatment or viral infection and their subsequent sysRNAi signalling mechanism(s), both in vivo and in vitro. This was investigated in species and cell lines that are known to have a robust (systemic) RNAi response: the migratory locust Locusta migratoria, the fruit fly Drosophila melanogaster S2 cells and the red flour beetle Tribolium castaneum TcA cells. First, we report on the production of dsRNA- and virus-derived siRNAs in L. migratoria in vivo, TcA cells and S2 cells. In addition, our data combined with the currently available literature, demonstrate a specific siRNA length preference in species belonging to five economically important insect orders (Lepidoptera, Diptera, Coleoptera, Orthoptera and Hymenoptera). Next, the presence of dsRNA- and virus-derived sRNAs was investigated in the cell-free haemolymph of L. migratoria and in the medium of cultured insect cell lines, as well as in specific extracellular fractions, such as extracellular vesicles (EVs), lipophorins and extracellular (ex)Ago2 containing complexes. In this context, clear interspecies differences were observed for both the dsRNA- and virus-derived sRNAs. In L. migratoria haemolymph, a clear dsRNA-derived siRNA profile was observed at different time points, which could not be detected in chromatographic fractions containing EVs or lipophorins. On the contrary, the culture medium of S2 cells and its derived EVs did not display a typical pattern of dsRNA-derived siRNAs and, subsequently, these EVs did not induce a significant silencing response in recipient cells. However, we have detected antisense siRNA peaks referring to two persistent viral infections in the culture medium and EVs of these cells. Moreover, virus-derived siRNAs were also identified in the cell-free culture medium, EVs and exAgo2 fractions of TcA cells. Lastly, we demonstrate for the latter that, upon dsRNA treatment, these cells process this dsRNA into siRNAs, after which their presence can be identified in EVs. Furthermore, these EVs were able to trigger a significant RNAi silencing response in recipient TcA cells, but not in other insect cell lines (i.e. Drosophila S2 cells, Bombyx mori BmN4 cells, Trichoplusia ni High Five cells and Spodoptera frugiperda Sf9 cells). In addition, other non-coding sRNAs (16-36 nt) were identified in EVs derived from these cells, including miRNAs, sRNAs corresponding to transposable elements, small nucleolar RNAs, small nuclear RNAs and both ribosomal RNA-derived and transfer RNA-derived sRNAs. Taken together, this doctoral thesis provides new fundamental insights in the RNAi-based antiviral immunity and the systemic spreading of the RNAi signal in insects.
Publication year:2020