Deciphering long dsRNA fate in mammalian cells
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Date
2020
Publication Type
Doctoral Thesis
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yes
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Abstract
In Arabidopsis thaliana, Caenorhabditis elegans and Drosophila, long dsRNA (ldsRNA) accumulation during virus infection, transposon activation or transgene expression, elicits a potent RNA interference (RNAi) response: ldsRNA is processed into small interfering RNAs by Dicer proteins, before loading into Argonaute (Ago) proteins which assemble into RNA-induced silencing complex (RISC) to slice complementary RNA or methylate DNA. The response can also be amplified and spread systemically throughout the organism. In mammals, specific RNAi can be observed in specific cell lines, but this effect is often mitigated by activation type I interferon (IFN) dsRNA binding protein (dsRBPs) which are responsible for cytotoxicity. In this PhD thesis, we aim to unravel these processes using state-of-the-art biochemical-, genetic- and molecular approaches. Here, we first show, that Dicer interactors protein activator of protein kinase R (PKR) (PACT) and TAR-RNA binding protein (TRBP) rapidly congregate in ldsRNA-rich granules (DRGs) upon ldsRNA stress. Mass spectrometry analysis reveals the recruitment of a compendium of additional RNA silencing- and type I IFN factors to these DRGs, thus providing evidence for an integrated cellular platform for recognition and processing of ldsRNA. Second, we confirm that modulating a key innate immunity receptor (PKR) in 293T cells dampens the ldsRNA-induced cytotoxic effects and uncovers a specific RNAi response on transgene- and endogenous targets in an Ago2- and a Dicer-dependent manner. Interestingly, our in-depth analysis reveals two modes of Dicer processing depending on the exogenous ldsRNA template used, suggesting ldsRNA sequence or structure specifically affects these processes. Furthermore, we also reveal that TRBP is a master regulator of Dicer-processing and Ago-loading during RNAi, most notably of ldsRNA templates which are fully processed by Dicer. Finally, we perform several genome-wide forward genetic screens to dissect the mechanisms leading to the uptake and sensing of exogenous ldsRNA. These experiments allowed us to identify a network of proteins involved in these processes, from cell surface binding to nuclear ldsRNA responses. By extensive validation of knock-outs from the type I IFN screen we managed to (i) molecularly dissect ldsRNA uptake by lipofection and (ii) describe a novel role of signal transducer and activator of transcription 1 (STAT1) in human RNAi. All together the work of this PhD thesis sheds new light on the cellular- and molecular impact of ldsRNA in mammalian cells, an important challenge in the fields of RNA therapeutics and virology.
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Examiner : Voinnet, Olivier
Examiner : Mateescu, Bogdan
Examiner : Werner, Sabine
Examiner : Brennecke, Julius
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ETH Zurich
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Subject
Innate immunity; RNAi; dsRNA binding proteins; PACT; PKR; TRBP; MAVS; dsRNA; siRNA; Argonaute
Organisational unit
03876 - Voinnet, Olivier / Voinnet, Olivier
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Related publications and datasets
Is supplemented by: https://doi.org/10.3929/ethz-b-000419986