In vitro studies provide insight into effects of Dicer-2 helicase mutations in Drosophila melanogaster
| Autor: | Loïc Talide, Emilie Lauret, Brenda L. Bass, Carine Meignin, Helen M. Donelick, Matthieu Bellet, Eric R.G.R. Aguiar, João Trindade Marques, P. Joseph Aruscavage |
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| Přispěvatelé: | Réponse immunitaire et developpement chez les insectes (RIDI - UPR 9002), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Modèles Insectes de l'Immunité Innée (M3I), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0303 health sciences
Small interfering RNA biology Point mutation viruses 030302 biochemistry & molecular biology fungi Helicase biology.organism_classification Cell biology 03 medical and health sciences RNA silencing RNA interference In vivo biology.protein [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Drosophila melanogaster Molecular Biology 030304 developmental biology Dicer |
| Zdroj: | RNA RNA, Cold Spring Harbor Laboratory Press, 2020, 26 (12), pp.1847-1861. ⟨10.1261/rna.077289.120⟩ |
| ISSN: | 1355-8382 1469-9001 |
| Popis: | In vitro, Drosophila melanogaster Dicer-2 (Dcr-2) uses its helicase domain to initiate processing of dsRNA with blunt (BLT) termini, and its Platform•PAZ domain to initiate processing of dsRNA with 3′ overhangs (ovrs). To understand the relationship of these in vitro observations to roles of Dcr-2 in vivo, we compared in vitro effects of two helicase mutations to their impact on production of endogenous and viral siRNAs in flies. Consistent with the importance of the helicase domain in processing BLT dsRNA, both point mutations eliminated processing of BLT, but not 3′ovr, dsRNA in vitro. However, the mutations had different effects in vivo. A point mutation in the Walker A motif of the Hel1 subdomain, G31R, largely eliminated production of siRNAs in vivo, while F225G, located in the Hel2 subdomain, showed reduced levels of endogenous siRNAs, but did not significantly affect virus-derived siRNAs. In vitro assays monitoring dsRNA cleavage, dsRNA binding, ATP hydrolysis, and binding of the accessory factor Loquacious-PD provided insight into the different effects of the mutations on processing of different sources of dsRNA in flies. Our in vitro studies suggest effects of the mutations in vivo relate to their effects on ATPase activity, dsRNA binding, and interactions with Loquacious-PD. Our studies emphasize the importance of future studies to characterize dsRNA termini as they exist in Drosophila and other animals. |
| Databáze: | OpenAIRE |
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