In vitro studies provide insight into effects of Dicer-2 helicase mutations in .

Fiche publication

Date publication

août 2020

Journal

RNA (New York, N.Y.)

Auteurs

Membres identifiés du Cancéropôle Est :
Dr MEIGNIN Carine

Tous les auteurs :
Donelick HM, Talide L, Bellet M, Aruscavage J, Lauret E, Aguiar E, Marques JT, Meignin C, Bass BL

Lien Pubmed

https://www.ncbi.nlm.nih.gov/pubmed/32843367

Résumé

In vitro, 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 and other animals.