Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila.
Short interfering RNAs (siRNAs) guide mRNA cleavage during RNA interference (RNAi). Only one siRNA strand assembles into the RNA-induced silencing complex (RISC), with preference given to the strand whose 5' terminus has lower base-pairing stability. In Drosophila, Dcr-2/R2D2 processes siRNAs from longer double-stranded RNAs (dsRNAs) and also nucleates RISC assembly, suggesting that nascent siRNAs could remain bound to Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing asymmetry of synthetic siRNAs and dictates strand selection by asymmetric binding to the duplex ends. During dsRNA processing, Dicer (Dcr) liberates siRNAs from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions. Because Dcr-2/R2D2 is unlikely to sense base-pairing asymmetry of an siRNA that is embedded within a precursor, it is not clear whether processed siRNAs strictly follow the thermodynamic asymmetry rules or whether processing polarity can affect strand selection. We use a Drosophila in vitro system in which defined siRNAs with known asymmetry can be generated from longer dsRNA precursors. These dsRNAs permit processing specifically from either the 5' or the 3' end of the thermodynamically favored strand of the incipient siRNA. Combined dsRNA-processing/mRNA-cleavage assays indicate that siRNA strand selection is independent of dsRNA processing polarity during Drosophila RISC assembly in vitro.
Preall
Jonathan B
JB
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, Illinois 60208, USA.
He
Zhengying
Z
Gorra
Jeffrey M
JM
Sontheimer
Erik J
EJ
D000818:Animals; D020029:Base Pairing / Q000235:genetics; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins / Q000378:metabolism; D008957:Models, Genetic; D010063:Ovum / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000378:metabolism; D043244:Ribonuclease III; D013816:Thermodynamics
D029721:Drosophila Proteins; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; C478069:DCR-2 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases