R2D2 organizes small regulatory RNA pathways in Drosophila.
Drosophila microRNAs (miRNAs) and small interfering RNAs (siRNAs) are generally produced by different Dicer enzymes (Dcr-1 and Dcr-2) and sorted to functionally distinct Argonaute effectors (AGO1 and AGO2). However, there is cross talk between these pathways, as highlighted by the recognition that Drosophila miRNA* strands (the partner strands of mature miRNAs) are generated by Dcr-1 but are preferentially sorted to AGO2. Here, we show that a component of the siRNA loading complex, R2D2, is essential both to load endogenously encoded siRNAs (endo-siRNAs) into AGO2 and to prevent endo-siRNAs from binding to AGO1. Northern blot analysis and deep sequencing showed that in the r2d2 mutant, all classes of endo-siRNAs were unable to load AGO2 and instead accumulated in the AGO1 complex. Such redirection was specific to endo-siRNAs and was not observed with miRNA* strands. We observed functional consequences of altered sorting in RNA interference (RNAi) mutants, since endo-siRNAs generated from cis-natural antisense transcripts (cis-NAT-siRNA) exhibited evidence for biased maturation as single strands in AGO1 according to thermodynamic asymmetry and a hairpin-derived endo-siRNA formed cleavage-competent complexes with AGO1 upon mutation of r2d2. Finally, we demonstrated a direct role for the R2D2/Dcr-2 heterodimer in sensing central mismatch positions that direct miRNA* strands to AGO2. Together, these data reveal new roles of R2D2 in organizing small RNA networks in Drosophila.
Sloan-Kettering Institute, Department of Developmental Biology, New York, NY 10065, USA.
Katsutomo
K
Okamura
Nicolas
N
Robine
Ying
Y
Liu
Qinghua
Q
Liu
Eric C
EC
Lai
D000818:Animals; D030801:Animals, Genetically Modified; D060565:Argonaute Proteins; D020137:Base Pair Mismatch; D001483:Base Sequence; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism; D017344:Genes, Insect; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D012313:RNA / Q000235:genetics* / Q000378:metabolism*; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism
C489654:AGO1 protein, Drosophila; C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D039642:Eukaryotic Initiation Factors; D035683:MicroRNAs; D009692:Nucleic Acid Heteroduplexes; C478068:R2D2 protein, Drosophila; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; D012313:RNA; C478069:DCR-2 protein, Drosophila; C515033:Dcr-1 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases