title: RNA helicase A is not required for RISC activity.
abstract: It has been shown that siRNAs can compete with each other or with endogenous miRNAs for RISC components. This competition may complicate the interpretations of phenotypes observed through siRNA-mediated knockdown of genes, especially those genes implicated in the RISC pathway. In this study, we re-examined the function of RNA helicase A (RHA), which has been previously proposed to function in RISC loading based on siRNA-mediated knockdown studies. Here we show that reduced RISC activity or loading of siRNAs was observed only in cells depleted of RHA using siRNA, but not using RNaseH-dependent antisense oligonucleotides (ASOs), suggesting that the impaired RISC function stems from the competition between pre-existing and newly transfected siRNAs, but not from reduction of the RHA protein. This view is further supported by the findings that cells depleted of a control protein, NCL1, using siRNA, but not ASO, exhibited similar defects on the loading and activity of a subsequently transfected siRNA. Transfection of RHA or NCL1 siRNAs, but not ASOs, reduced the levels of endogenous miRNAs, suggesting a competition mechanism. As a positive control, we showed that reduction of MOV10 by either siRNA or ASO decreased siRNA activity, confirming its role in RISC function. Together, our results indicate that RHA is not required for RISC activity or loading, and suggest that proper controls are required when using siRNAs to functionalize genes to avoid competition effects.
authors.affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Inc., 2855 Gazelle Court, Carlsbad, CA 92010, USA. Electronic address: lliang@isisph.com.
authors.forename: Xue-Hai
authors.initials: XH
authors.lastname: Liang
authors.forename: Stanley T
authors.initials: ST
authors.lastname: Crooke
mesh_terms: D015153:Blotting, Western; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D020365:RNA Helicases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000097796:Nucleolin
chemical_list: D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D020365:RNA Helicases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; D000097796:Nucleolin; C530476:Mov10 protein, human
keywords: ASOs; Ago2; Antisense oligonucleotides; Competition; RHA; RISC; RNA helicase A; RNA induced silencing complex; miRNA; siRNA