ATP-dependent human RISC assembly pathways.
The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1-4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3'-mid (guide position 12-15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.
Yoda
Mayuko
M
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Kawamata
Tomoko
T
Paroo
Zain
Z
Ye
Xuecheng
X
Iwasaki
Shintaro
S
Liu
Qinghua
Q
Tomari
Yukihide
Y
D000255:Adenosine Triphosphate / Q000378:metabolism*; D060565:Argonaute Proteins; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000302:isolation & purification / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases; D035683:MicroRNAs / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000096:biosynthesis* / Q000378:metabolism
C115848:AGO1 protein, human; C466422:AGO2 protein, human; C517143:AGO3 protein, human; C517144:AGO4 protein, human; D060565:Argonaute Proteins; D017931:DNA Primers; D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors; D035683:MicroRNAs; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D000255:Adenosine Triphosphate; D008156:Luciferases