C3PO, an endoribonuclease that promotes RNAi by facilitating RISC activation.
The catalytic engine of RNA interference (RNAi) is the RNA-induced silencing complex (RISC), wherein the endoribonuclease Argonaute and single-stranded small interfering RNA (siRNA) direct target mRNA cleavage. We reconstituted long double-stranded RNA- and duplex siRNA-initiated RISC activities with the use of recombinant Drosophila Dicer-2, R2D2, and Ago2 proteins. We used this core reconstitution system to purify an RNAi regulator that we term C3PO (component 3 promoter of RISC), a complex of Translin and Trax. C3PO is a Mg2+-dependent endoribonuclease that promotes RISC activation by removing siRNA passenger strand cleavage products. These studies establish an in vitro RNAi reconstitution system and identify C3PO as a key activator of the core RNAi machinery.
Liu
Ying
Y
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Ye
Xuecheng
X
Jiang
Feng
F
Liang
Chunyang
C
Chen
Dongmei
D
Peng
Junmin
J
Kinch
Lisa N
LN
Grishin
Nick V
NV
Liu
Qinghua
Q
D000595:Amino Acid Sequence; D000818:Animals; D060565:Argonaute Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism
C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C478068:R2D2 protein, Drosophila; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; D011994:Recombinant Proteins; C542957:Trax protein, Drosophila; C509504:trsn protein, Drosophila; C478069:DCR-2 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases