Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation.
MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.
Fabian
Marc R
MR
Department of Biochemistry, McGill University, Montreal, QC H3G 1Y6, Canada.
Mathonnet
Géraldine
G
Sundermeier
Thomas
T
Mathys
Hansruedi
H
Zipprich
Jakob T
JT
Svitkin
Yuri V
YV
Rivas
Fabiola
F
Jinek
Martin
M
Wohlschlegel
James
J
Doudna
Jennifer A
JA
Chen
Chyi-Ying A
CY
Shyu
Ann-Bin
AB
Yates
John R
JR
Hannon
Gregory J
GJ
Filipowicz
Witold
W
Duchaine
Thomas F
TF
Sonenberg
Nahum
N
D000818:Animals; D060565:Argonaute Proteins; D001201:Ascites / Q000235:genetics / Q000378:metabolism; D001324:Autoantigens / Q000378:metabolism; D001665:Binding Sites; D002287:Carcinoma, Krebs 2 / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism; D005095:Exoribonucleases; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism*; D054398:Receptors, CCR4 / Q000378:metabolism; D012097:Repressor Proteins; D012260:Ribonucleases; D014162:Transfection
C526735:Ago2 protein, mouse; D060565:Argonaute Proteins; D001324:Autoantigens; C515355:Ccr4 protein, mouse; D015852:Eukaryotic Initiation Factor-2; D039603:Eukaryotic Initiation Factor-4G; D035683:MicroRNAs; D039102:Poly(A)-Binding Proteins; D011506:Proteins; D012333:RNA, Messenger; D034743:RNA-Induced Silencing Complex; D054398:Receptors, CCR4; D012097:Repressor Proteins; C542687:mirnlet7 microRNA, mouse; C489778:Cnot7 protein, mouse; D005095:Exoribonucleases; D012260:Ribonucleases