Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system.
MicroRNAs (miRNAs) are incorporated into miRNP complexes and regulate protein expression post-transcriptionally through binding to 3'-untranslated regions of target mRNAs. Here we describe a recapitulation of let-7 miRNA-mediated translational repression in a cell-free system, which was established with extracts prepared from HEK293F cells overexpressing miRNA pathway components. In this system, both the cap and poly(A) tail are required for the translational repression, and let-7 directs the deadenylation of target mRNAs. Our work suggests that let-7 miRNPs containing Argonaute and GW182 impair the synergistic enhancement of translation by the 5'-cap and 3'-poly(A) tail, resulting in translational repression.
Wakiyama
Motoaki
M
RIKEN Genomic Sciences Center, Protein Research Group, Tsurumi, Yokohama 230-0045, Japan.
Takimoto
Koji
K
Ohara
Osamu
O
Yokoyama
Shigeyuki
S
D060565:Argonaute Proteins; D001324:Autoantigens / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics; D020868:Gene Silencing; D017930:Genes, Reporter; D006801:Humans; D066298:In Vitro Techniques; D049409:Luciferases, Firefly / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D026723:Polyadenylation; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D001324:Autoantigens; D015852:Eukaryotic Initiation Factor-2; D035683:MicroRNAs; D012315:RNA Caps; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C465663:TNRC6A protein, human; C487931:mirnlet7 microRNA, human; D049409:Luciferases, Firefly