Ago-TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps.
MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.
Chen
Chyi-Ying A
CY
Department of Biochemistry and Molecular Biology, The University of Texas Medical School, Houston, Texas, USA.
Zheng
Dinghai
D
Xia
Zhenfang
Z
Shyu
Ann-Bin
AB
D000818:Animals; D060565:Argonaute Proteins; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D015152:Blotting, Northern; D015153:Blotting, Western; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D041681:NIH 3T3 Cells; D011485:Protein Binding; D020871:RNA Stability / Q000235:genetics* / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism*
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D001324:Autoantigens; D015852:Eukaryotic Initiation Factor-2; D035683:MicroRNAs; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; C465663:TNRC6A protein, human