Systematic identification of C. elegans miRISC proteins, miRNAs, and mRNA targets by their interactions with GW182 proteins AIN-1 and AIN-2.
MicroRNAs (miRNAs) regulate gene expression for diverse functions, but only a limited number of mRNA targets have been experimentally identified. We show that GW182 family proteins AIN-1 and AIN-2 act redundantly to regulate the expression of miRNA targets, but not miRNA biogenesis. Immunoprecipitation (IP) and mass spectrometry indicate that AIN-1 and AIN-2 interact only with miRNA-specific Argonaute proteins ALG-1 and ALG-2 and with components of the core translational initiation complex. Known miRNA targets are enriched in AIN-2 complexes, correlating with the expression of corresponding miRNAs. Combining IP with pyrosequencing and microarray analysis of RNAs associated with AIN-1/AIN-2, we identified 106 previously annotated miRNAs plus nine new candidate miRNAs, but nearly no siRNAs, and more than 3500 potential miRNA targets, including nearly all known ones. Our results demonstrate an effective biochemical approach to systematically identify miRNA targets and provide valuable insights regarding the properties of miRNA effector complexes.
Zhang
Liang
L
Howard Hughes Medical Institute, University of Colorado at Boulder, Boulder, CO 80309, USA.
Ding
Lei
L
Cheung
Tom H
TH
Dong
Meng-Qiu
MQ
Chen
Jun
J
Sewell
Aileen K
AK
Liu
Xuedong
X
Yates
John R
JR
Han
Min
M
D020413:3' Untranslated Regions / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000166:cytology / Q000196:embryology / Q000201:enzymology / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D035683:MicroRNAs / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000378:metabolism; D018510:RNA, Helminth / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D013997:Time Factors
D020413:3' Untranslated Regions; C504319:AIN-1 protein, C elegans; C525939:AIN-2 protein, C elegans; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D039561:Eukaryotic Initiation Factor-4E; D035683:MicroRNAs; D012315:RNA Caps; D018510:RNA, Helminth; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D004722:Endoribonucleases