| MicroRNAs (miRNAs) regulate gene expression for diverse functions, but only a limited number of mRNA targets have been identified by experimental methods and the detailed mechanisms of miRNA regulation remains to be elucidated. Here we described the identification and characterization of a C.elegans ALG-1 interaction protein, AIN-2, which is a homolog of the previously identified AIN-1 protein and the mammalian GW182 family protein. Our genetic and biochemical analysis revealed that AIN-1 and AIN-2 act redundantly in miRNA effector complex to regulate development timing by regulating the expression of miRNA targets, lin-28 and hbl-1, but both AIN-1 and AIN-2 are not required for miRNA biogenesis.;Immunoprecipitation (IP) and mass spectrometric analysis indicate that both AIN-1 and AIN-2 interact only with miRNA-specific Argonaut proteins ALG-1 and ALG-2 and with components of the core translational initiation complex. RT-PCR analysis revealed that known miRNA targets are enriched in AIN-2 complexes, correlating with the expression of corresponding miRNAs. By IP purification and high throughput pyrosequencing of miRNAs associated with AIN-1 and AIN-2, we identified 106 previously annotated miRNAs and 9 likely new miRNAs, but no siRNAs or 21URNAs. Further microarray analysis of mRNAs associated with AIN-1 and AIN-2 complexes revealed more than 3500 potential targets of miRNA, including lin-28, hbl-1 and nearly all other known miRNA-regulated mRNAs.;Using the AIN IP method, we systematically analyzed the dynamics of miRNA-mediated regulations during C.elegans development. Thousands of miRNA targets, including 630 new miRNA targets, were indentified in AIN-2 associated miRISCs at different development stages. More than 30% of the newly identified miRNA targets are subject to dynamic miRNA regulations during development. In general, miRNA targets were significantly enriched in genes involved in signaling processes, whereas genes with housekeeping functions were significantly under-represented. In addition, differential assembly kinetics of different miRNAs into AIN-2 containing miRISC was observed, suggesting that the incorporation of mature miRNAs into functional miRISCs is a complexly regulated process. |
