SYSTEMATIC IDENTIFICATION OF C ELEGANS MIRISC PROTEINS, MIRNAS, AND MRNA TARGET

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 MicroRNAs(MiRNAs)调节基因表达具有不同的功能，但实验上只确定了有限数量的mRNA靶标。我们发现GW182家族蛋白AIN-1和AIN-2冗余地调控miRNA靶标的表达，但不调控miRNA的生物发生。免疫沉淀(IP)和质谱分析表明，AIN-1和AIN-2只与miRNA特异的ArgAerte蛋白ALG-1和ALG-2以及核心翻译起始复合体的成分相互作用。已知的miRNA靶标富含在AIN-2复合体中，与相应的miRNAs的表达相关。结合IP、焦磷酸测序和与AIN-1/AIN-2相关的RNA的微阵列分析，我们鉴定了106个先前注释的miRNAs和9个新的候选miRNAs，但几乎没有siRNAs，以及超过3500个潜在的miRNA靶标，包括几乎所有已知的miRNAs。我们的结果证明了一种有效的生化方法来系统地识别miRNA靶标，并为了解miRNA效应器复合体的性质提供了有价值的见解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.