AIN-1 and AIN-2::GFP IP of C.elegans miRNA targets

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 9 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. Keywords: IP microarray of miRNA targets微小RNA（microRNAs, miRNAs）可通过调控基因表达参与多种生物学功能，但目前经实验验证的mRNA靶点数量仍极为有限。本研究发现，GW182家族蛋白AIN-1与AIN-2能够以冗余方式调控miRNA靶点的表达，而非参与miRNA的生物发生过程。免疫沉淀（Immunoprecipitation, IP）结合质谱分析结果表明，AIN-1与AIN-2仅可与miRNA特异性Argonaute蛋白ALG-1、ALG-2以及核心翻译起始复合物的组分发生相互作用。已知的miRNA靶点在AIN-2复合物中显著富集，且该富集现象与对应miRNA的表达水平呈正相关。通过将IP技术与焦磷酸测序、AIN-1/AIN-2结合RNA的微阵列分析相结合，我们共鉴定出106个已注释的miRNA以及9个新的候选miRNA，几乎未检测到小干扰RNA（small interfering RNA, siRNA），同时筛选得到超过3500个潜在miRNA靶点，其中几乎涵盖了所有已报道的已知靶点。本研究证实了一种可系统性鉴定miRNA靶点的高效生化方法，并为阐明miRNA效应复合物的特性提供了重要见解。关键词：miRNA靶点免疫沉淀微阵列分析