Department of Pathology and Laboratory Medicine, University of Kentucky Medical Center, Lexington, Kentucky,40506-9983, USA.
RNA. 2010 Feb;16(2):394-404. doi: 10.1261/rna.1905910. Epub 2009 Dec 30.
MicroRNAs (miRNAs) play key roles in gene expression regulation by guiding Argonaute (AGO)-containing microribonucleoprotein (miRNP) effector complexes to target polynucleotides. There are still uncertainties about how miRNAs interact with mRNAs. Here we employed a biochemical approach to isolate AGO-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with a previously described anti-AGO antibody. Co-immunoprecipitated (co-IPed) RNAs were subjected to downstream Affymetrix Human Gene 1.0 ST microarray analysis. During rigorous validation, the "RIP-Chip" assay identified target mRNAs specifically associated with AGO complexes. RIP-Chip was performed after transfecting brain-enriched miRNAs (miR-107, miR-124, miR-128, and miR-320) and nonphysiologic control miRNA to identify miRNA targets. As expected, the miRNA transfections altered the mRNA content of the miRNPs. Specific mRNA species recruited to miRNPs after miRNA transfections were moderately in agreement with computational target predictions. In addition to recruiting mRNA targets into miRNPs, miR-107 and to a lesser extent miR-128, but not miR-124 or miR-320, caused apparent exclusion of some mRNAs that are normally associated with miRNPs. MiR-107 and miR-128 transfections also result in decreased AGO mRNA and protein levels. However, AGO mRNAs were not recruited to miRNPs after either miR-107 or miR-128 transfection, confirming that miRNAs may alter gene expression without stable association between particular mRNAs and miRNPs. In summary, RIP-Chip assays constitute an optimized, validated, direct, and high-throughput biochemical assay that provides data about specific miRNA:mRNA interactions, as well as global patterns of regulation by miRNAs.
微小 RNA（miRNAs）通过引导 Argonaute（AGO）含有 microribonucleoprotein（miRNP）效应复合物靶向靶多核苷酸，在基因表达调控中发挥关键作用。miRNAs 如何与 mRNAs 相互作用仍存在不确定性。在这里，我们采用生化方法，通过先前描述的抗 AGO 抗体的共免疫沉淀（co-IP）从人 H4 肿瘤细胞中分离含有 AGO 的 miRNP。共免疫沉淀（co-IPed）的 RNA 进行下游 Affymetrix Human Gene 1.0 ST 微阵列分析。在严格的验证过程中，“RIP-Chip”测定鉴定了与 AGO 复合物特异性相关的靶 mRNA。转染脑富集 miRNA（miR-107、miR-124、miR-128 和 miR-320）和非生理对照 miRNA 后进行 RIP-Chip，以鉴定 miRNA 靶标。正如预期的那样，miRNA 转染改变了 miRNPs 的 mRNA 含量。miRNA 转染后招募到 miRNPs 的特定 mRNA 种类与计算靶标预测中等程度一致。除了将 mRNA 靶标招募到 miRNPs 中外，miR-107 和在较小程度上 miR-128，但不是 miR-124 或 miR-320，导致与 miRNPs 正常相关的一些 mRNA 明显排除。miR-107 和 miR-128 转染也导致 AGO mRNA 和蛋白水平降低。然而，miR-107 或 miR-128 转染后，AGO mRNAs 未被招募到 miRNPs，这证实了 miRNA 可以改变基因表达，而特定 mRNAs 和 miRNPs 之间没有稳定的关联。总之，RIP-Chip 测定构成了一种优化、验证、直接和高通量的生化测定，提供了有关特定 miRNA:mRNA 相互作用以及 miRNA 调节的全局模式的数据。
