Anti-PABPC1 Co-Immunoprecipitation for Examining the miRNAs Directly Targeting the 3′-UTR of EED mRNA

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate post-transcriptional gene expression by base pairing with partially complementary sequences within target messenger RNAs (mRNAs). Although the target genes and the precise biological functions of individual miRNAs remain largely unknown, miRNAs have been implicated in diverse biological processes, including both normal and pathological states. As a single stranded mRNA can be directly targeted by multiple miRNAs, and as the target sites may exist in the 3′-untranslated region (UTR), 5′-UTR, or the coding regions, it is essential to develop an effective method to identify the full-scale miRNA regulatory pattern of each particular gene. In this study, we employed a biochemical approach to identify the miRNA profiles that regulate the expression of embryonic ectoderm development (EED) protein by using anti-PABPC1 ribonucleoprotein (RNP) co-immunoprecipitation (Co-IP). The full length EED mRNA was subcloned into an expression vector and transiently transfected into a Flag-PABPC1 stable expression cell line. Subsequent to cross-linking and an anti-Flag Co-IP, the miRNAs that directly targeted EED were identified. We found that the best time point to distinguish the positive miRNAs from the background was 18 hours after the plasmid transfection. As expected, the miRNAs that directly target EED were found to interact with EED mRNA through the miRNA-induced silencing complex (miRISC). Meanwhile, the EED mRNA was bound by Flag-PABPC1. This method depends on the integrity of the miRISC complex and achieves greater efficiency when ultraviolet irradiation is used for the process of cross-linking. By using anti-PABPC1 RIP, we identified EED to be a new target gene of miR-16; a finding further confirmed using a dual-luciferase assay. In summary, our data indicate that anti-PABPC1 RIP is a validated and direct biochemical method to provide data about specific miRNA-mRNA interactions, as well as global miRNA patterns regulating the mRNAs.微小RNA（MicroRNAs, miRNAs）是一类小型非编码RNA分子，通过与靶信使RNA（messenger RNAs, mRNAs）内部分互补序列碱基配对，调控转录后基因表达。尽管单个miRNA的靶基因及其确切生物学功能在很大程度上仍未阐明，但miRNA已被证实参与多种生物学过程，包括生理与病理状态。由于单链mRNA可被多个miRNA直接靶向，且靶位点可存在于3′非翻译区（3′-untranslated region, UTR）、5′非翻译区（5′-UTR）或编码区，因此开发有效方法以鉴定特定基因的全面miRNA调控模式至关重要。本研究采用生化手段，通过抗PABPC1核糖核蛋白（anti-PABPC1 ribonucleoprotein, RNP）免疫共沉淀（co-immunoprecipitation, Co-IP），鉴定调控胚胎外胚层发育蛋白（embryonic ectoderm development, EED）表达的miRNA谱。将全长EED mRNA亚克隆至表达载体，并瞬时转染至稳定表达Flag-PABPC1的细胞系中。在交联及抗Flag免疫共沉淀后，鉴定出直接靶向EED的miRNA。研究发现，区分阳性miRNA与背景信号的最佳时间点为质粒转染后18小时。如预期所示，直接靶向EED的miRNA可通过miRNA诱导沉默复合体（miRNA-induced silencing complex, miRISC）与EED mRNA结合。同时，EED mRNA可被Flag-PABPC1结合。该方法依赖miRISC复合体的完整性，且在交联过程中采用紫外照射时可获得更高效率。通过抗PABPC1 RIP实验，我们鉴定出EED是miR-16的新靶基因，这一发现经双荧光素酶报告实验进一步验证。综上，本研究数据表明，抗PABPC1 RIP是一种经过验证的直接生化方法，可获取特定miRNA-mRNA互作以及调控mRNA的全局miRNA模式相关数据。