Biochemical Identification of Targets of Individual MicroRNAs in Mouse Embryonic Stem Cells. Mus musculus

Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays, and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-Sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs, and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes, and upregulating pluripotency-associated genes such as Lin28. Overall design: For RNA-IP experiments, cells were transfected with 300 pmol of miR-294 or cel-239b control miRNA (Dharmacon) and harvested 12-16 hr after transfection for immunoprecipitation. Following RNA-immunoprecipitation of Ago2-myc, RNA from the INPUT (total RNA) and IP were subjected to library preparation and sequenced by SOLiD. There are 10 samples from Dicer1-null mouse ES cells, which are essentially 5 sample pairs of INPUT (A) and its corresponding RNA-IP (B): Ago2-myc transfected with miR-294 (sampe 1), 2 replicates of Ago2-myc-MUT (catalytically inactive) transfected with miR-294 (sample 2 & 5), 2 replicates of Ago2-myc transfected with cel-239b (sample 3 & 4).

小鼠胚胎干细胞（Mouse Embryonic Stem, ES）表达一组独特的微小RNA（microRNAs, miRNAs），即miR-290-295基因簇。阐明此类miRNAs的功能及其如何整合入ES细胞调控网络，亟需鉴定其直接调控靶点。然而该研究存在核心难点：多细胞动物的miRNAs与其靶点的互补结合程度有限，仅需miRNA种子序列的6个核苷酸即可介导二者的相互作用。
为鉴定miR-294的调控靶点，我们采用了缺失所有内源性成熟miRNAs的Dicer1基因敲除ES细胞，并仅向其中转染miR-294。随后我们通过两种策略探究小鼠ES细胞中的miR-294靶点：其一为利用微阵列开展转录组分析；其二为采用生化手段分离与RNA诱导沉默复合物（RNA Induced Silencing Complex, RISC）效应蛋白Argonaute2（Ago2）结合的mRNA靶点，随后进行RNA测序。由于Dicer1缺失时，RISC复合物几乎不含内源性成熟miRNAs，因此仅会包含转染的miR-294及其碱基配对的调控靶点。
我们的研究数据显示，miR-294或可通过调控c-Myc靶点基因的一个子集，并上调Lin28等多能性相关基因，进而促进细胞多能性。
总体实验设计：针对RNA免疫沉淀实验，我们向细胞中转染300 pmol的miR-294或cel-239b对照miRNA（购自Dharmacon），并于转染后12~16小时收获细胞用于免疫沉淀。对Ago2-myc完成RNA免疫沉淀后，分别对INPUT（总RNA）样本与免疫沉淀（IP）产物中的RNA进行文库制备，随后通过SOLiD平台完成测序。本研究共包含10个来自Dicer1基因敲除小鼠ES细胞的样本，本质上为5组INPUT（A）与对应RNA-IP（B）的样本对：转染miR-294的Ago2-myc样本（样本1）、转染miR-294的Ago2-myc-MUT（催化失活型）的2个生物学重复样本（样本2与5）、转染cel-239b的Ago2-myc的2个生物学重复样本（样本3与4）。