Genome-wide identification of targets and function of individual MicroRNAs in mouse embryonic stem cells.
Genome-wide identification of targets and function of individual MicroRNAs in mouse embryonic stem cells.
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DOI：
10.1371/journal.pgen.1001163
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发表时间：
2010-10-21
期刊：
影响因子：
4.5
通讯作者：
Surani MA
中科院分区：
文献类型：
--
作者：
Hanina SA;Mifsud W;Down TA;Hayashi K;O'Carroll D;Lao K;Miska EA;Surani MA
小鼠胚胎干细胞（ES细胞）表达一组独特的微小RNA（miRNA），即miR - 290 - 295簇。要阐明这些miRNA的作用以及它们如何整合到ES细胞调控网络中，需要鉴定它们的直接调控靶点。然而，困难在于后生动物miRNA与其靶点的互补性有限，这种相互作用仅需要miRNA种子序列的6个核苷酸。为了鉴定miR - 294的靶点，我们使用了Dicer1缺失的ES细胞，这种细胞缺乏所有内源性成熟miRNA，然后仅将miR - 294导入这些ES细胞。接着我们采用两种方法在小鼠ES细胞中发现miR - 294的靶点：使用微阵列进行转录组分析，以及一种生化方法来分离与RNA诱导沉默复合体（RISC）效应物Argonaute2（Ago2）蛋白相关的mRNA靶点，随后进行RNA测序。在缺乏Dicer1的情况下，RISC复合体基本上没有成熟的miRNA，因此应该只包含转染的miR - 294及其碱基配对的靶点。我们的数据表明，miR - 294可能通过调控c - Myc靶基因的一个子集以及上调多能性相关基因如Lin28来促进多能性。
植物和动物的干细胞含有许多小RNA，它们有助于调控分化为不同细胞类型。植物中小RNA成熟所必需的一个基因发生突变会导致干细胞（称为分生组织细胞）处于一种不确定的、过度增殖的状态。同样在蠕虫中，一种名为lin - 4 miRNA的小RNA可防止“类干细胞”在不适当的时候出现。因此，确定关键的单个小RNA在胚胎干细胞中的确切功能是很重要的。为了解决这个问题，我们构建了缺乏所有miRNA的胚胎干细胞，并向其中导入了一种单一的miRNA。我们发现一种单一的miRNA能够影响干细胞中许多基因的表达，而这些基因反过来又调控干细胞的关键特性。这些共同有助于在干细胞中建立一个复杂的基因调控网络，从而确定它们的特性。我们的发现具有广泛的意义，因为不同的miRNA在发育胚胎的不同细胞类型中具有关键功能。了解这些分子的功能也很重要，因为miRNA功能的失调是一些人类疾病（包括癌症）的基础。
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. Stem cells in plants and animals contain many small RNAs, which help to regulate differentiation into diverse cell types. Mutation in a gene necessary for the maturation of small RNAs in plants causes the stem cells (called meristem cells) to remain in an indeterminate, overproliferating state. Similarly in worms, a small RNA called lin-4 miRNA prevents “stem cell–like cells” appearing at inappropriate times. Thus, it is important to determine the precise functions of key individual small RNAs in embryonic stem cells. To address this, we created embryonic stem cells lacking all miRNAs into which we introduced a single miRNA. We discovered that a single miRNA could affect the expression of many genes in stem cells, which in turn regulate key properties of stem cells. These together help establish an intricate network of gene regulation in stem cells that defines their properties. Our findings are of broad interest because different miRNAs have critical functions in diverse cell types in developing embryos. It is important to understand the function of these molecules also because misregulation of miRNA function underlies some human diseases, including cancers.
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