Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach
Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach
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DOI：
10.1073/pnas.1218887109
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发表时间：
2012-12-11
影响因子：
11.1
通讯作者：
Goodman, Richard H.
中科院分区：
文献类型：
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作者：
Cambronne, Xiaolu A.;Shen, Rongkun;Goodman, Richard H.
识别靶标对于了解 microRNA (miRNA) 表达的生物学效应至关重要。挑战在于表征特定 miRNA 的靶标群，尤其是当靶标在 miRNA-RNA 诱导的沉默复合物 (RISC)-信使 RNA (mRNA) 复合物中被主动下调时。我们开发了一种强大且多功能的策略，称为 RISCtrap，以稳定和纯化这种瞬态相互作用中的目标。通过确定包含已知和先前未知转录本的 miR-124、miR-132 和 miR-181 的特定高置信度目标数据集，证明了其实用性。用 RISCtrap 鉴定出两个先前未知的 miR-132 靶标，即激酶 1 (CRK1) 的接头蛋白 CT10 调节剂和紧密连接相关蛋白 1 (TJAP1)，它们在成年小鼠前脑中受到 miR-132 的内源性调节。此外，这些数据集在靶标数量以及 microRNA 识别元件 (MRE) 基序的类型和频率方面存在差异，从而揭示了个体 miRNA 调节的靶标集的先前未被充分认识的特异性水平。
Identifying targets is critical for understanding the biological effects of microRNA (miRNA) expression. The challenge lies in characterizing the cohort of targets for a specific miRNA, especially when targets are being actively down-regulated in miRNA- RNA-induced silencing complex (RISC)-messengerRNA (mRNA) complexes. We have developed a robust and versatile strategy called RISCtrap to stabilize and purify targets from this transient interaction. Its utility was demonstrated by determining specific high-confidence target datasets for miR-124, miR-132, and miR-181 that contained known and previously unknown transcripts. Two previously unknown miR-132 targets identified with RISCtrap, adaptor protein CT10 regulator of kinase 1 (CRK1) and tight junction-associated protein 1 (TJAP1), were shown to be endogenously regulated by miR-132 in adult mouse forebrain. The datasets, moreover, differed in the number of targets and in the types and frequency of microRNA recognition element (MRE) motifs, thus revealing a previously underappreciated level of specificity in the target sets regulated by individual miRNAs.