Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in Drosophila and the identification of putative target genes
Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in Drosophila and the identification of putative target genes
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DOI：
10.1093/nar/gkg707
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发表时间：
2003-09-01
影响因子：
14.9
通讯作者：
Tabler, M
中科院分区：
文献类型：
--
作者：
Boutla, A;Delidakis, C;Tabler, M
微RNA是一类小型非编码调节性RNA，通过不完美的碱基配对与mRNA损害翻译。为了分析其细胞功能，我们在果蝇胚胎中注入了不同的miRNA特异性DNA反义寡核苷酸。在四种情况下，我们观察到对正常发育的严重干扰，一个人具有中等影响，六个寡核苷酸不会引起可检测的表型。我们进一步使用miR-13a DNA反义寡核苷酸作为cDNA库模板上的PCR底漆。我们以这种实验方式确定了九个果蝇基因，这些基因的特征是3'非翻译区域基序，这些基序允许与miR-13或相关miRNA的双层形成不完善。这些基因（包括SOS和MYD88）代表了miRNA调节的推定靶标。两个基因的靶基序的诱变，然后在果蝇Schneider 2（S2）细胞中转染和随后的报告基因分析证实了以下假设：miR-13的结合潜力与基因表达成反比。
Micro-RNAs are a class of small non-coding regulatory RNAs that impair translation by imperfect base pairing to mRNAs. For analysis of their cellular function we injected different miRNA-specific DNA antisense oligonucleotides in Drosophila embryos. In four cases we observed severe interference with normal development, one had a moderate impact and six oligonucleotides did not cause detectable phenotypes. We further used the miR-13a DNA antisense oligonucleotide as a PCR primer on a cDNA library template. In this experimental way we identified nine Drosophila genes, which are characterised by 3' untranslated region motifs that allow imperfect duplex formation with miR-13 or related miRNAs. These genes, which include Sos and Myd88, represent putative targets for miRNA regulation. Mutagenesis of the target motif of two genes followed by transfection in Drosophila Schneider 2 (S2) cells and subsequent reporter gene analysis confirmed the hypothesis that the binding potential of miR-13 is inversely correlated with gene expression.