A role for microRNAs in the Drosophila circadian clock
A role for microRNAs in the Drosophila circadian clock
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DOI：
10.1101/gad.1819509
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发表时间：
2009-09-15
影响因子：
10.5
通讯作者：
Rosbash, Michael
中科院分区：
文献类型：
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作者：
Kadener, Sebastian;Menet, Jerome S.;Rosbash, Michael
关于翻译控制对昼夜节律的影响，我们知之甚少。为了解决这个问题，特别是microRNAs(MiRNAs)的翻译控制，我们取消了果蝇昼夜节律组织中的miRNA生物发生途径。再加上昼夜节律介导的转录增加，这严重影响了果蝇的行为节律，表明miRNAs在昼夜节律保持中发挥作用。为了确定对这种调控重要的miRNA-mRNA对，Ago1的免疫沉淀和微阵列分析确定了miRNA介导的控制下的mRNAs。它们包括三个核心时钟信使核糖核酸-时钟(CLK)、VRILE(VRI)和时钟橙(CWO)。为了识别参与昼夜节律计时的miRNAs，我们利用了昼夜细胞特异性的miRNA生物发生途径的抑制，随后进行了平铺阵列分析。该方法鉴定了蝇头昼夜节律组织中表达的miRNAs。行为和分子实验表明，其中一个miRNAs，发育调节因子bantam，在核心昼夜节律起搏器中发挥作用。S2细胞生化实验表明，BANTAM通过与位于CLK 3‘非翻译区(UTR)内的三个靶点结合来调节CLK的翻译。此外，CLK转基因在其3‘UTRs中含有突变的BANTAM位点，对CLK突变果蝇的拯救节律比野生型CLK转基因要差得多。
Little is known about the contribution of translational control to circadian rhythms. To address this issue and in particular translational control by microRNAs (miRNAs), we knocked down the miRNA biogenesis pathway in Drosophila circadian tissues. In combination with an increase in circadian-mediated transcription, this severely affected Drosophila behavioral rhythms, indicating that miRNAs function in circadian timekeeping. To identify miRNA-mRNA pairs important for this regulation, immunoprecipitation of AGO1 followed by microarray analysis identified mRNAs under miRNA-mediated control. They included three core clock mRNAs-clock (clk), vrille (vri), and clockworkorange (cwo). To identify miRNAs involved in circadian timekeeping, we exploited circadian cell-specific inhibition of the miRNA biogenesis pathway followed by tiling array analysis. This approach identified miRNAs expressed in fly head circadian tissue. Behavioral and molecular experiments show that one of these miRNAs, the developmental regulator bantam, has a role in the core circadian pacemaker. S2 cell biochemical experiments indicate that bantam regulates the translation of clk through an association with three target sites located within the clk 3' untranslated region (UTR). Moreover, clk transgenes harboring mutated bantam sites in their 3' UTRs rescue rhythms of clk mutant flies much less well than wild-type CLK transgenes.