Department of Entomology, College of Plant Protection, Nanjing Agricultural University/Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing 210095, China.
Genomics. 2011 May;97(5):294-303. doi: 10.1016/j.ygeno.2011.02.004. Epub 2011 Feb 17.
MicroRNA (miRNA) transcription is still not well understood until now. To increase the miRNA abundance, we stimulated miRNA transcription with CuSO(4) and knocked down Drosha enzyme using dsRNA in Drosophila S2 cells. The full length transcripts of bantam, miR-276a and miR-277, the 5'-end of miR-8, the 3'-end of miR-2b and miR-10 were obtained. We also conducted a series of miRNA promoter analysis to prove the reliability of RACE results. Luciferase-reporter assays proved that both bantam and miR-276a promoters successfully drove the expressions of downstream luciferase genes. The promoter activities were impaired by introducing one or multiple mutations at predicted transcription factor binding sites. Chromatin immunoprecipitation analysis confirmed that hypophosphorylated RNA polymerase II and transcription factor c-Myc physically bind at miRNA promoter. RNA interference of transcription factors Mad and Prd led to down-expression of bantam, miR-277 and miR-2b but not miR-276a, whereas RNAi of Dorsal had the opposite effect.
miRNA（微 RNA）转录至今仍未被充分了解。为了增加 miRNA 的丰度，我们使用 CuSO4 刺激 miRNA 转录，并在果蝇 S2 细胞中使用 dsRNA 敲低 Drosha 酶。我们获得了 bantam、miR-276a 和 miR-277 的全长转录本、miR-8 的 5'端、miR-2b 和 miR-10 的 3'端。我们还进行了一系列 miRNA 启动子分析，以证明 RACE 结果的可靠性。荧光素酶报告基因分析证实，bantam 和 miR-276a 启动子都成功地驱动了下游荧光素酶基因的表达。在预测的转录因子结合位点处引入一个或多个突变会损害启动子活性。染色质免疫沉淀分析证实，低磷酸化 RNA 聚合酶 II 和转录因子 c-Myc 可物理结合在 miRNA 启动子上。转录因子 Mad 和 Prd 的 RNA 干扰导致 bantam、miR-277 和 miR-2b 的表达下调，但 miR-276a 不受影响，而 Dorsal 的 RNAi 则产生相反的效果。
