Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu 210093, China.
Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu 210093, China.
J Biol Chem. 2014 Apr 11;289(15):10270-10275. doi: 10.1074/jbc.C113.541417. Epub 2014 Mar 4.
Mature microRNAs (miRNAs), ∼ 22-nucleotide noncoding RNAs regulating target gene expression at the post-transcriptional level, have been recently shown to be transported into the nucleus where they modulate the biogenesis of other miRNAs or their own expression. However, the mechanism that governs the transport of mature miRNAs from cytoplasm to nucleus remains unknown. Here, we report that importin 8 (IPO8), a member of the karyopherin β (also named the protein import receptor importin β) family, plays a critical role in mediating the cytoplasm-to-nucleus transport of mature miRNAs. Specifically knocking down IPO8 but not other karyopherin β family proteins via siRNA significantly decreases the nuclear transport of various known nucleus-enriched miRNAs without affecting their total cellular levels. IPO8-mediated nuclear transport of mature miRNAs is also dependent on the association of IPO8 with the Argonaute 2 (Ago2) complex. Cross-immunoprecipitation and Western blot analysis show that IPO8 is physically associated with Ago2. Knocking down IPO8 via siRNA markedly decreases the nuclear transport of Ago2 but does not affect the total cellular Ago2 level. Furthermore, dissociating the binding of miRNAs with Ago2 by trypaflavine strongly reduces the IPO8-mediated nuclear transport of miRNAs.
成熟的 microRNAs（miRNAs）是一类约 22 个核苷酸的非编码 RNA，可以在转录后水平调节靶基因的表达，最近研究表明它们可以被运输到细胞核内，从而调节其他 miRNAs 的生物发生或自身表达。然而，调控成熟 miRNAs 从细胞质到细胞核运输的机制仍不清楚。本文报道了 importin 8（IPO8），一种核输入蛋白β（也称为蛋白输入受体 importin β）家族成员，在介导成熟 miRNAs 的细胞质到细胞核运输中起着关键作用。具体来说，通过 siRNA 特异性敲低 IPO8 而不是其他核输入蛋白β家族蛋白，显著减少了各种已知富含核的 miRNAs 的核内运输，而不影响它们的总细胞水平。IPO8 介导的成熟 miRNAs 的核内运输也依赖于 IPO8 与 Argonaute 2（Ago2）复合物的结合。免疫沉淀和 Western blot 分析表明，IPO8 与 Ago2 存在物理关联。通过 siRNA 敲低 IPO8 会显著减少 Ago2 的核内运输，但不影响总细胞 Ago2 水平。此外，用 trypaflavine 解离 miRNAs 与 Ago2 的结合会强烈降低 IPO8 介导的 miRNAs 的核内运输。
