Structural requirements for pre-microRNA binding and nuclear export by Exportin 5
Structural requirements for pre-microRNA binding and nuclear export by Exportin 5
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DOI：
10.1093/nar/gkh824
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Cullen, BR
中科院分区：
文献类型：
--
作者：
Zeng, Y;Cullen, BR
成熟人类 microRNA 的生物发生和功能依赖于 Exportin 5 (Exp5) 的前 microRNA 前体的核输出。人类 miR-30 microRNA 的前体是一个 63 nt 长的 RNA 发夹，带有 2 nt 3' 突出端，与 Exp5 和 Ran-GTP 辅因子形成特定复合物。在这里，我们检查了 Exp5 结合 pre-microRNA 的结构要求。我们的数据表明 pre-miR-30 结合需要 >16 bp 的 RNA 茎，并通过 3' 突出来促进。尽管 pre-miR-30 茎环的平端衍生物仍然能够结合 Exp5，但 5' 突出端具有抑制性。失去有效结合 Exp5 能力的 miR-30 变体无法有效地从细胞核输出，并且表达水平也降低。此外，pre-microRNA/Exp5/Ran-GTP 复合物的形成抑制了 pre-miRNA 的体外核酸外切消化。总之，这些数据表明 Exp5 结合前 microRNA 涉及识别几乎所有 RNA 发夹（末端环除外）。此外，这些结果表明 Exp5 结合不仅介导 pre-microRNA 核输出，而且还可以防止核 pre-microRNA 降解。
The biogenesis and function of mature human microRNAs is dependent on the nuclear export of pre-microRNA precursors by Exportin 5 (Exp5). The precursor for the human miR-30 microRNA, which is a 63 nt long RNA hairpin bearing a 2 nt 3' overhang, forms a specific complex with Exp5 and the Ran-GTP cofactor. Here, we have examined the structural requirements for pre-microRNA binding by Exp5. Our data indicate that pre-miR-30 binding requires an RNA stem of >16 bp and is facilitated by a 3' overhang. Although a blunt-ended derivative of the pre-miR-30 stem-loop remained capable of binding Exp5, 5' overhangs were inhibitory. miR-30 variants that had lost the ability to bind Exp5 effectively were not efficiently exported from the nucleus and were also expressed at reduced levels. Furthermore, formation of a pre-microRNA/Exp5/Ran-GTP complex inhibited exonucleolytic digestion of the pre-miRNA in vitro. Together, these data demonstrate that pre-microRNA binding by Exp5 involves recognition of almost all of the RNA hairpin, with the exception of the terminal loop. Moreover, these results argue that Exp5 binding not only mediates pre-microRNA nuclear export but also prevents nuclear pre-microRNA degradation.