A LIN28-dependent structural change in pre-let-7g directly inhibits dicer processing.
A LIN28-dependent structural change in pre-let-7g directly inhibits dicer processing.
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DOI：
10.1021/bi200851d
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发表时间：
2011-09-06
期刊：
影响因子：
2.9
通讯作者：
Balasubramanian, Shankar
中科院分区：
文献类型：
--
作者：
Lightfoot, Helen L.;Bugaut, Anthony;Armisen, Javier;Lehrbach, Nicolas J.;Miska, Eric A.;Balasubramanian, Shankar
最近的几项研究提供了证据表明，细胞质RNA结合蛋白LIN 28在哺乳动物和C.优美的然而，抑制的确切机制仍然知之甚少。在这里，我们报告的体外研究，结合RNA酶足迹，凝胶迁移结合试验和加工试验，调查的分子基础和功能之间的相互作用的天然let-7 g前体（pre-let-7 g）和LIN 28。我们已经绘制了pre-let-7 g的结构，并确定了与LIN 28物理相互作用的pre-let-7 g末端环的区域。我们还确定了LIN 28结合后的构象变化，其导致在pre-let-7 g的Dicer加工位点处的另外双链区域的解旋。此外，我们表明，显示开放的上部茎的突变体pre-let-7 g抑制pre-let-7 g Dicer加工至与LIN 28相同的程度。该数据支持LIN 28可在切粒机加工步骤直接抑制let-7 g生物发生的机制。
Several recent studies have provided evidence that LIN28, a cytoplasmic RNA-binding protein, inhibits the biogenesis of members of the let-7 microRNA (miRNA) family at the Dicer step in both mammals and C. elegans. However, the precise mechanism of inhibition is still poorly understood. Here we report on an in vitro study, which combined RNase footprinting, gel shift binding assays and processing assays, to investigate the molecular basis and function of the interaction between the native let-7g precursor (pre-let-7g) and LIN28. We have mapped the structure of pre-let-7g and identified regions of the terminal loop of pre-let-7g that physically interact with LIN28. We have also identified a conformational change upon LIN28 binding that results in the unwinding of an otherwise double-stranded region at the Dicer-processing site of pre-let-7g. Furthermore, we showed that a mutant pre-let-7g that displays an open upper stem inhibited pre-let-7g Dicer processing to the same extent as LIN28. The data supports a mechanism by which LIN28 can directly inhibit let-7g biogenesis at the Dicer processing step.
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