The Crystal Structure of the NHL Domain in Complex with RNA Reveals the Molecular Basis of Drosophila Brain-Tumor-Mediated Gene Regulation
The Crystal Structure of the NHL Domain in Complex with RNA Reveals the Molecular Basis of Drosophila Brain-Tumor-Mediated Gene Regulation
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DOI：
10.1016/j.celrep.2015.09.068
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发表时间：
2015-11-10
期刊：
影响因子：
8.8
通讯作者：
Meister, Gunter
中科院分区：
文献类型：
--
作者：
Loedige, Inga;Jakob, Leonhard;Meister, Gunter
TRIM-NHL蛋白在后生动物中是保守的，并控制各种干细胞谱系中的细胞命运决定。果蝇TRIM-NHL蛋白脑肿瘤（Brat）通过抑制自我更新因子指导神经干细胞的分化。Brat是一种RNA结合蛋白，具有翻译阻遏物的功能。然而，目前尚不清楚Brat调节哪些RNA以及如何实现RNA结合特异性。使用RNA免疫沉淀和RNAcompete，我们确定在果蝇胚胎中的Brat结合的mRNA，并确定共识结合基序的Brat以及一些额外的TRIM-NHL蛋白，表明TRIM-NHL蛋白是保守的，序列特异性RNA结合蛋白。我们证明，布拉特介导的镇压和直接RNA结合依赖于所确定的基序，并显示，结合的本地化因子米兰达的布拉特-NHL域抑制布拉特活动。最后，为了解开NHL结构域的序列特异性，我们将BratNHL结构域与RNA复合物结晶，并呈现这种折叠的高分辨率蛋白质-RNA结构。
TRIM-NHL proteins are conserved among metazoans and control cell fate decisions in various stem cell linages. The Drosophila TRIM-NHL protein Brain tumor (Brat) directs differentiation of neuronal stem cells by suppressing self-renewal factors. Brat is an RNA-binding protein and functions as a translational repressor. However, it is unknown which RNAs Brat regulates and how RNA-binding specificity is achieved. Using RNA immunoprecipitation and RNAcompete, we identify Brat-bound mRNAs in Drosophila embryos and define consensus binding motifs for Brat as well as a number of additional TRIM-NHL proteins, indicating that TRIM-NHL proteins are conserved, sequence-specific RNA-binding proteins. We demonstrate that Brat-mediated repression and direct RNA-binding depend on the identified motif and show that binding of the localization factor Miranda to the Brat-NHL domain inhibits Brat activity. Finally, to unravel the sequence specificity of the NHL domain, we crystallize the BratNHL domain in complex with RNA and present a high-resolution protein-RNA structure of this fold.