Laboratory for RNA Biology, Biochemistry Center Regensburg, University of Regensburg, 93053 Regensburg, Germany.
Laboratory for RNA Biology, Biochemistry Center Regensburg, University of Regensburg, 93053 Regensburg, Germany.
Cell Rep. 2015 Nov 10;13(6):1206-1220. doi: 10.1016/j.celrep.2015.09.068. Epub 2015 Oct 29.
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 Brat-NHL domain in complex with RNA and present a high-resolution protein-RNA structure of this fold.
TRIM-NHL 蛋白在后生动物中保守，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来指导神经元干细胞的分化。Brat 是一种 RNA 结合蛋白，作为翻译抑制剂发挥作用。然而，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 RNA 竞争，我们鉴定了果蝇胚胎中 Brat 结合的 mRNA，并定义了 Brat 以及许多其他 TRIM-NHL 蛋白的一致结合基序，表明 TRIM-NHL 蛋白是保守的、序列特异性的 RNA 结合蛋白。我们证明 Brat 介导的抑制和直接 RNA 结合依赖于鉴定出的基序，并表明定位因子 Miranda 与 Brat-NHL 结构域的结合抑制 Brat 活性。最后，为了解析 NHL 结构域的序列特异性，我们将 Brat-NHL 结构域与 RNA 进行结晶，并呈现该折叠的高分辨率蛋白-RNA 结构。
