Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany;
Genes Dev. 2014 Apr 1;28(7):749-64. doi: 10.1101/gad.236513.113.
The Drosophila protein brain tumor (Brat) forms a complex with Pumilio (Pum) and Nanos (Nos) to repress hunchback (hb) mRNA translation at the posterior pole during early embryonic development. It is currently thought that complex formation is initiated by Pum, which directly binds the hb mRNA and subsequently recruits Nos and Brat. Here we report that, in addition to Pum, Brat also directly interacts with the hb mRNA. We identify Brat-binding sites distinct from the Pum consensus motif and show that RNA binding and translational repression by Brat do not require Pum, suggesting so far unrecognized Pum-independent Brat functions. Using various biochemical and biophysical methods, we also demonstrate that the NHL (NCL-1, HT2A, and LIN-41) domain of Brat, a domain previously believed to mediate protein-protein interactions, is a novel, sequence-specific ssRNA-binding domain. The Brat-NHL domain folds into a six-bladed β propeller, and we identify its positively charged top surface as the RNA-binding site. Brat belongs to the functional diverse TRIM (tripartite motif)-NHL protein family. Using structural homology modeling, we predict that the NHL domains of all TRIM-NHL proteins have the potential to bind RNA, indicating that Brat is part of a conserved family of RNA-binding proteins.
果蝇蛋白脑肿瘤（Brat）与 Pumilio（Pum）和 Nanos（Nos）形成复合物，在早期胚胎发育过程中在后极抑制 hunchback（hb）mRNA 的翻译。目前认为，复合物的形成是由 Pum 启动的，它直接结合 hb mRNA，随后招募 Nos 和 Brat。在这里，我们报告 Brat 还直接与 hb mRNA 相互作用。我们确定了 Brat 结合 hb mRNA 的位点与 Pum 一致基序不同，并表明 Brat 的 RNA 结合和翻译抑制不需要 Pum，这表明 Brat 具有迄今为止尚未被识别的 Pum 非依赖性功能。我们还使用各种生化和生物物理方法证明，Brat 的 NHL（NCL-1、HT2A 和 LIN-41）结构域，以前被认为介导蛋白质-蛋白质相互作用，是一个新的、序列特异性的 ssRNA 结合结构域。Brat-NHL 结构域折叠成一个六叶 β 螺旋桨，我们确定其带正电荷的顶面为 RNA 结合位点。Brat 属于功能多样的 TRIM（三部分基序）-NHL 蛋白家族。使用结构同源建模，我们预测所有 TRIM-NHL 蛋白的 NHL 结构域都有可能结合 RNA，这表明 Brat 是一个保守的 RNA 结合蛋白家族的一部分。
