Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Nucleic Acids Res. 2019 Sep 19;47(16):8770-8784. doi: 10.1093/nar/gkz583.
PUF proteins, named for Drosophila Pumilio (PUM) and Caenorhabditis elegans fem-3-binding factor (FBF), recognize specific sequences in the mRNAs they bind and control. RNA binding by classical PUF proteins is mediated by a characteristic PUM homology domain (PUM-HD). The Puf1 and Puf2 proteins possess a distinct architecture and comprise a highly conserved subfamily among fungal species. Puf1/Puf2 proteins contain two types of RNA-binding domain: a divergent PUM-HD and an RNA recognition motif (RRM). They recognize RNAs containing UAAU motifs, often in clusters. Here, we report a crystal structure of the PUM-HD of a fungal Puf1 in complex with a dual UAAU motif RNA. Each of the two UAAU tetranucleotides are bound by a Puf1 PUM-HD forming a 2:1 protein-to-RNA complex. We also determined crystal structures of the Puf1 RRM domain that identified a dimerization interface. The PUM-HD and RRM domains act in concert to determine RNA-binding specificity: the PUM-HD dictates binding to UAAU, and dimerization of the RRM domain favors binding to dual UAAU motifs rather than a single UAAU. Cooperative action of the RRM and PUM-HD identifies a new mechanism by which multiple RNA-binding modules in a single protein collaborate to create a unique RNA-binding specificity.
PUF 蛋白以果蝇 Pumilio（PUM）和秀丽隐杆线虫 fem-3 结合因子（FBF）命名，识别其结合和控制的 mRNA 中的特定序列。经典 PUF 蛋白的 RNA 结合由特征性 PUM 同源结构域（PUM-HD）介导。Puf1 和 Puf2 蛋白具有独特的结构，并在真菌物种中构成高度保守的亚家族。Puf1/Puf2 蛋白包含两种类型的 RNA 结合结构域：一个分歧的 PUM-HD 和一个 RNA 识别基序（RRM）。它们识别含有 UAAU 基序的 RNA，通常成簇存在。在这里，我们报告了一种真菌 Puf1 的 PUM-HD 与双 UAAU 基序 RNA 复合物的晶体结构。两个 UAAU 四核苷酸中的每一个都被一个 Puf1 PUM-HD 结合，形成 2:1 的蛋白质-RNA 复合物。我们还确定了 Puf1 RRM 结构域的晶体结构，该结构域确定了一个二聚化界面。PUM-HD 和 RRM 结构域协同作用以确定 RNA 结合特异性：PUM-HD 决定与 UAAU 结合，而 RRM 结构域的二聚化有利于与双 UAAU 基序而不是单个 UAAU 结合。RRM 和 PUM-HD 的协同作用确定了一种新的机制，即单个蛋白质中的多个 RNA 结合模块协同作用，创建独特的 RNA 结合特异性。
