Mukherjee Chandrama, Bakthavachalu Baskar, Schoenberg Daniel R
Center for RNA Biology, The Ohio State University, Columbus, Ohio, United States of America; Department of Molecular & Cellular Biochemistry, The Ohio State University, Columbus, Ohio, United States of America.
PLoS Biol. 2014 Aug 19;12(8):e1001933. doi: 10.1371/journal.pbio.1001933. eCollection 2014 Aug.
Cytoplasmic capping is catalyzed by a complex that contains capping enzyme (CE) and a kinase that converts RNA with a 5'-monophosphate end to a 5' diphosphate for subsequent addition of guanylic acid (GMP). We identify the proline-rich C-terminus as a new domain of CE that is required for its participation in cytoplasmic capping, and show the cytoplasmic capping complex assembles on Nck1, an adapter protein with functions in translation and tyrosine kinase signaling. Binding is specific to Nck1 and is independent of RNA. We show by sedimentation and gel filtration that Nck1 and CE are together in a larger complex, that the complex can assemble in vitro on recombinant Nck1, and Nck1 knockdown disrupts the integrity of the complex. CE and the 5' kinase are juxtaposed by binding to the adjacent domains of Nck1, and cap homeostasis is inhibited by Nck1 with inactivating mutations in each of these domains. These results identify a new domain of CE that is specific to its function in cytoplasmic capping, and a new role for Nck1 in regulating gene expression through its role as the scaffold for assembly of the cytoplasmic capping complex.
细胞质加帽反应由一种复合物催化，该复合物包含加帽酶（CE）和一种激酶，该激酶将具有5'-单磷酸末端的RNA转化为5'-二磷酸，以便随后添加鸟苷酸（GMP）。我们确定富含脯氨酸的C末端是CE的一个新结构域，它参与细胞质加帽反应是必需的，并表明细胞质加帽复合物在Nck1上组装，Nck1是一种在翻译和酪氨酸激酶信号传导中起作用的衔接蛋白。这种结合对Nck1具有特异性，且不依赖于RNA。我们通过沉降和凝胶过滤表明，Nck1和CE存在于一个更大的复合物中，该复合物可以在体外重组Nck1上组装，并且Nck1的敲低会破坏该复合物的完整性。CE和5'激酶通过与Nck1的相邻结构域结合而并列，并且在这些结构域中具有失活突变的Nck1会抑制帽稳态。这些结果确定了CE在细胞质加帽反应中特有的一个新结构域，以及Nck1通过作为细胞质加帽复合物组装支架的作用在调节基因表达方面的新作用。
