Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742, USA.
Nucleic Acids Res. 2011 Mar;39(6):2445-57. doi: 10.1093/nar/gkq1131. Epub 2010 Nov 17.
Nucleolar Essential Protein 1 (Nep1) is required for small subunit (SSU) ribosomal RNA (rRNA) maturation and is mutated in Bowen-Conradi Syndrome. Although yeast (Saccharomyces cerevisiae) Nep1 interacts with a consensus sequence found in three regions of SSU rRNA, the molecular details of the interaction are unknown. Nep1 is a SPOUT RNA methyltransferase, and can catalyze methylation at the N1 of pseudouridine. Nep1 is also involved in assembly of Rps19, an SSU ribosomal protein. Mutations in Nep1 that result in decreased methyl donor binding do not result in lethality, suggesting that enzymatic activity may not be required for function, and RNA binding may play a more important role. To study these interactions, the crystal structures of the scNep1 dimer and its complexes with RNA were determined. The results demonstrate that Nep1 recognizes its RNA site via base-specific interactions and stabilizes a stem-loop in the bound RNA. Furthermore, the RNA structure observed contradicts the predicted structures of the Nep1-binding sites within mature rRNA, suggesting that the Nep1 changes rRNA structure upon binding. Finally, a uridine base is bound in the active site of Nep1, positioned for a methyltransfer at the C5 position, supporting its role as an N1-specific pseudouridine methyltransferase.
核仁必需蛋白 1（Nep1）是小亚基（SSU）核糖体 RNA（rRNA）成熟所必需的，并且在 Bowen-Conradi 综合征中发生突变。尽管酵母（酿酒酵母）Nep1 与 SSU rRNA 三个区域中的保守序列相互作用，但相互作用的分子细节尚不清楚。Nep1 是一种 SPORT RNA 甲基转移酶，可催化假尿嘧啶 N1 的甲基化。Nep1 还参与 SSU 核糖体蛋白 Rps19 的组装。导致供甲基供体结合减少的 Nep1 突变不会导致致死性，这表明酶活性可能不是功能所必需的，而 RNA 结合可能发挥更重要的作用。为了研究这些相互作用，确定了 scNep1 二聚体及其与 RNA 复合物的晶体结构。结果表明，Nep1 通过碱基特异性相互作用识别其 RNA 结合位点，并稳定结合 RNA 中的茎环。此外，观察到的 RNA 结构与成熟 rRNA 中 Nep1 结合位点的预测结构相矛盾，这表明 Nep1 在结合后改变 rRNA 结构。最后，一个尿嘧啶碱基结合在 Nep1 的活性位点上，位于 C5 位置的甲基转移位置，支持其作为 N1 特异性假尿嘧啶甲基转移酶的作用。
