Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709.
Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709.
J Biol Chem. 2020 May 1;295(18):5857-5870. doi: 10.1074/jbc.RA119.011193. Epub 2020 Mar 27.
The ribosome biogenesis factor Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RφH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. Underscoring the significance of Las1 function in the cell, mutations in the human (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of experiments in and assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN' chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage.
核糖体生物发生因子 Las1 是一种必需的内切核糖核酸酶，在真核生物中高度保守，是新建立的高等真核生物和原核生物核苷酸结合（HEPN）结构域内切核酸酶家族的成员。HEPN 内切核酸酶参与多种 RNA 切割途径，具有一个短的 HEPN 内切核酸酶基序（RφH），对于 RNA 切割很重要。大多数 HEPN 内切核酸酶参与应激激活的 RNA 切割途径；Las1 在加工前 rRNA 中起着重要作用。Las1 功能在细胞中的重要性突显出来，人类（LAS1 样）基因中的突变与神经功能障碍有关。两个相邻的 HEPN 内切核酸酶基序构成了 Las1 的复合内切核酸酶活性位点，但单个 HEPN 基序残基的作用尚未明确。在这里，我们使用和实验相结合的方法，表明两个 HEPN 内切核酸酶基序都需要 Las1 内切核酸酶活性和保真度。通过深入的序列分析和系统的诱变，我们确定了 Las1 亚家族的保守 HEPN 基序，并揭示了其典型和特殊的元件。使用重组的 Las1 HEPN-HEPN'嵌合体，我们定义了 Las1 内切核酸酶切割 RNA 的分子要求。有趣的是，Las1 HEPN 基序的两个拷贝对于内切核酸酶功能都很重要，这表明两个 HEPN 基序都参与协调 Las1 活性位点内的 RNA。我们还确定了两个 HEPN 结构域的构象灵活性对于适当的内切核酸酶功能很重要。我们工作的结果揭示了关于双 HEPN 结构域如何协同作用以驱动 Las1 介导的 RNA 切割的关键信息。
