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It takes two (Las1 HEPN endoribonuclease domains) to cut RNA correctly.
Journal of Biological Chemistry ( IF 5.5 ) Pub Date : 2020-03-27 , DOI: 10.1074/jbc.ra119.011193
Monica C Pillon 1 , Kevin H Goslen 1 , Jacob Gordon 1 , Melissa L Wells 1 , Jason G Williams 2 , Robin E Stanley 1
Affiliation  

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 (RφXXXH) 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 LAS1L (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 in vivo experiments in Saccharomyces cerevisiae and in vitro 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.

中文翻译:

正确切割RNA需要两个(Las1 HEPN内切核糖核酸酶结构域)。

核糖体生物发生因子Las1是一种必需的核糖核酸内切酶,在整个真核生物中都是高度保守的,并且是一个新成立的高级真核生物和原核生物核苷酸结合(HEPN)域含核酸酶家族的成员。HEPN核酸酶参与各种RNA裂解途径,并共享一个短RNA裂解重要的HEPN核酸酶基序(RφXXXH)。大多数HEPN核酸酶都参与应激激活的RNA裂解途径。Las1在处理pre-rRNA中起基本作用。强调了Las1功能在细胞中的重要性,人类LAS1L(类LAS1)基因的突变与神经功能障碍有关。两个并列的HEPN核酸酶基序创建了Las1的复合核酸酶活性位点,但单个HEPN基序残基的作用定义不清。在这里,结合使用酿酒酵母的体内实验和体外测定,我们显示了两个HEPN核酸酶基序都是Las1核酸酶活性和保真度所必需的。通过深入的序列分析和系统诱变,我们确定了Las1子家族中的HEPN共有基序,并揭示了其规范和专门的元素。使用重构的Las1 HEPN-HEPN'嵌合体,我们定义了RNA切割的分子要求。有趣的是,Las1 HEPN基序的两个拷贝对于核酸酶功能都很重要,揭示了两个HEPN基序都参与协调Las1活性位点内的RNA。我们还建立了两个HEPN域的构象灵活性对于适当的核酸酶功能很重要。
更新日期:2020-05-01
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