Germline Maintenance Through the Multifaceted Activities of GLH/Vasa in Caenorhabditis elegans P Granules
Germline Maintenance Through the Multifaceted Activities of GLH/Vasa in Caenorhabditis elegans P Granules
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DOI：
10.1534/genetics.119.302670
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发表时间：
2019-11-01
期刊：
影响因子：
3.3
通讯作者：
Updike, Dustin L.
中科院分区：
文献类型：
--
作者：
Marnik, Elisabeth A.;Fuqua, J. Heath;Updike, Dustin L.
VASA同源物是依赖于ATP的死盒解旋酶、多能因子，以及指定和保护生殖系的关键成分。它们调节翻译，放大piwi相互作用的RNAs(PiRNAs)，并作为RNA溶剂；然而，突变衍生的等位基因的有限可用性及其广泛的表型使它们的分析变得复杂。现在，有了成簇的规则间隔的短回文重复(CRISPR/Cas9)，这些限制可以被缓解，以确定为什么蛋白质结构域在整个进化过程中丢失或保留。在这里，我们使用28个内源突变等位基因来定义秀丽线虫中/Vasa的功能基序。我们发现‘S解旋酶活性是维持其与P颗粒的结合所必需的。当解旋酶和侧翼结构域之外发生变化时，仍然留在P颗粒中，但生育能力仍然受到影响。从GLH蛋白中去除富含甘氨酸的重复序列会逐渐减少核周的P颗粒润湿样相互作用。相关蛋白质的质谱学表明一个瞬时的piRNA扩增复合体是保守的，并揭示了三个结构保守的PCI(26S蛋白酶体LID、COP9和eIF3)复合体之间的新的亲和力，以及对组装的核糖体和26S蛋白酶体的相互厌恶。这些结果表明，P颗粒划分细胞质以排除大的蛋白质组装，有效地保护相关转录本免受翻译和相关蛋白质的周转。在胚芽颗粒内，VASA同系物可以作为溶剂，通过小RNA监视和扩增途径确保mRNA的可及性，并通过胚芽颗粒促进mRNA输出以启动翻译。
Vasa homologs are ATP-dependent DEAD-box helicases, multipotency factors, and critical components that specify and protect the germline. They regulate translation, amplify piwi-interacting RNAs (piRNAs), and act as RNA solvents; however, the limited availability of mutagenesis-derived alleles and their wide range of phenotypes have complicated their analysis. Now, with clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), these limitations can be mitigated to determine why protein domains have been lost or retained throughout evolution. Here, we define the functional motifs of /Vasa in Caenorhabditis elegans using 28 endogenous, mutant alleles. We show that 's helicase activity is required to retain its association with P granules. remains in P granules when changes are made outside of the helicase and flanking domains, but fertility is still compromised. Removal of the glycine-rich repeats from GLH proteins progressively diminishes P-granule wetting-like interactions at the nuclear periphery. Mass spectrometry of -associated proteins implies conservation of a transient piRNA-amplifying complex, and reveals a novel affinity between and three structurally conserved PCI (26S Proteasome Lid, COP9, and eIF3) complexes or "zomes," along with a reciprocal aversion for assembled ribosomes and the 26S proteasome. These results suggest that P granules compartmentalize the cytoplasm to exclude large protein assemblies, effectively shielding associated transcripts from translation and associated proteins from turnover. Within germ granules, Vasa homologs may act as solvents, ensuring mRNA accessibility by small RNA surveillance and amplification pathways, and facilitating mRNA export through germ granules to initiate translation.