Vasa homologs are ATP-dependent DEAD-box helicases, multipotency factors, and critical components that specify and protect the germline. They regulate translation, amplify piRNAs, and act as RNA solvents; but, the limited availability of mutagenesis-derived alleles and their wide... 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 GLH-1/Vasa in Caenorhabditis elegans using 28 endogenous, mutant alleles. We show that GLH-1's helicase activity is required to retain its association with P granules. GLH-1 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 GLH-1-associated proteins implies conservation of a transient piRNA-amplifying complex, and reveals a novel affinity between GLH-1 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.

中文翻译：

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通过秀丽隐杆线虫 P 颗粒中 GLH/Vasa 的多方面活性维持种系。

Vasa 同源物是 ATP 依赖性死盒解旋酶、多能因子以及指定和保护种系的关键组件。它们调节翻译、扩增 piRNA 并充当 RNA 溶剂；但是，诱变衍生的等位基因及其广泛的可用性有限……Vasa 同源物是 ATP 依赖性死盒解旋酶、多能因子以及指定和保护种系的关键组件。它们调节翻译、扩增 piwi 相互作用 RNA (piRNA) 并充当 RNA 溶剂；然而，诱变衍生等位基因的可用性有限及其广泛的表型使他们的分析变得复杂。现在，通过成簇的规则间隔的短回文重复序列 (CRISPR/Cas9)，可以减轻这些限制，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定义了秀丽隐杆线虫中 GLH-1/Vasa 的功能基序。我们表明，GLH-1 的解旋酶活性是保持其与 P 颗粒结合所必需的。当解旋酶和侧翼结构域之外发生变化时，GLH-1 仍保留在 P 颗粒中，但生育力仍然受到损害。从 GLH 蛋白中去除富含甘氨酸的重复序列会逐渐减少核外围的 P 颗粒润湿样相互作用。GLH-1 相关蛋白的质谱分析表明瞬时 piRNA 扩增复合物的保守性，并揭示了 GLH-1 与三个结构保守的 PCI（26S 蛋白酶体盖、COP9 和 eIF3）复合物或“zomes”之间的新型亲和力，以及对组装的核糖体和 26S 蛋白酶体具有相反的厌恶感。这些结果表明，P 颗粒将细胞质分隔开来排除大的蛋白质组装体，从而有效地保护相关转录物免受翻译和相关蛋白质的周转。在胚芽颗粒内，Vasa 同系物可以充当溶剂，通过小 RNA 监视和扩增途径确保 mRNA 的可及性，并促进 mRNA 通过胚芽颗粒输出以启动翻译。