Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA.
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Genetics. 2023 Aug 31;225(1). doi: 10.1093/genetics/iyad126.
Cullin-RING ubiquitin ligases (CRLs) are the largest class of ubiquitin ligases with diverse functions encompassing hundreds of cellular processes. Inactivation of core components of the CRL4 ubiquitin ligase produces a germ cell defect in Caenorhabditis elegans that is marked by abnormal globular morphology of the nucleolus and fewer germ cells. We identified DDB1 Cullin4 associated factor (DCAF)-1 as the CRL4 substrate receptor that ensures proper germ cell nucleolus morphology. We demonstrate that the dcaf-1 gene is the ncl-2 (abnormal nucleoli) gene, whose molecular identity was not previously known. We also observed that CRL4DCAF-1 is required for male tail development. Additionally, the inactivation of CRL4DCAF-1 results in a male-specific lethality in which a percentage of male progeny arrest as embryos or larvae. Analysis of the germ cell nucleolus defect using transmission electron microscopy revealed that dcaf-1 mutant germ cells possess significantly fewer ribosomes, suggesting a defect in ribosome biogenesis. We discovered that inactivation of the sperm-fate specification gene fog-1 (feminization of the germ line-1) or its protein-interacting partner, fog-3, rescues the dcaf-1 nucleolus morphology defect. Epitope-tagged versions of both FOG-1 and FOG-3 proteins are aberrantly present in adult dcaf-1(RNAi) animals, suggesting that DCAF-1 negatively regulates FOG-1 and FOG-3 expression. Murine CRL4DCAF-1 targets the degradation of the ribosome assembly factor periodic trptophan protein 1 (PWP1). We observed that the inactivation of Caenorhabditis elegansDCAF-1 increases the nucleolar levels of PWP1 in the germ line, intestine, and hypodermis. Reducing the level of PWP-1 rescues the dcaf-1 mutant defects of fewer germ cell numbers and abnormal nucleolus morphology, suggesting that the increase in PWP-1 levels contributes to the dcaf-1 germline defect. Our results suggest that CRL4DCAF-1 has an evolutionarily ancient role in regulating ribosome biogenesis including a conserved target in PWP1.
Cullin-RING 泛素连接酶 (CRLs) 是最大的泛素连接酶家族，具有多种功能，涵盖了数百种细胞过程。CRL4 泛素连接酶的核心成分失活会导致秀丽隐杆线虫的生殖细胞缺陷，其特征是核仁的异常球状形态和生殖细胞减少。我们鉴定出 DDB1 Cullin4 相关因子 (DCAF)-1 是 CRL4 泛素连接酶的底物受体，可确保正确的生殖细胞核仁形态。我们证明 dcaf-1 基因是 ncl-2（异常核仁）基因，其分子身份以前未知。我们还观察到 CRL4DCAF-1 是雄性尾巴发育所必需的。此外，CRL4DCAF-1 的失活会导致雄性特异性致死，其中一部分雄性后代停滞在胚胎或幼虫阶段。使用透射电子显微镜分析生殖细胞核仁缺陷发现，dcaf-1 突变体生殖细胞核糖体数量明显减少，提示核糖体生物发生缺陷。我们发现，精子命运指定基因 fog-1（生殖系 1 的雌性化）或其蛋白相互作用伴侣 fog-3 的失活可挽救 dcaf-1 核仁形态缺陷。FOG-1 和 FOG-3 蛋白的表位标记版本在成年 dcaf-1(RNAi)动物中异常存在，表明 DCAF-1 负调控 FOG-1 和 FOG-3 的表达。鼠类 CRL4DCAF-1 靶向核糖体组装因子周期性色氨酸蛋白 1 (PWP1) 的降解。我们观察到，秀丽隐杆线虫 DCAF-1 的失活会增加生殖系、肠和皮下组织中线粒体的 PWP1 核仁水平。降低 PWP-1 的水平可挽救 dcaf-1 突变体生殖细胞数量减少和核仁形态异常的缺陷，表明 PWP-1 水平的增加导致了 dcaf-1 生殖系缺陷。我们的结果表明，CRL4DCAF-1 在调节核糖体生物发生中具有古老的进化作用，包括在 PWP1 中有一个保守的靶标。
