The role of SPE-11 in C. elegans egg activation

SPE-11 在秀丽隐杆线虫卵激活中的作用

• 批准号: 9148884
• 负责人: Andy Golden
• 金额: $ 23.74万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9148884
• 关键词: Alleles; Amino Acids; Animals; Biochemical; Biological; C-terminal; Caenorhabditis elegans; Cell Nucleus; Cells; Chromosome Mapping; Chromosomes; Co-Immunoprecipitations; Complex; DNA; Defect; Development; Embryo; Embryonic Development; Enzymes; Essential Amino Acids; Event; Female; Fertilization; Genetic; Germ Lines; Goals; Haploidy; Human; Lesion; Lethal Genes; Mass Spectrum Analysis; Meiosis; Molecular; Mutate; Mutation; Nuclear; Oocytes; Orthologous Gene; Partner in relationship; Phenotype; Process; Proteins; Reporter; Role; Scheme; Series; Single Nucleotide Polymorphism Map; Spermatocytes; Structure; Techniques; Tertiary Protein Structure; Topoisomerase II; Transgenic Organisms; Work; egg; genome sequencing; male; mutant; novel; protein protein interaction; rapid technique; research study; sperm cell

In the past year, we found that using a combination of whole-genome sequencing and SNP (WGS/SNP) mapping is an efficient and relatively rapid technique for the identification of molecular lesions, even in cases where gene mapping is not precise (Jaramillo-Lambert et al., 2015 G3). Using this technique we screened mutants previously characterized as maternal-effect embryonic-lethal but were rescued for viability when mated to wild-type males, suggesting either a sperm or zygotic defect. We identified a new mutation that causes sperm-specific embryonic lethality, it7; mutant males sire dead progeny when mated to wild-type females. Through cell biological analysis we have shown that it7 animals make anucleate sperm. The primary spermatocytes appear to attempt the meiotic divisions but the chromosomes fail to segregate, resulting in anucleate sperm that are capable of fertilizing oocytes; such haploid embryos die during early development. With the help of WGS/SNP mapping we discovered that it7 is an allele of top-2, the C. elegans ortholog of human topoisomerase II, an enzyme that resolves DNA entanglements. We now aim to characterize the male-specific function of TOP-2 during meiosis. Alongside the top-2 work we have continued to study the only strict paternal-effect lethal (PEL) mutant previously identified in C. elegans, SPE-11, through structure function analysis. SPE-11 is a 299 amino acid protein with no obvious protein domains. SPE-11 does have five predicted nuclear localization sequences (NLS) and localizes to germ-line nuclei of the male. We used a GFP::SPE-11 reporter construct that was generated in our lab to analyze mutated NLS and C-terminal deletion constructs for proper SPE-11 localization and rescue of a spe-11 deletion mutant. Previously, we found that NLS1 was neither required for proper localization of SPE-11 nor required for proper function of SPE-11 in the embryo. However, when NLS5 is mutated, SPE-11 is mislocalized and fails to rescue the spe-11 deletion mutant. Interestingly, NLS5 resides in the C-terminus of the SPE-11 protein. Curiously, the majority of existing spe-11 mutant alleles result in C-terminal truncations of the SPE-11 protein, suggesting that the C-terminus is very important for function. To determine which part of the C-terminus is essential for early embryogenesis, we created transgenic lines carrying constructs with truncated versions of SPE-11. We previously showed that two constructs with truncated versions of SPE-11 (GFP::SPE-11 1-289 and GFP::SPE-11 1-294) fail to rescue the embryonic lethality of the spe-11 deletion mutant suggesting that even the most C-terminal end of the protein (i.e. the last five amino acids) are essential for function. Further analysis has identified three critical amino acids in the C-terminus that are required for function. We are now working on identifying SPE-11 interacting proteins through co-immunoprecipitation experiments with the known components of the egg-activation complex and through mass spectrometry. Once interacting proteins are identified we will ask whether the critical C-terminal residues are required for protein-protein interactions.

在过去的一年里，我们发现，使用全基因组测序和 SNP (WGS/SNP) 作图相结合是一种有效且相对快速的分子病变识别技术，即使在基因作图不精确的情况下也是如此 (Jaramillo-兰伯特等人，2015 G3)。使用这种技术，我们筛选了以前被定性为母体效应胚胎致死的突变体，但在与野生型雄性交配时获得了生存能力，这表明精子或合子缺陷。我们发现了一种新的突变，它会导致精子特异性胚胎致死，it7；突变的雄性与野生型雌性交配时会产生死亡的后代。通过细胞生物学分析，我们已经证明动物可以产生无核精子。初级精母细胞似乎试图进行减数分裂，但染色体无法分离，从而产生能够使卵母细胞受精的无核精子；这种单倍体胚胎在早期发育过程中死亡。在 WGS/SNP 作图的帮助下，我们发现 it7 是 top-2 的等位基因，top-2 是人类拓扑异构酶 II（一种解决 DNA 缠结的酶）的线虫直系同源物。我们现在的目标是表征减数分裂期间 TOP-2 的雄性特异性功能。 除了 top-2 工作之外，我们还通过结构功能分析继续研究先前在秀丽隐杆线虫中发现的唯一严格父系效应致死 (PEL) 突变体 SPE-11。 SPE-11是一种299个氨基酸的蛋白质，没有明显的蛋白质结构域。 SPE-11确实有五个预测的核定位序列（NLS）并定位于雄性的种系细胞核。我们使用我们实验室生成的 GFP::SPE-11 报告构建体来分析突变的 NLS 和 C 端删除构建体，以实现正确的 SPE-11 定位和拯救 spe-11 删除突变体。此前，我们发现 NLS1 既不是 SPE-11 正确定位所必需的，也不是 SPE-11 在胚胎中正常发挥功能所必需的。然而，当NLS5突变时，SPE-11定位错误，无法拯救spe-11缺失突变体。 有趣的是，NLS5 位于 SPE-11 蛋白的 C 末端。奇怪的是，大多数现有的 spe-11 突变等位基因都会导致 SPE-11 蛋白的 C 端截短，这表明 C 端对于功能非常重要。为了确定 C 末端的哪一部分对于早期胚胎发生至关重要，我们创建了携带 SPE-11 截短版本构建体的转基因系。我们之前表明，具有 SPE-11 截短版本的两个构建体（GFP::SPE-11 1-289 和 GFP::SPE-11 1-294）未能挽救 spe-11 缺失突变体的胚胎致死性，这表明即使蛋白质的最 C 末端（即最后五个氨基酸）对于功能至关重要。进一步分析确定了 C 末端功能所需的三个关键氨基酸。我们现在正致力于通过卵子激活复合物已知成分的免疫共沉淀实验和质谱分析来鉴定 SPE-11 相互作用蛋白。一旦鉴定出相互作用的蛋白质，我们将询问蛋白质-蛋白质相互作用是否需要关键的 C 末端残基。