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，我们发现结合使用全基因组测序和SNP(WGS/SNP)图谱是一种有效和相对快速的分子病变识别技术，即使在基因图谱不精确的情况下也是如此(Jaramillo-Lambert等人，2015年G3)。使用这项技术，我们筛选出了以前被描述为母体效应胚胎致死的突变体，但当与野生型雄性交配时，它们被挽救了生存能力，这表明要么是精子缺陷，要么是受精性缺陷。我们发现了一种导致精子特异性胚胎致死的新突变，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，我们发现有7种动物能产生无核精子。初级精母细胞似乎试图进行减数分裂，但染色体未能分离，导致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。通过WGS/SNP定位，我们发现IT7是线虫拓扑异构酶II的同源基因top-2的等位基因，Top2是一种解决DNA缠结的酶。我们现在的目标是描述TOP-2在减数分裂过程中男性特有的功能。 在TOP-2工作的同时，我们继续通过结构功能分析研究之前在线虫中发现的唯一严格的父系效应致死(PEL)突变体SPE-11。SPE-11是一个由299个氨基酸组成的蛋白质，没有明显的蛋白结构域。SPE-11确实有五个预测的核定位序列(NLS)，并定位于雄性生殖系的细胞核。我们使用了我们实验室中产生的GFP：：SPE-11报告结构来分析突变的NLS和C末端缺失结构，以便正确地定位SPE-11和拯救SPE-11缺失突变体。以前，我们发现NLS1既不是SPE-11在胚胎中正确定位所必需的，也不是SPE-11在胚胎中正常功能所必需的。然而，当NLS5发生突变时，SPE-11被错位定位，无法挽救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末端截断，这表明C末端对功能非常重要。为了确定C末端的哪一部分对早期胚胎发育是必不可少的，我们创建了携带SPE-11截短版本的构建物的转基因株系。我们以前证明了两个带有SPE-11截短版本的构建体(GFP：：SPE-11 1-289和GFP：：SPE-11 1-294)不能挽救spe-11缺失突变体的胚胎致死性，这表明即使是蛋白质的最末端(即最后五个氨基酸)也是功能所必需的。进一步的分析已经确定了C末端的三个关键氨基酸，它们是功能所必需的。我们现在正致力于通过与鸡蛋激活复合体的已知成分进行免疫共沉淀实验和通过质谱分析来鉴定SPE-11相互作用的蛋白质。一旦鉴定出相互作用的蛋白质，我们就会问蛋白质之间的相互作用是否需要关键的C末端残基。