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

在过去的一年中，我们发现，即使在基因定位不精确的情况下，使用全基因组测序和SNP （WGS/SNP）作图的组合也是一种高效且相对快速的分子病变鉴定技术（Jaramillo-Lambert等人，2015 G3）。使用这种技术，我们筛选了以前被认为是母体效应胚胎致死性的突变体，但当与野生型雄性交配时，这些突变体被拯救出来，表明要么是精子缺陷，要么是合子缺陷。我们发现了一种新的突变，它会导致精子特异性的胚胎致命性。突变的雄性在与野生型雌性交配时产生死亡的后代。通过细胞生物学分析，我们已经证明，有7种动物产生无核精子。原代精细胞似乎试图进行减数分裂，但染色体无法分离，导致无核精子无法与卵母细胞受精；这种单倍体胚胎在早期发育过程中死亡。在WGS/SNP图谱的帮助下，我们发现it7是秀丽隐杆线虫的top-2等位基因，top-2是人类拓扑异构酶II的同源基因，拓扑异构酶II是一种解决DNA纠缠的酶。我们现在的目标是描述TOP-2在减数分裂期间的男性特异性功能。

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.