mRNAs that co-purify with OMA-1 in the C. elegans germline (microarray)

The oocytes of most animals arrest at diplotene or diakinesis, but resume meiosis (meiotic maturation) in response to hormones. In C. elegans, maturation of the 1 oocyte requires the presence of sperm, Gas-adenylate cyclase-PKA signaling in the gonadal sheath cells, and germline function of two Tis11-like CCCH zinc-finger proteins, OMA-1 and OMA-2 (OMA proteins). Prior studies indicate that the OMA proteins redundantly repress the translation of specific mRNAs in oocytes (zif-1, mom-2, nos-2, glp-1) and early embryos (mei-1). We purified OMA-1-containing ribonucleoprotein particles (RNPs) and identified mRNAs that associate with OMA-1 in oocytes using microarrays. We examined the relative abundances of mRNAs in OMA-1 RNPs using high-throughput RNA sequencing. Previously identified targets of OMA-dependent translational repression in oocytes were found to be both enriched (>2-fold relative to input RNA) and abundant in purified OMA-1 RNPs. Furthermore, we verified that some of the newly identified mRNAs that share these characteristics are translationally repressed by OMA-1/2 in oocytes through sequences in their 3UTRs. Although meiotic maturation is stimulated by sperm, we found that the mRNAs copurifying with OMA-1 are not significantly different in the presence and absence of sperm, suggesting that sperm-dependent signaling does not modify the suite of mRNAs stably associated with OMA-1. Further, several tested OMA-1-associated mRNAs were shown to be translationally repressed in both the presence and absence of sperm.多数动物的卵母细胞会停滞于双线期（diplotene）或终变期（diakinesis），但可在激素刺激下恢复减数分裂，即减数分裂成熟（meiotic maturation）。在秀丽隐杆线虫（C. elegans）中，一号卵母细胞的成熟过程需要精子的参与、性腺鞘细胞内的Gas蛋白-腺苷酸环化酶-PKA信号通路（Gas-adenylate cyclase-PKA signaling），以及两种类似Tis11的CCCH型锌指蛋白（CCCH zinc-finger proteins）——OMA-1与OMA-2（OMA蛋白）的生殖系功能。既往研究显示，OMA蛋白可在卵母细胞（靶mRNA包括zif-1、mom-2、nos-2、glp-1）及早期胚胎（靶mRNA为mei-1）中冗余性抑制特定mRNA的翻译。我们通过纯化获取了携带OMA-1的核糖核蛋白颗粒（RNPs），并借助基因芯片（microarrays）鉴定了卵母细胞中与OMA-1结合的mRNA；随后采用高通量RNA测序（high-throughput RNA sequencing）分析了OMA-1 RNPs内mRNA的相对丰度。结果发现，此前已报道的受OMA依赖型翻译抑制的卵母细胞靶mRNA，不仅在纯化得到的OMA-1 RNPs中显著富集（较输入RNA富集倍数≥2），且在颗粒内丰度较高。此外，我们验证了部分新鉴定的、符合上述特征的mRNA，其翻译可通过其3'非翻译区（3'UTR）中的序列元件，被OMA-1/2在卵母细胞中抑制。尽管精子可触发减数分裂成熟，但我们的实验显示，无论是否存在精子，与OMA-1共纯化的mRNA谱均无显著差异，这提示精子依赖的信号通路并不会改变与OMA-1稳定结合的mRNA集合。进一步的检测表明，多款经筛选的OMA-1结合mRNA，在有、无精子的培养条件下均受到翻译抑制。