RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes

Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.Staufen2（Stau2）是一种双链RNA结合蛋白，通过调控mRNA转运、mRNA稳定性、蛋白质翻译及核糖核蛋白组装参与细胞命运决定。目前对于减数分裂过程中哺乳动物卵母细胞内Stau2的表达与功能尚缺乏深入研究。本研究报道了Stau2在小鼠卵母细胞减数分裂中的亚细胞分布与功能。蛋白质免疫印迹（Western blot）分析显示，从生发泡（germinal vesicle, GV）期到第二次减数分裂中期（metaphase II, MII）的卵母细胞中，Stau2均呈现高表达且表达量稳定。免疫荧光染色结果表明，GV期卵母细胞内Stau2呈均匀分布状态，而生发泡破裂（germinal vesicle breakdown, GVBD）后Stau2组装为丝状结构；尤其在第一次减数分裂中期（MI）和MII期，Stau2与纺锤体存在共定位现象。当用诺考达唑（nocodazole）破坏微管结构时，Stau2的组装状态被解除；反之，当用紫杉醇（taxol）稳定微管时，Stau2并未与稳定后的微管共定位，而是聚集于染色体排列区域周围。上述结果表明，Stau2的组装及与微管的共定位依赖于微管的完整性及其正常动力学特性。在BHK与MEF细胞的间期及有丝分裂过程中，Stau2并未分布于微管上，而是与顺式高尔基体标记蛋白GM130共定位，提示在完全分化的体细胞中，Stau2与高尔基体复合物相关联，而非与纺锤体存在关联。通过特异性吗啉代寡核苷酸介导的Stau2敲低，会破坏卵母细胞内的纺锤体形成、染色体排列及微管-动粒附着过程。大多数卵母细胞停滞于MI期，且动粒处的MAD1信号明亮，表明纺锤体组装检验点（spindle assembly checkpoint, SAC）被激活；部分卵母细胞滞留于第一次减数分裂末期（TI），提示第一极体排出受到抑制。综上，本研究数据证实Stau2对于小鼠卵母细胞的纺锤体形成及减数分裂进程的适时推进具有不可或缺的作用。