RNA结合蛋白PABPN1L在调节小鼠早期胚胎发育中的作用及机制研究

Defect of decay of maternal mRNA, leads to an arrest of embryogenesis. Sability and clearance of mRNA depends on regulation of mRNA poly(A) tail by RNA binding protein. However, the mechanism of mRNA clearance during maternal-zygotic transition (MZT) is still unclear. We focus on a ovary specific RNA binding protein, PABPN1L (poly(A)binding protein nuclear 1-like), which is based on proteomic study from State Key Laboratory of Reproductive Medicine. PABPN1L-null females produced morphologically normal oocytes but were infertile, owing to early developmental arrest. We found an interaction of PABPN1L and EIF4G, which suggests PABPN1L bridged EIF4G-BTG4-CNOT7, a deadenylase, and facilitated decay of maternal mRNA. We will find the details of female infertile by PABPN1L defect. We will profile and identify target RNAs of PABPN1L and investigate the role of PABPN1L-RNA complex during embryo development,thus contributing to reducing early embryonic development block and improving the success rate of IVF.

母源mRNAs清除异常可导致胚胎发育阻滞，母源mRNAs的稳定依赖于poly(A) 尾的调控，已知RNA结合蛋白可参与mRNA 的poly(A) 尾部调节，然而母源mRNAs清除过程中RNA结合蛋白的调节机制尚未完全阐明。PABPN1L是我们前期关注到的卵巢特异的RNA结合蛋白。敲除小鼠模型发现缺失该蛋白导致雌性不孕、早期胚胎发育异常；RNA深度测序结果提示PABPN1L敲除后母源mRNAs降解异常。CO-IP分析发现PABPN1L可与EIF4G相互作用，提示其可能参与去腺苷酸化酶EIF4G-BTG4-CNOT7通路介导的母源mRNAs清除。本题拟对PABPN1L敲除后潜在的未降解母源mRNAs进行验证和功能研究，探索该蛋白与EIF4G相互作用及调节mRNA稳定的机制，全面阐明PABPN1L在小鼠早期胚胎发育中的作用，为降低早期胚胎发育阻滞、提高IVF 成功率提供理论基础。

不孕症影响全球10%-15%的家庭。然而，早期胚胎发育异常引起的女性不孕的发病机制尚不清楚。我们建立了一个模拟人PABPN1L剪接异常的小鼠模型（Pabpn1l -/-），发现雌性不育，雄性可育。Pabpn1l-/-卵母细胞可以正常产生、排卵和受精，但不能超过2细胞期。利用RNA-Seq，我们发现Pabpn1l-/-MII卵母细胞中RNA的大规模上调。在Pabpn1l-/-MII卵母细胞上调的2401个转录本中，有1523个转录本（63.4%）在Btg4-/-MII卵母细胞中上调，而在Ythdf2-/-MII卵母细胞中只有53个转录本（2.2%）上调。我们发现到来自Pabpn1l-/-卵母细胞的合子转录物的poly（A）尾比对照组长，这一现象与Btg4-/-小鼠相似。令人惊讶的是，这些mRNAs的poly（A）尾在Pabpn1l-/-MII卵母细胞中明显短于Pabpn1l+/+卵母细胞。这些结果表明，Pabpn1l参与了BTG4介导的母体mRNA降解，并且在MII期卵母细胞中，可能独立于参与母体mRNA降解而拮抗卵母细胞poly（A）尾缩短。因此，我们证实PABPN1L变异可能是女性不育的遗传标记。