RNA降解相关蛋白Lsm1在多能性调控过程中的作用和机制研究

RNA degradation plays an important role in early embryo development and embryonic stem cell differentiation, especially for the process of zygotic gene activation. However, the key molecule and detail regulation network is unclear. This project will dissect the function and mechanism of RNA degradation in pluripotency regulation by studying the function RNA degradation related protein Lsm1 on mouse early embryo and embryonic stem cells. Our precious results show Lsm1 was dynamically changed in early embryos and embryonic stem cells, Lsm1 knock down decreased embryo development competency and induced embryonic stem cells differentiate to endoderm and mesoderm, Lsm1 bind to H2A.Z and H3.3 mRNA and induced their degradation. We will further find Lsm1 downstream genes by RNA-seq, illustrate how Lsm1 targeted histone variant affects its downstream genes by ChIP-seq, screen Lsm1 co-factors by IP-MS to interpret its specificity. This project will confirm the hypothesis that Lsm1 regulate pluripotency through changing genome epigenetic modification base on histone variant mRNA degradation, dissect the mechanism of RNA degradation in pluripotency regulation, and further modify the mechanism of pluripotency regulation.

RNA降解在早期胚胎发育和胚胎干细胞分化过程中发挥重要作用，尤其是对合子基因激活至关重要，但其核心分子和调控网络尚未深入研究。本项目以RNA降解相关蛋白Lsm1为切入点，以小鼠早期胚胎和胚胎干细胞为研究对象，揭示RNA降解在多能性调控过程中的作用机制。我们前期研究发现Lsm1在早期胚胎和胚胎干细胞中呈动态表达，敲降该基因降低早期胚胎发育能力，促进胚胎干细胞向中内胚层方向分化，初步证实Lsm1参与H2A.Z和H3.3的RNA降解过程。在此基础上，利用RNA测序寻找Lsm1下游效应基因，通过组蛋白变体ChIP测序揭示调控下游效应基因的机制。采用IP-MS寻找Lsm1共同作用蛋白，阐明特异性识别靶分子的机制。由此证实Lsm1通过调控组蛋白变体RNA降解，改变基因表观修饰水平而影响其表达，从而调控多能性这一科学问题。本研究阐明RNA降解在多能性调控中的作用，为进一步完善多能性调控机制奠定基础。

哺乳动物精子与卵子结合后完成受精过程，形成合子开启胚胎发育过程。精子和卵子受精后分别形成雌雄原核，二者在染色质结构、转录活性、表观修饰水平上均存在不对称性，其中H3K9me3修饰表现出显著的雌原核特异性，但其形成的分子机制有待深入研究。早期胚胎胚胎发育过程中，有序的表观遗传修饰对基因表达调控具有重要作用，是维持胚胎正常发育必不可少的因素。表观调节的不正常会显著降低胚胎发育能力，甚至导致胚胎致死。因此探究胚胎发育过程中表观修饰建立维持机制及其对胚胎发育的影响具有重要意义。.本研究以实验室先前早期胚胎发育蛋白质谱数据为研究基础，发现RNA降解相关蛋白Lsm1可能参与表观调控并影响胚胎发育，以此分子为研究切入点。我们发现Lsm1敲降会导致雄原核染色质出现H3K9me3修饰，在此基础上利用RNA测序和免疫荧光染色方法发现，Lsm1通过调控组蛋白H3.1/H3.3蛋白定位而非表达发挥作用。由于Lsm1为RNA结合蛋白，我们采用微量细胞RIP-seq方法检测了合子中Lsm1直接作用分子，发现其靶分子近着丝粒RNA，并调控其降解。随后我们利用敲降和过表达实验证明，近着丝粒RNA参与雌雄原核不对称H3K9me3修饰的调控，并影响早期胚胎发育。同时发现近着丝粒RNA过表达会造成雄原核H3.1比例增加而H3.3比例减少，利用RNA pull-down实验进一步证明近着丝粒RNA对H3.1具有更强的亲和性，由此我们推测近着丝粒RNA通过调控雄原核中H3.1/H3.3的比例，影响H3K9me3修饰水平。.根据以上实验，我们提出小鼠合子通过Lsm1/近着丝粒RNA/H3变体通路调控雌雄原核不对称H3K9me3修饰建立，从而调控早期胚胎发育能力这一分子机制，进一步完善早期胚胎表观修饰建立机制。