Functional analysis of Smg6/Est1 in stem cell maintenance and differentiation

EST1，端粒1越来越短，最初被发现是端粒酶机制的辅助因子。在酵母中，est1的缺失会导致端粒缩短和细胞衰老。脊椎动物est1，Smg6，也参与了非正义介导的mRNA衰退(NMD)，降解带有提前终止密码子(PTC)的异常mRNAs，这可能是通过选择性剪接(AS)产生的。因为这些事件在各种细胞过程中具有深远的作用，包括细胞增殖和细胞死亡以及胚胎发育。超过90%的人类多外显子基因是交替剪接的；超过60%的人类致病突变影响剪接而不是改变编码序列。据推测，NMD平衡基因异构体的储存库，并有助于发育和组织动态平衡的生物过程。我们最近的研究表明，由于胚胎干细胞分化受阻，Smg6缺失会导致早期胚胎死亡。Smg6是如何调节干细胞的自我更新和分化发育的，目前尚不清楚。该项目的目标是：(1)研究Smg6的生物学功能；(2)确定Smg6-NMD调控干细胞维持和分化的特异性靶点；(3)利用小鼠和细胞模型了解Smg6在干细胞维持和衰老中的功能。该项目将有助于我们实验室在小鼠模型和干细胞方面的有趣的初步结果和专业知识。我们将采用以下方法：(1)带有Smg6基因敲除等位基因的小鼠、ES和体细胞的产生和鉴定；(2)RNA-seq和PAR-CLIP测序以确定Smg6的特异性靶点；(3)体外和体内分化实验；(4)特定干细胞室，即中枢神经系统中Smg6的缺失。这项研究将促进我们对干细胞维持、组织动态平衡、再生和健康衰老的理解。

Est1, ever shorter telomere 1, is originally discovered as a co-factor of the telomerase machinery. Loss of Est1 in yeast causes telomere shortening and cell senescence. Vertebrate Est1, Smg6, is also involved in non-sense mediated mRNA decay (NMD) degrading aberrant mRNAs with premature termination codon (PTC), which may be produced by alternative splicing (AS). AS events have a profound role in various cellular processes, including cell proliferation and cell death as well as embryonic development. Over 90% of human multi-exonic genes are alternatively spliced; over 60% of human disease-causing mutations affect splicing rather than changing coding sequences. It is hypothesised that NMD balances the reservoir of gene isoforms, and contributes to the biological processes of development and tissue homeostasis. Our recent study shows that Smg6 deletion causes early embryonic lethality due to blocked differentiation of embryonic stem cells. How Smg6 regulates stem cell self-renewal and differentiation and development remains unknown. The objectives of the project: (1) To study the biological function of Smg6; (2) To characterize the Smg6-NMD specific targets in regulating stem cell maintenance and differentiation; (3) To understand the functions of Smg6 in stem cell maintenance and ageing using mouse and cellular models.This project will benefit the interesting preliminary results and the expertise of our lab in mouse model and stem cells. We will employ the following approaches: (1) The generation and characterization of mice, ES and somatic cells, with Smg6 knock-out alleles; (2) RNA-seq and PAR-CLIP sequencing to identify Smg6 specific targets; (3) in vitro and in vivo differentiation assays; (4) Deletion of Smg6 in specific stem cell compartment, i.e. central nervous system. This study will advance our understandings of the stem cell maintenance, tissue homeostasis, regeneration and healthy ageing.