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

Est1，更短的端粒1，最初是作为端粒酶机制的辅助因子被发现的。酵母中Est1的缺失导致端粒缩短和细胞衰老。脊椎动物Est1， Smg6也参与非义介导的mRNA衰变（NMD），降解带有过早终止密码子（PTC）的异常mRNA，这可能是由选择性剪接（AS）产生的。AS事件在包括细胞增殖、细胞死亡和胚胎发育在内的各种细胞过程中具有深远的作用。超过90%的人类多外显子基因是选择性剪接的；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平衡了基因同种异构体的储存库，并有助于发育和组织稳态的生物过程。我们最近的研究表明，由于胚胎干细胞分化受阻，Smg6缺失导致早期胚胎死亡。Smg6调控干细胞自我更新、分化和发育的机制尚不清楚。项目目标：(1)研究Smg6的生物学功能；(2)研究Smg6-NMD调控干细胞维持和分化的特异性靶点；(3)通过小鼠和细胞模型了解Smg6在干细胞维持和衰老中的功能。这个项目将受益于有趣的初步结果和我们实验室在小鼠模型和干细胞方面的专业知识。我们将采用以下方法：(1)具有Smg6敲除等位基因的小鼠、胚胎干细胞和体细胞的产生和表征；(2) RNA-seq和PAR-CLIP测序鉴定Smg6特异性靶点；(3)体外和体内分化试验；(4)在特定干细胞区室，即中枢神经系统中缺失Smg6。本研究将促进我们对干细胞维持、组织稳态、再生和健康衰老的认识。

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.