出芽酵母中RNA剪接依赖的无义介导的mRNA衰变的机理研究

Nonsense mediated mRNA decay (NMD) is a conserved mRNA degradation pathway in eukaryotes. NMD is induced by a nonsense mutation in mRNA coding region, requires ribosomes and couples to translation. Currently we still do not understand exactly how a ribosome differentiates a premature termination condon (PTC) from the normal/native stop codon. In S. cerevisiae, many studies revealed that poly(A) tail, the downstream proximal sequence of the stop codon, plays a pivot role, which is supported by recent studies from Drosophila melanogaster, C. elegans, and human cells. It was believed that cytoplasmic poly(A) binding protein (PABP), docking on poly(A) tail, helps ribosome to confirm if the stop codon is native or prematured. However, contrastly, many studies from human cells suggested that a downstream intron has a key function in efficient NMD, via an exon junction complex(EJC), which binds to mRNA after intron spliced, acting as a platform complex to recruit key NMD factors. In our previous study, it was observed that splicing did enhance NMD in fission yeast, S. pombe. The splicing enhancement does require an intron, but regardless of the location downstream or upstream of an PTC. More interestingly, the intron enhancement in S. pombe NMD is independent of the presence of EJC. The initial experiment in S. cerevisiae also suggested that the splicing does enhance NMD. We are going to investigate the mechanism of how the splicing functions in NMD in S. cerevisiae, and whether splicing affects the eukaryotic translation or not. Furthermore, by studying the relationship between splicing and translation, we are able to reveal the important function of RNA process in the regulation of gene expression.

无义介导的核糖核酸衰减（NMD）是一种存在于真核生物细胞内高度保守的核糖核酸降解现象。NMD由信使核糖核酸的编码区中存在的无义突变所引发，需要核糖体结合到核糖核酸且依赖翻译的进行。对NMD机理的阐释一直是一个研究热点，但是在酵母和人类细胞中的研究结果仍然存在一定的不同。在出芽酵母及其他低等生物中，该降解过程取决于无义突变与下游poly（A）尾的距离；而在人类细胞的研究中表明，位于无义突变下游的内含子所导致的核糖核酸剪接，对NMD的发生有决定性的影响。 我们在裂殖酵母的相关研究中，发现核糖核酸的剪接的确可以诱发NMD；但是RNA剪接对降解的增强效应无需在无义突变的下游发生。我们在出芽酵母中也有类似的发现，拟在出芽酵母中，进一步研究核糖核酸剪接与NMD之间的关系来揭示核糖核酸剪接对翻译的影响，寻找参与该过程的剪接因子，阐述RNA剪接依赖的NMD机理，表明RNA加工在基因表达调控中的重要作用。

无义突变介导的mRNA降解（nonsense mediated mRNA decay，NMD）是在真核生物体内广泛存在的一种mRNA质量控制模式，其主要生物学功能是通过调节生物体内不同状态的mRNA的丰度，从而影响多种生物学途径。NMD途径与多种人类疾病密切相关，机制性阐释已经广泛展开，但是在高等哺乳动物和出芽酵母模式中存在诸多差异，主要集中在内含子是否在该降解过程中发挥功能，并且关键的科学问题即核糖体如何分辨提前终止密码子（pre-mature termination codon，PTC）仍然有待回答。.本项目以出芽酵母为主要模型生物，以GFP和内源性基因构建NMD报告系统，尝试多个内源性内含子，观察到RNA剪接对NMD的增强作用。确定RNA剪接与PTC的距离能够影响剪接依赖NMD的效率。从建立的剪接因子敲除菌株库，筛选确认剪接因子Prp17p参与剪接依赖的NMD过程；通过核糖体分析手段证明Prp17p与核糖体存在相关性，可能潜在地通过与核糖体地作用，影响剪接依赖的NMD效应。进行蛋白质与核酸互作的方法学研究，发现Sp1等转录因子可以通过与特殊的核酸区间结合，从而影响细胞色素基因的本底表达；确认Prp17和NMD核心复合物并不具有直接相互作用；细胞核内也未观察到相互作用。建立mRNP的串联亲和层析方法，基于MSII-MBP-TAP的纯化模式结合质谱分析，发现鉴定OLA1、TDA1和TAE1等蛋白因子可以结合mRNA从而抑制NMD，功能性验证初步表明，OLA1和TDA1可能通过相同的途径影响mRNP的组成从而影响NMD。.本项目的研究结果说明当前低等生物中的NMD模型存在不足之处，为后续关联性研究RNA剪接和NMD提供了很好的范例和证据支持，同时表明在真核生物细胞内，核内mRNA加工过程与胞质中蛋白合成存在广泛、紧密的联系。在此提示下，本研究拓展性探索了mRNP结构组成与mRNA降解途径之间的联系，创新性的例证了mRNP对NMD的实质性影响，初步说明不同的mRNP组成对NMD的效应存在强烈差异，增加了对NMD机器的方向性理解。