SMG1-UPF1-eRF1-eRF3复合体参与贾第虫无义介导的mRNA降解激活

无义介导的mRNA降解（NMD）是一种重要的真核生物mRNA质量监控途径。NMD可识别并降解含有提前终止密码子（PTC）的异常mRNA（PTC-mRNA）。但NMD途径对PTC-mRNA的识别和降解机制尚无阐明。蓝氏贾第虫（Giardia lamblia）是一种寄生性的原生动物，进化上处于真核生物基部，对其NMD途径的研究有利于了解NMD途径的机制与进化。本研究通过双分子荧光互补实验、酵母双杂交实验和体外pull-down实验，分析了贾第虫的UPF1 (GlUPF1)、SMG1 (GlSMG1)和肽链释放因子（GleRF1、GleRF3）之间的相互作用关系。结果表明，贾第虫的肽链释放因子都能够与GlUPF1发生相互作用，且GlUPF1的CH结构域与GleRF3能够形成较稳定的复合体，而GlSMG1的激酶结构域PIKK能与UPF1的C端和N端结构域相互作用。进一步研究证实，GlSMG1的PIKK结构域能使GlUPF1两种截短体GlUPF1（1~500 aa）和GlUPF1（501~1 304 aa）发生磷酸化修饰，说明GlUPF1 的N端和C端均有GlSMG1的磷酸化位点。进一步分析证实，T111是GlUPF1上的1个磷酸化位点。我们的研究结果表明，贾第虫NMD途径起始阶段，首先在mRNA的PTC处的核糖体上形成SMG1-UPF1-eRF1-eRF3(SURF)复合体，并且GlSMG1磷酸化修饰GlUPF1，由此激活NMD途径，可能招募XRN1和SKI7d等酶参与无义mRNA的降解。

SMG1-UPF1-eRF1-eRF3 Complex Is Involved in the Nonsense-mediated mRNA Decay in Protozoan Giardia lamblia

Nonsense-mediated mRNA decay (NMD) is an important mRNA quality-control mechanism in eukaryotes. It can identify and degrade abnormal mRNAs containing premature termination codon (PTC). However, we have known insufficiently the mechanism about recognition and degradation of PTC-mRNA in NMD pathway. Giardia lamblia is a kind of parasitic protozoan, located on the root in eukaryotic evolution. The studies on NMD of G. lamblia are useful to understand the mechanism and evolution of NMD pathway. In this study, the interaction between GlUPF1, GlSMG1 and polypeptide release factors of G. lamblia was confirmed by using bimolecular fluorescence complementation (BiFC) assays, yeast two hybrid assays and the in vitro pull-down experiments. The results showed that polypeptide release factors of G. lamblia could interact with GlUPF1, and the CH domain of GlUPF1 and GleRF3 could form a stable complex. The PIKK domain, the kinase domain of GlSMG1, could also interact with both C-terminal and N-terminal domains of GlUPF1. In addition, we confirmed that the truncated UPF1 (1-500 aa) and UPF1 (501-130 4 aa) could be phosphorylated by PIKK. Furthermore, we identified that T111 might be a phosphorylation site of GlUPF1. Altogether, our results showed that a SMG1-UPF1-eRF1-eRF3 (SURF) complex was firstly formed on the ribosome pausing on the PTC-mRNA in G. lamblia, and then GlSMG1 phosphorylated GlUPF1 to activate the NMD pathway. Finally, RNAases such as XRN1 and SKI7 might be recruited to participate in mRNA decay process.