The Initiative Mechanism Of Nonsense-mediated MRNA Decay Pathway In Protozoan Giardia Lamblia

Nonsense-mediated mRNA decay(NMD)is a translation-dependent mRNA quality-control mechanism in eukaryotes.It can identify and degrade abnormal mRNAs containing premature termination codon(PTC),protecting the cells from the harm of truncated proteins.The present researches of NMD mechanism mainly focused on the identification of PTC-mRNAs,the activation mechanism of NMD pathway and the degradation mechanism of abnormal mRNA.There were many researches of higher eukaryotic NMD pathway,but the research of protozoan NMD pathway was scarce.In order to study the evolution and molecular mechanism of NMD pathway,we chose Giardia lamblia,located on the root of eukaryotic evolution,as experimental material to study the mechanism of its NMD pathway.This study aims to clarify characteristics of NMD mechanism of protozoan Giardia lamblia,which will provid data support for the evolution of the eukaryotic NMD pathway.In this study,we cloned genes of GlUPF1,GlSMG1 and polypeptide release factors of G.lamblia.The activation mechanism of NMD of G.lamblia was investigated by using analysis of the interaction relationship among these NMD factors and function of GlSMG1 as a kinase.Firstly,the recombinant plasmids containing GleRF1,GleRF3 and GlUPF1 were constructed.These recombinant plasmids were used for bimolecular fluorescence complementation,yeast two-hybrid and pull-down experiments.The interactions between GlUPF1 and polypeptide release factors of G.lamblia were analyzed subsequently.The experimental results showed that GlUPF1 could interact with GleRF1 and GleRF3.However,GleRF1 couldn't interact with truncated GlUPF1,but GleRF3 could interact with GlUPF1(1~500 aa).These results suggested that the interaction between GleRF1 and GlUPF1 might depend on the complete structure of GlUPF1.The region of the interaction between GleRF3 and GlUPF1 is GlUPF1(1~500 aa)which contained CH domain.The above results confirmed that GlUPF1,GleRF1 and GleRF3 could form a stable complex.Then,we cloned genes of each domain of GlSMG1(GlHEAT,GlFAT+FRB,GlPIKK).The recombinant plasmids were constructed for using in BiFC,yeast two-hybrid and pull-down experiment.The interactions between GlUPF1 and each domain of GlSMG1 were analyzed by using these experiments.The results showed that GlUPF1 could interact with each domain of GlSMG1 and GlPIKK,and the kinase domain of GlSMG1 could interact with truncated forms of GlUPF1.The quatitive experiments were conducted by using measurement the activity of ?-galactosidase.The results indicated that the interaction between GlPIKK and GlUPF1 was strongest,and the interaction between GlUPF1(1~500 aa)and GlPIKK,and the interaction between GlUPF1(501~1 304 aa)and GlPIKK were higher.It suggested that GlSMG1 could interact with GlUPF1,and GlPIKK was the main domain of the interaction between GlUPF1 and GlSMG1.GlPIKK interacted mainly with C-terminal and N-terminal domain of GlUPF1.Above results suggested that GlSMG1 could interacted with GlUPF1.Then,we would like to know the biological significance of the interaction between UPF1 and SMG1 in G.lamblia cells.The experiments of phosphorylation modification of GlUPF1 were conducted.We displayed that GlPIKK phosphorylated truncated forms of GlUPF1 by using in vitro phosphorylation reaction system,and we measured the phosphorylation level of truncated forms of GlUPF1 by using phosphoprotein phosphate estimation kit.The results showed that GlPIKK could phosphorylated GlUPF1(1~500 aa)and GlUPF1(501~1 304 aa).In order to study the phosphorylation sites in GlUPF1,we further predicted the potential phosphorylation sites of GlUPF1 online by using NetPhos 3.1 software.Predicted higher score residues Ser 72,Thr 111 and Ser 1271 in GlUPF1 were changed into Ala by site-specific mutagenesis.The mutant proteins were generated by prokaryotic expression and purification.Then,we detected the phosphorylation level of mutant proteins after in vitro phosphorylation reaction.The results showed that the phosphorylation levels of GlUPF1(1~500 aa)T111A decreased significantly,suggesting that T111 might be a phosphorylation site of GlUPF1.All of our results showed that at the initial stage of G.lamblia,the SMG1-UPF1-eRF1-eRF3(SURF)complex firstly formed at the ribosome located on the PTC of mRNA,and then GlSMG1 phosphorylated GlUPF1 to trigger NMD pathway.XRN1 enzymes might be recruited to involving in mRNA decay process by phosphorylated GlUPF1.Our results might lay the foundation for the future studies of the mechanism of NMD downstream in G.lamblia,and contribute to further understand the origins and evolutions of NMD pathway,revealed the diversity of NMD mechanism during evolution,and further provided data support for charifing NMD mechanism.