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

Gene expression involves many complex and delicate regulatory processes in eukaryotes.Nonsense-mediated m RNA decay(NMD)pathway can recognize and degrade premature termination codon(PTC)containing m RNAs,prevent the synthesis of truncated proteins that could be harmful to the cell.It can also regulate the expression level of about 10%-20% normal physiological m RNA or non-coding RNA to ensure the homeostasis of cells.Meantime,it is closely related to embryonic development,cellular immunity and nervous system development.Therefore,it is of great significance to study the mechanism of NMD pathway.The core mechanism of the NMD pathway is the recognition and degradation of m RNAs containing nonsense codons.So far,there are three models that tend to be mainstream: the exon junction complex(EJC)model represented by mammals;the 3'-UTR(untranslated sequence)model represented by fruit flies;the downstream sequence elements(DSE)model represented by Saccharomyces cerevisiae.However,the exact mechanism is stll unclear.By studying the NMD pathway in protozoa Giardia lamblia,based on the particularity of its evolutionary status,it will provide theoretical and data support for elucidation of the detailed molecular mechanism of the NMD pathway in higher organisms in terms of evolution and molecular mechanism.Previous report in our laboratory showed that after the formation of SURF(SMG1-UPF1-e RF1-e RF3)complex at PTC of m RNA,UPF1 was phosphorylated and the NMD pathway was activated,but the degradation mechanism of downstream nonsense m RNA after its activation has not be studied.In this paper,the related factors involved in the NMD pathway of G.lamblia were obtained with the support of bioinformatics data,like G.lamblia up-frameshift 1(Gl UPF1),G.lamblia HRP1(Gl HRP1),G.lamblia 5'-3' exoribonuclease 1(Gl XRN1)and G.lamblia 3'-5' superkiller protein 7(Gl Ski7p).By constructing Gl UPF1,Gl UPF1(1-500 aa)and Gl UPF1(501-1 304 aa)proteins with different structural domains,we analyzed the interaction between NMD factors in G.lamblia by yeast two hybridization experiment in vivo.The results showed that the full length Gl UPF1 could interact with Gl HRP1,Gl XRN1 and Gl Ski7 p.The CH domain and C termination domain could also interact with Gl HRP1,Gl XRN1(1-500 aa)and Gl Ski7 p.Taking ONPG as the substrate,the activity of ?-galactosidase was tested to analyze the interaction strength of Gl UPF1(1-500 aa)and Gl UPF1(501-1 304 aa)with Gl HRP1,Gl XRN1(1-500 aa)and Gl Ski7 p,respectively.The results showed that the interaction strength of Gl UPF1(501-1 304 aa)with Gl HRP1,Gl XRN1(1-500 aa)and Gl Ski7 p was stronger than that of Gl UPF1(1-500 aa).Further in vitro experiments were conducted to analyse the interactions between NMD pathway factors,the recombinant plasmid p ET-28a-Gl Ski7 p,p ET-28a-Gl HRP1,p ET-28 aGl XRN1(1~500aa),p GEX-6P-1-UPF1(1~500aa)and p GEX-6P-1-UPF1(501~1 304 aa)were constructed.Then expressed and purified in E.coli BL21.The interaction between the factors was further verified by Ni-NAT column pull-down experiment.The results indicated that Gl UPF1(1-500 aa)and Gl UPF1(501-1 304 aa)had strong interactions with Gl HRP1,Gl XRN1(1-500 aa)and Gl Ski7 p.Combined with the previous research results of this laboratory,we proposed a NMD pathway in protozoan G.lamblia: SURF(SMG1-UPF-e RF)complex was first formed on PTC-m RNA,then the Gl UPF1 was phosphorylated by PI3K-related kinase 1 Gl SMG1 and NMD pathway was activated;In the next step,interaction between Gl UPF1 and Gl HRP1 marked transcripts as an NMD target,then Gl UPF1 recruited 5'-3' exoribonuclease Gl XRN1 and 3'-5' exoribonuclease Gl Ski7 p to degrade aberrant transcripts in two directions.