Fuctional Analysis Of UPF Factors Of NMD Pathway From Euplotes Octocarinatus,Protozoa

The importance of gene expression for organisms is exemplified by the existence of diverse molecular mechanisms that detect errors and thereby ensure the accuracy of gene expression.Nonsense-mediated mRNA decay(NMD) is a quality control mechanism for eukaryotic gene expression,which identifies and degrades nonsense mRNAs containing premature termination codons(PTCs).mRNA decay is a process dependent on intracellular protein translation that has been known to regulate the expression level of transcripts in plants,animals,fungi and ciliates.The recognition of nonsense mRNAs is the core of NMD mechanism,and UPF(up-frameshift) proteins are a group of key factors involved in the identification and degradation of nonsense mRNA.Previous studies had shown that only UPF1 and UPF2 were identified in Euplotes octocarinatus,and UPF3 conserved in human and yeast was not found.Bioinformatics analysis showed that the EoUPF2 contained three tandem MIF4G(middle domain of translation initiation factor 4G)domains and a C terminal UPF1 binding region.Most eukaryotes share the core NMD factors,however,the molecular nature of the NMD pathway varies between organisms.In this paper,in order to study the evolutionary relationships of EoUPF1 and EoUPF2 with other species,we constructed a phylogenetic tree of 26 species and 24 species in several.From the analysis of their evolutionary relationships,we found that the evolutionary relationships between EoUPF1 and EoUPF2,respectively,close to those of Saccharomyces cerevisiae.Inter Pro database analyses show that the core domains of the two are very similar.Secondly,the interaction between UPF1 and UPF2 and eukaryotic polypeptide release factor 3(eRF3) was verified by pull down experiment.We speculate that EoUPF1,EoUPF2 and eRF3 formed eRF3/EoUPF1/EoUPF2 complex in Euplotes octocarinatus.Finally,we further confirmed that the MIF4 G domain of EoUPF2 can interact with the component factors Y14a/Y14b of exon-exon junction complex(EJC) by using experiments of yeast two hybrid and pull down.In higher eukaryotic cells,UPF3 mediates the interaction of UPF2 with EJC core factor Y14,while UPF3 is absent in E.octocarinatus.How does EoUPF2 function without UPF3? The study confirmed that EoUPF2 directly interacts with EJC component factors Y14 a and Y14b through its MIF4 G domain.However,we found that the interaction between EoUPF2 and Y14 a was more significant in ?-galactosidase assay.Real-time fluorescence quantitative PCR analysis excluded the interaction between EoUPF2 and Y14a/Y14b is related to the copies of themselves.Therefore,we propose hypotheses: when the translating ribosome encounters and stalls at the premature termination codon,and the peptide chain releasing factor recognizes the termination codon.eRF3 interacts with EoUPF1 and EoUPF2 to form the eRF3/EoUPF1/EoUPF2 complex.By interacting with EoUPF1 and Eo Y14,EoUPF2 mediates the coupling between eRF3/EoUPF1/EoUPF2 complex and EJC,and initiates the recognition process of nonsense mRNA.The experimental results in this paper provide data support for the future elucidation the molecular mechanism of NMD in E.octocarinatus.