Preliminary Analysis Of NMD Pathway In Ciliates Euplotes Octocarinatus

The eukaryotic gene expression involves many processes.mRNA is a key intermediate during the process from DNA transcription to protein translation,which may occur in any link of DNA replication,transcription and post-transcription processing.Therefore,eukaryotic cells have evolved a series of mechanisms for gene repair and mRNA quality monitoring,which ensure the accuracy of gene expression by monitoring and degrading abnormal transcripts at the translation level.The nonsense-mediated mRNA decay is an important mechanism for mRNA quality monitoringNMD can distinguish and degrade aberrant transcripts harbouring premature termination codons(PTCs),to avoid the accumulation of truncated protein and potential damage to cells.Although NMD pathway exists from fungal yeast to mammalian cells,the detailed molecular mechanism of NMD pathway is still not fully elucidated.Through the study of NMD pathway in protozoa(Euplotes octocarinatus),on the one hand,we can better explain and understand NMD from the perspective of evolution;On the other hand,as an early branch of eukaryotes,the study on NMD of protozoa will help to elucidate the molecular mechanism of NMD in higher eukaryotes.Firstly,we identified the five NMD factors from Euplotes genome,including EuUPF1,EuUPF2,EuY14 a,EuY14b and EuMAGO.Among them,EuUPF1 and EuUPF2 are the core factors of NMD pathway,and EuY14 a,EuY14b and EuMAGO are the components of exon-exon junction complex(EJC).Based on bioinformatics analysis,using experiments of protein interaction in vivo(Yeast Two-hybrid),we confirmed the interacting relationship between EuUPF1 and EuUPF2-CT through its CH domain,EuY14-RRM(RNA recognition motif)and EuMAGO,EuUPF1-CH domain and EuY14a-RRM,EuUPF2-CT and EuMAGO.The intensity of interaction between EuY14a/EuMAGO and EuY14b/EuMAGO were determined byβ-galactosidase activity using ONPG as substrate,showing that the interaction between EuY14 b and EuMAGO was stronger than that of EuY14 a and EuMAGO,suggesting EuY14 b might play more key role in NMD pathway.To further confirm the interacting relationship among these factors,the in vitro experiments were conducted.A series of recombinant expression plasmids were constructed and expressed in E.coli.The purified proteins were used to in vitro Pull down experiments.The results further confirmed the interaction between EuUPF1-CH and EuUPF2-CT,EuUPF1-CH and EuY14a-RRM,EuY14(EuY14a and EuY14b)and EuMAGO,EuUPF2-CT and EuMAGO.Based on the experimental results,we preliminarily established the relationship factors and the pathway of NMD in E.octocarinatus.The data of GenBank indicated that the introns content in Euplotes genome is lower than that in higher eukaryotes genome,and far higher than that in yeast genome.We speculated that the EJC-independent and EJC-dependent pathways might coexist in ciliates Euplotes cell.EJC-independent NMD pathway suggested that the CH domain of EuUPF1 could directly interact with EuY14 a,and EuY14 a through its RRM domain combined with abnormal mRNA.On the other hand,EJC-dependent pathways suggested that EuUPF1 were recruited to abnormal mRNA through EuUPF2,EuMAGO,and EuY14(EuY14a and EuY14b)factors.In general,EuUPF1 recognize and combine with PTC-mRNA mediated by two ways to recruite various nucleases to degrade abnormal mRNA.However,the detailed molecular mechanism of the NMD pathways in Euplotes remaines to be further studied.