| Eukaryotic translation initiation factor 5A(eIF5A)is currently the only protein in cells known to contain hypusination modification.It was originally thought to function in the translation initiation,however,subsequent research has shown that it also plays an important role in the elongation and termination of translation.eIF5 A is a highly conserved translation factor and has been found to be widespread in eukaryotes,archaea and bacteria,and it is a key factor in the process of protein synthesis,but its biological function is not very clear so far.Euplotes octocarinatus is a freshwater ciliate,a single-celled eukaryotic organism.It exhibits nuclear dimorphism,and it has a unique way of m RNA translation,the universal stop codon UGA is translated into cysteine and high frequency programmed ribosomal frameshifting(PRF)occurs during translation.Previous studies have showed that eIF5 A of slime molds,alfalfa,fruit flies and humans can substitute for eIF5 A in yeast.As a singlecelled eukaryotic ciliate,can its eIF5 A function as a substitute for yeast eIF5A? In order to explore the conservation and specificity of the biological functions of E.octocarinatus eIF5 A and analyze its role in the process of protein translation,we cloned the eIF5 A gene of E.octocarinatus,and analyzed its sequence,studied its expression level,and transferred it to temperature-sensitive yeast strains to discuss the conservation of its biological functions.The main results are as follows:1.Identification and expression analysis of the eIF5 A gene of E.octocarinatus.Two Eo-eIF5 A genes were searched in the E.octocarinatus genomic database.Further analysis found that the Eo-eIF5A2 gene may need to occur-1 PRF to produce a complete protein.The sliding sequence is "AAA AAG UAG".Using single-cell PCR and sequencing analysis,this phenomenon was preliminarily verified at the nucleic acid level.The absolute quantitative results of real-time fluorescent quantitative PCR(q PCR)showed that the copy numbers of Eo-eIF5A1 and Eo-eIF5A2 were basically the same,and there is a significant difference in the expression level of the two at the transcription level,the m RNA level of Eo-eIF5A1 was about 1.5 times that of Eo-eIF5A2.Hypoxia stress was applied to E.octocarinatus and it was found that the relative expression of Eo-eIF5A1 and Eo-eIF5A2 genes increased.2.Analysis of the functional conservation of eIF5 A of E.octocarinatusThrough multiple sequence alignment of eIF5 A of different species,it is found that the sequence identity of Eo-eIF5A1 and Eo-eIF5A2 is low.Compared with other eukaryotes,the N-terminus is about 10 amino acids less.The phylogenetic tree analysis of 34 different species of eIF5 A revealed that the eIF5 A of Euplotes gathered into a single group,which has a certain degree of particularity.However,the analysis of its high-level structure revealed that the tertiary structure of Eo-eIF5 A is highly similar to that of other eukaryotes.Amplify the Eo-eIF5A1 gene and Eo-eIF5A2 gene by PCR,and connect them to the p RS315 vector to construct yeast-related expression plasmids and transfer them into temperature-sensitive yeast strains tif51A-1(both 25? and 37? can grow normally)and tif51A-3 for analysis and tif51A-3(it can grow normally at 25?,but cannot grow at 37?)for analysis.the results show that Eo-eIF5 A cannot remedy the growth defect phenotype of temperature-sensitive yeast strains,indicating that Eo-eIF5 A cannot functionally replace SceIF5 A,and the function of Eo-eIF5 A is not very conservative.Using the same temperaturesensitive yeast strain to study the eIF5 A of the other two ciliates(Oxytricha trifallax,Ot and Tetrahymena thermophile,Tt),it was found that Ot-eIF5 A could not remedy the growth defect phenotype of temperature-sensitive yeast strains,while Tt-eIF5 A can remedy the growth defect phenotype of temperature-sensitive yeast strains,indicating that this phenomenon is not a special case of E.octocarinatus.Later,the protein expression of its yeast strains was analyzed,and the results showed that the temperature was 25°C and 37°C,Eo-eIF5A1,Ot-eIF5 A and Tt-eIF5 A proteins in the tif51A-1 yeast strain can be expressed normally.It is suggested that the eIF5 A of E.octocarinatus and Oxytricha trifallax can't remedy the growth defect of temperature-sensitive yeast strains in yeast,which may be due to the modification defect after expression.3.Identification and analysis of key sites function of eIF5 A of E.octocarinatusIn order to analyze the reasons of Eo-eIF5 A could not remedy the growth defect of temperature-sensitive yeast strains,the key sites related to the function of E.octocarinatus eIF5 A were identified and analyzed.First,the N-terminal deletion sequence of Eo-eIF5 A was filled,and transferred it to temperature-sensitive yeast strains for research,and it was found that the N-terminal deletion sequence had no effect on the function of Eo-eIF5 A in yeast;then the 12 amino acids near the modification site of Eo-eIF5 A hypusination were mutated,and the results showed that this site does not affect the function of Eo-eIF5 A in yeast.Finally,the two sites were mutated at the same time,and the results show no difference from a single mutation.By constructing a chimera of the eIF5 A gene of yeast and E.octocarinatus,and studying the effect of the N-terminal and C-terminal domains on the function of Eo-eIF5 A,it was found that neither of the two chimeras could remedy the function defects of yeast,it suggests that there may be key sites on these two domains that affect their functions at the same time.In summary,in this study,two eIF5 A genes were identified and analyzed in E.octocarinatus,and both have transcription activity.Sequence analysis shows that EoeIF5A2 may need to occur-1 PRF,and through single-cell PCR and sequence alignment,preliminary verification from the nucleic acid level,and then using q PCR found that the expression levels of Eo-eIF5A1 and Eo-eIF5A2 are significantly different at the transcription level,and after hypoxia stress,the expression of both has increased.By constructing the yeast-related expression plasmids of Eo-eIF5 A and transferred it into temperature-sensitive yeast strains,and observing its growth,it was found that Eo-eIF5 A could not remedy the growth defect of temperature-sensitive yeast strains,it indicated that the function of Eo-eIF5 A is not very conservative.Subsequently,the N-terminal deletion sequence of the Eo-eIF5 A gene and the mutation of the hypusination modification site were studied and found that only mutating these two sites in Eo-eIF5 A to the corresponding sequence of yeast eIF5 A cannot substitute for eIF5 A in yeast.Moreover,the chimera formed by the yeast eIF5 A gene and Eo-eIF5A1 gene still cannot remedy the yeast defect,it indicated that there may be key sites in the N-terminal and C-terminal domains that affect the function of Eo-eIF5 A at the same time.This study provides experimental data for the follow-up study of the biological functions of Eo-eIF5 A. |
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