Preliminary Study On The Function Of Retrotransposon LINE1 In Cold Adaptation Of Cells

Retrotransposons are reverse transcriptase-mediated replicative nucleotide elements that are abundant in metazoan genomes and play important roles in genome evolution.One group of the retrotransposons,the long interspersed nucleotide elements?LINEs?arise early in evolution which date back at least 600 million years and are very popular in vertebrate genomes.They account for 21%of the human genome and more than 10%of the zebrafish genome.LINE1?L1?RNA contains two main open reading frames:ORF1encodes nucleic binding protein and ORF2 encodes an endonuclease and reverse transcriptase.LINE1 propagate through a copy-and-paste mechanism that consists of several consecutive steps:transcription,transporting to the cytoplasm,translation,formation of protein ribonucleic complex,returning to nucleus,target-site primed reverse transcription and insertion in the new site.Previous studies have found that propagation of some of the retrotransposons in Antarctica Dissostichus mawsoni mRNA have been increased and may be related to the low temperature environment.A complete and biologically active retrotransposon sequence was analyzed and named Dissostichus mawsoni LINE1?dmL1?.We first constructed the expression vector pc-l1-1FH containing human L1?hmL1?fragment.Through western blot and immunofluorescence analysis,we found that hmL1 had retrotransposition activity and high expression in cytoplasm.Some differentially expressed genes and cell-substrate junction cellular component were analyzed by RNA-seq for Hela cells transfected pc-l1-1FH and control plasmid.Then we employed a reporter system to investigate the cold inducibility of LINE propagation using LINE elements derived from fishes and human.In this system we can assess retrotransposition levels by relative expression of EGFP RNA.In this work we attempted overexpression of dmL1 and hmL1in Hela cells then treated cells in the low temperature for 7 days.The RT-qPCR results showed that the level of L1 mRNA and retrotransposition was increased when facing low temperature.We conclude that L1retrotransposition activity was cold-induced.We found that ROS?Reactive oxygen species,ROS?accumulation was accompanied by a fracture of the DNA strand and L1 retrotransposition activity increment.Studies have shown that ROS production is associated with retrotransposon.To analyze the relationship between the L1retrotransposition and ROS accumulation under cold,we added the H₂O₂ to the medium.As the H₂O₂ gradient increases and we found that propagation of L1 in the cells was increased and reached maximum at 0.5mM.Then we removed ROS by adding anti-oxidative agent?N-acetylcysteine,NAC?,and found that expression of exogenous L1 decreased in the cells.We conclude that the retrotransposition of L1 under low temperature was mediated by the release of ROS within the cell.We also found that the survival rate of Hela cells transfected with L1was increased in our lab's preliminary work.By immunofluorescence staining of the G3BP1 and found that dmL1 retrotransposition can also reduce stress granules in the cells under low temperature pressure and eventually lead to alleviating the intracellular stress.Finally,to illustrate whether the phenomenon of cold-induced dmL1retrotransposition found in human HeLa cells is repeatable in fish,we conducted further experiments in zebrafish ZF4 cells and zebrafish and got the same conclusions in the cells.We found that zebrafish endogenous ZFL2-1 mRNA showed high expression in brain and gonad after the hypothermic stimulation.The zebrafish acclimated in 20?showed doubled copy number in gonad.We've already come to conclusion that the phenomena cold-induce retrotransposition goes for zebrafish as well.In conclusion,in this study the cold-induced dmL1 retrotransposition was found to be mediated by ROS/DNA breaks pathway.In addition,the cold-induced LINE propagation may in turns alleviate the intracellular stress.The study of L1 propagation and its effects on physiological state of cells provides us with a new insight of adaptive evolution under low temperature.