Reversed. L1 Transposon Coding Sequence Of ORF1 Function

It is found recently that amount of repeat sequences in human genome, which were considered as"junk sequence", have significant functions. Most of them are composed of mobile elements, including transposon and retrotransposon. Retrotransposon are categorized into long terminal repeat (LTR) sequences and non-long terminal repeat sequences which including SINEs (short interspersed nuclear elements) and LINEs (long interspersed nuclear elements). In the LINEs, LINE-1 is the major member and appears frequently in the form of 5'truncated. The full-length LINE-1 gene encoded two proteins, termed ORF1p and ORF2p, which are required for its retrotransposition. The mechanism termed Target-primed reverse transcription (TPRT) is considered to be responsible for this process.The activity of LINE-1 in the normal differentiation cells is constrained by promoter hypermethylations, whereas, in the developing germ cells, early embryonic tissues and tumor cells, LINE-1 is active. It is demonstrated that LINE-1 is activated in almost all tumor cells tested so far,suggesting that it may involve common mechanisms of tumorigenesis. Apparently, it will help us to understand the regulation of tumor cells and also give a clue for developing molecular target drugs through LINE-1 investigation.Among two proteins encoded by LINE-1, ORF2p encompasses nucleic acid enzymes and exonuclease activity, while ORF1p has the activity of nucleic acid binding with its own RNA to form RNP. Compared to ORF2p, the facts of excessive expression of ORF1p in tumor cells and homology lack with all known protein suggests that ORF-1p may play an important role beyond traditional retransposition in tumor cells. In this study, we plan to explore the function of ORF-1p as follow:(1) The characterization of ORF-1p by overexpression in transfected cells.To character ORF-1p in regulation of cells, the full-length L1-ORF1(human and mouse) and different mutants were prepared and constructed. These recombinant expression vectors were transfected HEK293 and NIH3T3 respectively depended gene derivation. The expressions were confirmed by the approaches of RT-PCR, Western Blot and flow cytometry. The results show that ORF1p in both human and mouse can significantly promote cell proliferation, whereas the mutants (L1(ORF1)48 encompassed a stop code in 48th amino acid of full-length sequence and L1(ORF1)m encompassed many mutant codes in full-length sequence) didn't exhibit any contribution to promote cell proliferation However, the mutant of L1(ORF1)109 encompassed a stop code in 109th amino acid of full-length sequence can promote cell proliferation significantly and this phenomenon fails to show in transfected cells with the gene of N-terminal 109 amino acids With transfected cells, the characters in cell cycle were assessed and shown that the expressions of ORF1p (either human or mouse) and L1(ORF1)109 can significantly reduce the S phase in NIH3T3 cells and HEK293 respectively, whereas the mutants have no significant effect on the cell cycle. This result is consistent with the cell proliferation assay. So the expression of ORF1 gene can promote cell proliferation and RNA with correct conformation is probably responsible for its function.(2) The identification of cis regulation of L1-ORF1 sequences by recombinant construction with reporter gene :Based on the rheostat function of LINE-1 acting in genome, we investigate its cis regulation upon expression of adjacent gene. With the reporter gene system, different lengths of L1-ORF1 gene, which included full-length gene, 2/3 5'truncated gene, 1/3 5'truncated gene,were inserted into the upstream and downstream of. luciferase gene respectively.. The expression of luciferase were detected in transfected cells and shown that L1-ORF1 gene with different lengths could inhibit the expression of the reporter gene located in either upstream or downstream of reporter gene. In downstream of reporter gene, L1-ORF1 suppresses the expression of luciferase with a length-dependent manner, but however, in upstream of reporter gene, 1/3 5'truncated gene exhibits the most significant suppression to reporter gene cpmpared to other constructs.(3) The investigation of mechanism of L1-ORF1 for its cis regulation.Given the significant suppression of L1-ORF1to expression of agjacent gene, the mechanism was investigated by epigenetics approach. By treating with 5-aza-dc (the inhibitor of the methytransferase) in transfected cells, the suppression of expression of reporter gene elicited by L1-ORF1 was attenuated significantly. So it is suggested that the cis regulation of L1-ORF1 sequence to adjacent gene may attribute to DNA methylation.Taken together, given the ubiquitious activity in tumor cells, L1-ORF1 apparently contribute to tumorigenesis; Beyond traditional retrotransposition, L1-ORF1 has some important roles through cis-and trans-regulation in tumor cells; What's more, L1-ORF1 probably acts as both coding and non-coding sequence in the process of regulation.So, as a promising molecular target, L1-ORF1 open a new avenue for cancer therapy.