The Investigation Upon Functions Of AGO2and L1-ORF1P In The Negative Feedback Regulation Of LINE-1

There are many repetitive sequences in the eukaryotic genomes, most of them are non-coding sequences. They play an important role in the regulation of gene transcription and translation, and the activation of mobile elements can affect the expression of adjacent genes. For example, it can cause gene mutation, expression alteration and genome remodeling if it inserts in or near the coding sequence. Among these repetitive sequences,one retrotransposon named LINE-1(L1) plays a critical role in cell proliferation and differentiation, tumorigenesis and development. Lls are inactivated in normal differentiated cells, but they are activated in the development of embryonic cells, germs and tumor cells, the activation of L1may involve in the molecular mechanisms of tumorigenesis and development.The full-length L1gene has two open reading frames, ORF1and ORF2, encodes two proteins named L1-ORF1p and L1-ORF2p. L1-ORF1p can bind its own nucleic acid and form RNP, while L1-ORF2p has endonulease and excision enzymes activity, both of them are required for its retrotransposition. From the results of our lab, ORF1is the key factor of the regulation of cell proliferation and tumorigenesis; It is reported that the first endo-siRNA is derived from the transcription of L1. L1is also considered as a gene of long non-coding RNA (lncRNA), since the5’UTR of L1encompasses both sense and antisense promoters by which generates a double string RNA (dsRNA). Given that many IncRNAs have the conservative secondary structure, particular splicing forms and diversified subcellular localization, implicating that L1may play an important role in the regulation of cell proliferation.Regarding dsRNA mediated regulation, RISC (RNA Inducing Silence Complex) is an important step for silencing of target gene. Argonaute, a kind of conservative basic proteins with weights about100kd in RISC, is considered to involve in selection and mature of siRNA/miRNA. There are four proteins named AGO1, AGO2, AGO3and AGO4in AGO protein family. AGO2is the only one which has the activity of RNase H. It can combine with siRNA to form RNA-induced-silencing-complex (RISC) and regulate the expression of the target gene. Given the co-localization of L1-ORF1p with RISC, we hypothesized that L1-ORF1p probably interacts with some components of RISC. The interaction of L1-ORF1p with Argronaute family members (AGO1/AGO2/AGO3/AGO4) was investigated and the results indicated that AGO1/AGO2/AGO4, but not AGO3, form a immuecomplex with L1-ORF1p. AGO2, a key component in RISC for siRNA regulation, was investigated further. With RNAi approach, AG02was down-regulated and its effect on the promoter activity of L1-5’UTR was observed by luciferase report system. It is indicated that the promoter activities of L1-5’UTR with full-length but not with other truncated mutants were increased, suggesting a siRNA-mediated mechanism. With overexpression of L1-ORF1p, we found that L1-ORF1p could suppress the promoter activity ofll L1-5’UTR in different constructs regardless its length and AG02can not modulate this inhibition. It is indicated that in spite of the interaction of L1-ORF1p with AG02, but, however, AG02is not involved in L1-ORFlp-mediated negative regulation to L1-5’UTR and the mechanism need to be investigated further.L1, a parasite element located in human genome, is recruited by host cell to participate some important regulations including its feedback regulation during evolution. With the feature of widespread distribution in human genome and activation in tumor, L1will facilitate us to understand the mechanism of tumorigenesis and develop a new anti-tumor drug.