L1-ORF1p and Ago2 are involved in a siRNA-mediated regulation for promoter activity of L1-5’UTR
Abstract
Introduction. Long interspersed nuclear elements-1 (L1), as the only one self-active retrotransposon of the mobile element, was found to be generally activated in tumor cells. The 5‘UTR of L1 (L1-5’UTR) contains both sense and antisense bidirectional promoters, transcription products of which can generate double-strand RNA (dsRNA). In addition, L1-ORF1p, a dsRNA binding protein encoded by L1, is considered to engage in some RNA-protein (RNP) formation. Ago2, one of the RISC components, can bind to dsRNA to form RISC complex, but its role in L1 regulation still remains unclear. Due that the 5‘UTR of L1 (L1-5’UTR) contains both sense and antisense bidirectional promoters, so the activities in both string were identified. A dsRNA-mediated regulation of L1-5’UTR, with the feedback regulation of L1-ORF1p as well as other key molecules engaged (Ago1–4) in this process, was also investigated.
Material and methods. Genomic DNA was extracted from HEK293 cells and subjected to L1-5’UTR preparation by PCR. Report gene system pIRESneo with SV40 promoter was employed. The promoter activities of different regions in L1-5’UTR were identified by constructing these regions into pIRESneo, which SV40 region was removed prior, to generate different recombinant plasmids. The promoter activities in recombinant plasmids were detected by the luciferase expression assay. Western blot and co-immunoprecipitation were employed to identify proteins expression and protein-protein interaction respectively.
Results. Ago2 is a member of Agos family, which usually forms a RISC complex with si/miRNA and is involved in post- transcriptional regulation of many genes. Here L1-ORF1p and Ago2 conducts a regulation as a negative feedback for L1-5'UTR sense promoter. L1-ORF1p could form the immune complexes with Ago1, Ago2 and Ago4, respectively.
Conclusions. L1-5’UTR harbors both sense and antisense promoter activity and a dsRNA-mediated regulation is responsible for L1-5’UTR regulation. Agos proteins and L1-ORF1p were engaged in this process.
Keywords: L1-5’UTRAgo2L1-ORF1pantisense promotersiRNAHEK293 cellsprotein interactions
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