Preliminary Clonal Analysis Of Extracellular RNA And Screening MiRNA-483 Precursor Associated Proteins By Yeast Three-Hybrid Technology

RNA is widely involved in every process of life, and plays an important role in it. However, the extracelluar RNA (eRNA) is seldom known to us. Although the discovery of microRNA (miRNA) gave us a thorough insight to the regulation of gene expression, the exact process of the pre-miRNA is not clear.In order to elucidate the biology function of eRNA, this study confirmed the existence of cell surface RNA and analyzed the change of RNA content in culture medium, using the HepG2 cell model, through the straggling effect of RNase A on cell and cell immunofluorescence experiment using RNA antibody. We enriched the RNA in culture medium by cryochem, and cloned them by our laboratory established method of RNA cloning. At the same time, we used microRNA-483 precursor which was identified by our laboratory as the bait, to screen the fetal liver cDNA library for its copartner. Major progresses as follows:1. RNA antibody detection (Immunofluorescence assay) confirmed the fact of presence cell surface RNA directly.2. RNase A was found to enhance the migration capacity and straggling activity of HepG2 cell, and increase the amount of pseudopod on cell surface.3. The dynamic change of extracellular RNA content in HepG2 cell culture medium as well as on cell surface was studied.4. The method of free-dried concentration of RNA in culture medium was established, and the clone method of RNA in culture medium was set up, some RNA in culture medium were acquired, including rRNA fragments, human elongation factor 1 a subunit, mitochondrial RNA fragment and rickettsiae RNA, one 28nt RNA on the 15th chromosome in CpG island with the high similaritywith the newly discovered piRNA.5. A candidate of microRNA-483 precursor interaction was acquired by yeastthree-hybrid technology, and cl4orf108 and ZNF43 has been verified by rotatingcertification.The role of RNA in life activities has been recognized. Extracellular RNA research has also shown signs of emerging, and this study confirmed the presence of extracellular RNA, and established the method of extracellular RNA extraction and cloning, acquiring a number of featured RNA; a new method of research on eRNA was established. It can be used for the detection of diseases, as well as lay a good foundation on in-depth studies of RNA in life activities. The microRNA-483 interaction protein was obtained through the yeast three-hybrid screening technology, providing clues on microRNA-483 processing mechanism as well as microRNA function in a new way.