Studies On The Localization Of RNA Polymerase Ⅲ Transcription In HeLa Cell Nuclei

The nuclei of eukaryotic cells contain three types of RNA polymerases: polymerase I, II and III. They perform different functions in the cells. RNA polymerase I synthesizes the three largest rRNAs. RNA polymerase II mainly produces mRNA encoding proteins and most of sn RNAs, and RNA polymerase III makes 5S rRNA and tRNA, as well as a few small nuclear RNAs.The transcription sites of the polymerases have been studied since early 1980s. The results of these early research work showed that RNA polymerase III transcription was localized in the nucleoplasm. However, with the development and the application of new technologies since 1990s, the controversy arose on the transcription sites of RNA polymerase III. In recent years, more and more scientists presumed that RNA polymerase III transcription might not occur in the nucleoplasm but in the nucleoli. Nevertheless, the possibility has not been proved directly.In this dissertation, the plasmids containing 5s promoter were transfected into HeLa cells,The transcription sites of RNA polymerase III and its transcripts were detected by fluorescence in situ hybridization(FISH) to DNA, RNA and DNA-RNA, respectively. It was showed under the laser scanning confocal microscopy that: for DNA level FISH, 81% of the DNAs were in the nucleoli and at the periphery of the nucleoli and 19% in the nucleoplasm; for RNA level FISH, 22% of the RNAs were in the nucleoli, 78% in the nucleoplasm and at the boundary between it and the nucleoli; for DNA-RNA level FISH, 25%, 46% and 29% of the DNAs or RNAs were in the nucleoli, at the periphery of the nucleoli and in the nucleoplasm, respectively. From these results, it is inferred that the active transcription of pol III occurs in the nucleoli and its periphery region, but not the nucleoplasm. The result will help us to further comprehend the mechanism of RNA polymerase III transcription, the way of its transcripts processing and transport, and the structural and functional relationship among the three RNApolymeraes.